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Voyager User`s Guide

1. 4100 Current Spec XX 2 50 0 0 0 400 800 1200 1600 2000 2 400 Accum Spec XX S 50 P 0 T r T 7 T r T r 0 0 400 800 1200 1600 2000 100 Not Used X X 2 amp 50 0 0 0 400 800 1200 1600 2000 2 100 Not Used XX S 50 m 0 T T T T T r T r 0 0 400 800 1200 1600 2000 Mass m z Figure 4 5 Adding Traces When you add a specific type of trace the label of the trace changes from Not Used to the label for the type of trace created Removing traces To remove a trace from the Spectrum window 1 Select click on the trace 2 Click IM in the toolbar The trace is removed Voyager Biospectrometry Workstation User s Guide 4 15 Chapter 4 Voyager Instrument Control Panel Basics 4 3 4 Annotating Traces Two ways to You can add text annotation to traces by annotate Copying ASCII text from any source then pasting on the trace e Typing text on the trace Copying text from To copy ASCII text ASCII source 1 Openthe ASCII text file 2 Select the line of text to copy then right click and select Copy from the menu displayed NOTE If you select more than one line of text only the first line is pasted when you annotate the trace Annotating To annotate the trace the trace 4 Click on the trace at the location at which you want to insert text 2 Right click then select Paste text if you copied results or Add text annotation if you wa
2. Figure 2 13 Instrument Control Panel Installing Software Starting To start the Sequence Control Panel from the Windows NT Sequence Control desktop Panel 1 Make sure the Instrument Control Panel is running NOTE If you start the Instrument Control Panel using Sequence the Sequence Control Panel icon it functions the same Control Panel way as if you started it using the Instrument Control Panel icon with two exceptions Warning and error messages are not displayed during operation and the Instrument Control Panel will close when you close the Sequence Control Panel 2 Double click the Sequence Conirol Panel icon on the desktop NOTE You can also click a toolbar button in the Instrument Control Panel to start the Sequence Control Panel The Sequence Control Panel opens Figure 2 14 Figure 2 14 Sequence Control Panel Voyager Biospectrometry Workstation User s Guide 2 33 2 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 5 3 Exiting the Software CAUTION If you are using the Voyager Workstation and you exit the Voyager Instrument Control software you can no longer control the workstation Do not exit the Voyager software until you have finished using the workstation Sequence Control 2 34 Panel Instrument Control Panel Applied Biosystems To exit the Sequence Control software 1 In the Sequence Control window selec
3. 0 1164 1170 T 1188 1194 Mass m z 100 90 s0 70 60 50 40 Intensity 304 204 10 fr 0 T Precursor lon Selector on ec 1 BP 1296 8 538852 3161 8 1164 LS 1176 Mass m z T 1170 Figure 8 17 Comparison of Spectra with Precursor lon Selector Off and On 8 70 Applied Biosystems Exploring PSD Mode Observing prompt When operating under conditions that yield high resolution for angiotensin and when the Precursor lon Selector is turned and PSD fragments Effect of Precursor lon Selector on prompt and PSD fragments off you should observe the following Prompt fragments Well resolved fragment ions generated in the source before acceleration The sharp peak at 1 181 7 Da is the y9 ion Prompt fragments appear at the expected mass in reflector mode because their flight time is the same as a molecular ion of the same mass accelerated from the source They do not appear at the expected mass in PSD mode PSD fragments A poorly resolved peak cluster that is a mixture of the y9 fragment 1 181 7 Da and the b9 H O fragment 1 183 6 Da PSD fragments are generated in the flight tube after acceleration PSD fragments appear at a higher mass than expected in a reflector calibrated spectrum They appear at the expected mass in PSD mode See PSD fragment ions on page 8 21 These higher than expected masses are corrected
4. Acquisition status Processing status Figure 2 27 OptiPlate Dialog Box 2 Inthe Instrument Settings BIC field select the OptiPlate_Linear1 BIC or OptiPlate_Reflector1 BIC in which you set laser intensities that yield signal intensity between 20 000 and 60 000 counts described in Verifying laser intensity on page 2 60 NOTE It is good practice to determine the laser intensity that yields acceptable signal intensity before running OptiPlate Due to variations in sample preparation and crystallization it may be necessary to determine the laser intensity each time you run OptiPlate 2 64 Applied Biosystems Setting data storage Setting reference position Selecting positions to optimize Running OptiPlate to Optimize Mass Accuracy Click El next to the Data File Path and Root Name field Specify the directory in which to store the data files collected by this procedure then type the base name to use for the data files Click Open Data file names are automatically numbered autosequenced when they are acquired Under Acquisition Pattern select the position of the reference position you spotted in step 2 in Preparing the plate to optimize on page 2 58 The reference position is indicated with a black dot in the center of the position Under Acquisition Pattern select Include All Positions or Include Selected Positions to specify the positions to optimize If you select the Inc
5. All selected segments are automatically acquired and appended to the currently open PSD DAT file e The experiment is automatically closed when all selected segments are acquired and saved Before acquiring PSD spectra in Automatic Control mode become familiar with the information in Section 8 4 Acquiring PSD Data with Standard BIC Files in Manual Control Mode The steps to acquire PSD data in Automatic Control mode are PSD data Step Described on page Determine the precursor ion mass Same procedure as in Manual 8 38 Control mode Determine the number of segments to acquire for a complete 8 40 composite spectrum same procedure as in Manual Control mode Set the PSD Acquisition parameters for Automatic Control mode 8 58 Fill in the segment list Same procedure as in Manual Control mode 8 47 Set laser increment and save the BIC file 8 62 Acquire PSD segments in Automatic mode 8 63 Voyager Biospectrometry Workstation User s Guide 8 57 Chapter 8 PSD Analysis 8 5 1 Setting PSD Acquisition Parameters for Automatic Control Mode To set PSD Acquisition parameters e Display the PSD Acquisition control page e Set voltages and external calibration for the precursor spectrum e Set precursor mass and PSD calibration for fragment spectra Displaying the To display the PSD Acquisition control page PSD Acquisition 4 Open the Angiotensin PSD_Auto BIC file
6. 7 25 7 7 Automatic Calibration During a Sequence RUN serr ienis r renien i iinr anaE 7 32 7 8 Customizing the Sequence Control Panel 7 43 Voyager Biospectrometry Workstation User s Guide 7 1 Chapter 7 Acquiring Spectra from the Sequence Control Panel 7 1 Overview The Voyager Sequence Control Panel Figure 7 1 allows e Acquisition of multiple samples using different instrument settings BIC files Selection of macros for advanced processing to apply before or after calibration You can use the macros supplied or create your own in the Data Explorer software e External internal and internal update calibration options described in Section 7 7 Automatic Calibration During a Sequence Run Before you begin Before you begin read e Starting Sequence Control Panel on page 2 33 e Section 4 6 Sequence Control Panel e Section 4 7 How the Instrument and Sequence Control Panels Interact y Sequence Control Panel Untitled seq oj x Eile Edit View Control Window Desktop Help isasele muay Sequence Status Untitled seq Sample z Instrument Data Explorer Set Pre Internal External z a 2 iniedi sea SS Position Data File Settings File File Macro Calibration Calibration Fil Sequence File Name Unttled seq 1 cal_mix2 Angiotensin_Jine scptest set 4 External Default cal cal_mix2 Angictensin_line soptest set External Default cal cal_
7. NOTE For typical applications use the Grid Voltage in the standard BIC file provided with the software This section is a demonstration of the impact of Grid Voltage Effect of Grid The resolution of an ion is affected by the amount of time it Voltage on spends in the reflector After an ion is accelerated and is product ion velocity focused ions of the same mass align in time it defocuses as it moves down the flight tube When an ion resolution re spends an equal amount of time refocusing in the reflector resolution is optimized Figure 8 19 D Focused higher mass ion D Unfocused mid mass ion Reflector Unfocused lower mass ion Flight tube 10 usec From source 10 usec Point of initial focus Figure 8 19 lon Focusing Voyager Biospectrometry Workstation User s Guide 8 73 Chapter 8 PSD Analysis By fine tuning the Grid Voltage you can alter the point of initial time focus of the ions which decreases the amount of time the ion spends defocusing This allows you to balance the defocusing time with the refocusing time in the reflector Figure 8 20 The goal in optimizing the Grid Voltage is to find a suitable setting that optimizes resolution in the middle of the mass range of interest Notice that as you bring lower mass ions into focus you compromise the focus of higher mass ions p Accepta
8. Voyager Biospectrometry Workstation User s Guide 5 49 Chapter 5 Optimizing Instrument Settings Parameter Mode Impact Guide Wire Voltage Linear Decreasing improves resolution NOTE Guide Wire Reflector Increasing improves sensitivity for higher Voltage is not mass available on Voyager DE STR models with serial number 4154 and later Grid Voltage Linear Critical parameter with optimum value for Reflector maximum resolution Digitizer Bin Size Linear Decreasing improves resolution nanoseconds Reflector NOTE Decreasing Bin Size increases file size Digitizer Input Linear Decreasing may improve signal to noise Bandwidth not Reflector ratio available with Signatec Increasing may improve the resolution digitizers Accelerating Voltage Linear Increasing improves sensitivity and resolution but is limited by other factors such as the digitization rate Reflector Decreasing to 10 000 to 15 000 V can improve resolution for compounds lt 2 000 Da Laser intensity Linear Increasing improves signal to noise ratio Reflector Increasing too high increases fragmentation and resolution is sacrificed due to saturation Decreasing too low decreases sensitivity Shots Linear Increasing improves signal to noise ratio Spectrum Reflector 5 50 Applied Biosystems Impact of Changing Instrument Settings Parameters 5 3 2 Understanding Grid Voltage NOTE
9. Plate Date Page of Path and File Name Samp Matrix Sample Linear Reflector 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Voyager Biospectrometry Sample Log Plate Date Page of Path and File Name Samp Matrix Sample Linear Reflector Appendix D Log Sheets D 8 Applied Biosystems E Grid Voltage and Delay Time Settings The following figures illustrate the relationship between Grid Voltage and Delay Time parameters These values are not absolute values for all systems Optimum settings may be slightly different for each system Voyager DE and Voyager DE PRO Linear Mode Pulse Delay nsec m z 2000 m z 15000 m z 5000 600 m z 25000 m z 50000 400 F 200 F m z 1000 0 L ji L L L L L L f L L i 87 88 89 90 91 92 93 94 95 96 Grid Voltage Voyager Biospectrometry Workstation User s Guide E 1 Appendix E Grid Voltage and Delay Time Settings Voyager DE STR Linear Mode Pulse Delay nsec m z a m z 5000 600 d k 2000 400 F m z 50000 200 m z 25000 m z 100 0 i n ji L i L 8
10. Hint To determine the macro assigned to the button number display the Data Explorer software Place the cursor over the macro button to display the macro name Macro buttons are numbered sequentially from left to right If the Macro toolbar is not displayed in the Data Explorer software select Toolbar from the View menu select Macros then click Close 3 Save the sequence file by selecting Save Sequence from the File menu Recommended To ensure that the correct peak is identified as the Pre Macro monoisotopic peak before peak matching calibration create a macro that performs peak deisotoping Specify this macro in the Pre Macro column in the Sequence For more information see the Data Explorer Software User s Guide Section 3 4 Deisotoping a Spectrum 7 10 Applied Biosystems Before Creating a Sequence Macros for To perform processing on data files that contain multiple multispectrum Voyager spectra you must create advanced macros data files Figure 7 4 is an example macro that deisotopes and prints all spectra in a multispectrum Voyager data file Standard macros created with the macro recorder process only the first spectrum in a data file Sub MultiSpecProcessing Dim NumSpectra As Long NumSpectra ActiveDocument SpecView TotalSpectrum Dim J As Long For J 1 To NumSpectra ActiveDocument SpecView ActiveSpectrum J Insert ActiveDocument SpecSetup DeisotopeAdductFormula H per spect
11. Voyager Biospectrometry Workstation User s Guide 2 41 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 6 5 Laser Configuration To check the laser configuration 1 Inthe Instrument Control Panel select Hardware Configuration from the Instrument menu 2 Click the Laser tab to display the Laser page Figure 2 19 Timed lon Selector High Voltage Instrument Digitizer Vacuum Control Stick Laser Extemal T Installed Manual Intensity Adjustment Small Large Figure 2 19 Laser Configuration 2 42 Applied Biosystems Hardware Configuration 3 Check the following fields as needed e External laser Read only Checked if an external laser is installed e Manual Intensity Adjustment Determines the increments in which the laser attenuator moves when using the Fine and Coarse laser controls on the Manual Laser Sample Positioning control page Small Determines laser adjustment increments when you click the Fine laser controls ET or press Ctrl PgUp Ctrl PgDn Large Determines laser adjustment increments when you click the Coarse laser controls NOTE You set laser firing rate in the Mode Digitizer dialog box described on page 5 26 4 Click OK to exit Voyager Biospectrometry Workstation User s Guide 2 43 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 6 6 Digitizer Configuration To check the configuration of the digitizer 1
12. 1 of more spectra from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 100 shots 50 shots 2 spectra Spectrum Acceptance Laser Intensity Adjustment Criteria r Sample Positioning Random Search Pattern C Search Pattem File IV Use Automated Sample Positioning Uniform EdgeBias Center Bias voyager defaut sp El 20 Positions r Stop Conditions 7 J Stop etter E consecutive failing acquisitions when using acceptance criteria J Stop etter F laser intensity adjustment failures when manual sample positioning Figure 5 7 Automatic Control Dialog Box Voyager Biospectrometry Workstation User s Guide Ceres 5 33 Chapter 5 Optimizing Instrument Settings Automatic Control parameters are described in Table 5 9 through Table 5 11 For information on setting Instrument Settings for Automatic Control mode see Section 6 6 2 Setting Instrument Settings for Automatic Control Mode Table 5 9 Automatic Control Parameters Laser Parameter Description Laser Intensity Adjustment Use Automated Laser Intensity Adjustment Enables or disables automated laser intensity adjustment For more information see Laser on page 6 39 Minimum and Maximum Controls the minimum and maximum laser intensity when Automated Laser Intensity Adjustment is enabled To specif
13. Chapter 1 Introducing the Voyager Biospectrometry Workstations Additional Parts unique to the Voyager DE PRO mass spectrometer parts of the Collision Cell Provided with CID Collision Induced Voyager DE PRO Dissociation option for enhanced fragmentation in PSD analysis e Reflector A single stage gridded mirror that focuses energy In reflector mode a uniform electric field is applied to the mirror to reflect ions lon reflection Filters out neutral molecules e Corrects time dispersion due to initial kinetic energy distribution e Provides greater mass accuracy and resolution The single stage design provides high mass resolution across a wide mass range and highly accurate mass measurements Accurate calibration formulas for the single stage reflector allow you to vary instrument conditions without degrading mass accuracy Also easy calibration of PSD data is facilitated by single stage reflectors For more information see Chapter 8 PSD Analysis e Reflector detector The reflector detector measures ions reflected by the mirror The reflector detector sends a signal to the digitizer for conversion Two multichannel plate reflector detectors optimized for response time are included 1 24 Applied Biosystems Parts of the Voyager DE and Voyager DE PRO Systems e Timed lon Selector A device that allows only ions of a selected mass of interest to pass to the detector The Timed lon Selector TIS dev
14. For more information on the remaining instrument settings parameters see Section 5 2 Instrument Settings Parameter Descriptions 5 66 Applied Biosystems Optimizing Instrument Settings Parameters 5 4 2 Determining the Laser Setting Overview Laser intensity and matrix This section includes e Overview e Laser intensity and matrix e Adjusting laser intensity e Signal saturation Adjust laser intensity as described below to obtain a setting that allows you to optimize resolution and signal to noise ratio as described in the following sections See Section 6 3 2 Laser Intensity for more information To Optimize Adjust Signal intensity Laser intensity LA Figure 5 21 Optimizing Signal Intensity Optimum laser intensity is sample and matrix dependent You can obtain an estimate of the starting intensity for an unknown sample by analyzing a standard in the same mass range using the same matrix The order of needed laser intensity from low to high for each matrix is as follows o cyano 4 hydroxycinnamic acid Sinapinic acid e 2 5 Dihydroxybenzoic acid e 3 Hydroxypicolinic acid Voyager Biospectrometry Workstation User s Guide 5 67 Chapter 5 Optimizing Instrument Settings Adjusting laser To efficiently optimize the laser setting increase or decrease intensity the laser settings to the mid setting of a continuously decreasing laser window To adjust the lase
15. Turn cursor on and off Medium Gray Y Y Label XX Label Absolute Size 10 E width fi a fo eod Teles Ts Wrderine Graphic Compression Local Max Average C Sum Figure 4 6 Graph and Plot Options Dialog Box For additional graphic and plot option descriptions see the Data Explorer Software User s Guide Section 1 5 Setting Graphic Options Voyager Biospectrometry Workstation User s Guide 4 23 Chapter 4 Voyager Instrument Control Panel Basics 4 5 Controlling the Workstation This section includes e Using toolbar buttons and Instrument menu commands e Adjusting laser intensity and selecting sample position 4 5 1 Using Toolbar Buttons and Instrument Menu Commands Instrument buttons in the toolbar Figure 4 7 and Instrument menu commands allow you to control the software and the Voyager mass spectrometer 2l el l 2 8 ele Figure 4 7 Instrument Toolbar Use toolbar buttons and Instrument menu commands in the Instrument Control Panel to e Turn high voltage on and off e Load eject the sample plate e Select the active sample position e Turn acquisition laser on and off e Save data e Accumulate and clear spectra 4 24 Applied Biosystems Turning high voltage on and off Loading and ejecting the sample plate Selecting the active sample position Turning acquisition laser on and off Saving data Controlling the Workstation Click a in
16. Workstations 1 Voyager DE PRO The Voyager DE PRO Biospectrometry Workstation is shown in Figure 1 12 Mass spectrometer Video monitor PB100466 Lu Control stick Figure 1 12 Voyager DE PRO Biospectrometry Workstation 1 18 Applied Biosystems Parts of the Voyager DE and Voyager DE PRO Systems Parts of the Voyager DE and Voyager DE PRO systems include e Mass spectrometer A time of flight mass spectrometer described in Section 1 5 2 Mass Spectrometer The high vacuum system of the mass spectrometer is described in Section 1 5 3 Vacuum System e Computer Data System A computer that operates the Voyager control software and the Voyager processing software You control the mass spectrometer using the computer Digitizer not shown An analog to digital converter that allows the signal from the mass spectrometer to be transferred to the computer The standard digitizers provided are e Voyager DE 500 MHz e Voyager DE RP 2 GHz Optional 2 to 4 GHz oscilloscopes are also available e Video monitor A monitor that displays a real time video image of the sample spot for examination during acquisition e Control stick A device that controls position of the sample plate i
17. You can manually accumulate spectra from different acquisitions to improve resolution and signal to noise ratio of your data To manually accumulate spectra from multiple acquisitions 1 Acquire a spectrum and evaluate the data as described in Section 6 2 1 Manually Acquiring Evaluating and Saving Spectra When acauisition is complete do not save the data If the spectrum is acceptable select Accumulate Spectrum from the Acquisition menu or click A to accumulate the spectrum The software adds an Accumulated trace to the Spectrum window and copies the Current trace in the Accumulated trace If the spectrum is not acceptable start a new acquisition The Current trace will be overwritten by the new acquisition Voyager Biospectrometry Workstation User s Guide 6 19 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 20 Applied Biosystems CAUTION If you change any instrument settings with the exception of Shots Spectrum and laser intensity the Accumulated trace is automatically cleared before the next acquisition Repeat step 1 though step 3 to acquire and accumulate additional spectra Each spectrum you accumulate is averaged with the existing Accumulated trace NOTE If needed select Clear Accumulated trace from the Acquisition menu or click When the data is acceptable select click on the Accumulated trace Select Save Spectrum from the Acquisition menu Obtaini
18. gt AH t B MH t gt A BH If MH 1 000 Da AH 700 Da and BH 300 Da Figure 8 4 shows the projected flights of these ions within the reflector portion of the mass spectrometer Voyager Biospectrometry Workstation User s Guide 8 21 Chapter 8 PSD Analysis Reflector I Mirror Ratio 1 00 MH MW 1 000 correctly focused AH MW 700 poorly focused BH MW 300 poorly focused Figure 8 4 Molecular and Fragment lon Flight in the Reflector The AH fragment has 70 percent of the kinetic energy of the MH ion and the BH ion has 30 percent With a Mirror Ratio setting of 1 0000 ions with lower kinetic energy are reflected quickly and are not focused by the mirror Fragment ions with a lower energy than that of the precursor ion can be focused by adjusting the voltage applied to the mirror Mirror Voltage is equal to Mirror _ Mirror Mirror to Accelerating Accelerating Voltage Ratio Voltage Ratio Voltage where Mirror Ratio is the value you set in PSD Acquisition Settings to focus fragment ions Mirror to Accelerating Voltage Ratio is a value set in the Advanced tab of Mode Digitizer settings in Instrument Settings to adjust the voltage of the mirror to reflect intact ions Do not change this value unless instructed to do so by Applied Biosystems Accelerating Voltage is the value you set in Instrument Settings You adjust the Mirror Voltage by decre
19. 8 1 1 PSD Analysis of Angiotensin Generating the Reflector mode precursor spectrum Generating an external calibration for the precursor ion Setting PSD acquisition parameters 1 Open the PSD Precursor BIC file provided with the software This is a reflector mode instrument settings BIC file All other instrument settings should be identical to the settings in the PSD mode Angiotensin_PSD BIC you use later to acquire segments 2 Acquire using a laser intensity that does not saturate the reflector spectrum 3 Save the precursor ion DAT file by clicking EJ in the toolbar To obtain maximum mass accuracy for the precursor ion follow the steps below to generate a single point external calibration file to use when you perform the PSD acquisition NOTE This is not a PSD calibration that affects fragment ion masses It ensures accurate mass of the precursor 1 Click in the Instrument Control Panel toolbar to open the precursor ion data file in the Data Explorer software 2 Create a single point calibration using the precursor ion mass then save the calibration file as ANGIO CAL by exporting the calibration constants from the data file For more information see the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating 1 Open the Angiotensin_PSD BIC file provided with the software This is a PSD mode BIC file The PSD Acquisition Settings control page Figure 8 1 is automatically
20. O 400 800 1200 1600 2000 Mass m z Wy SPEC cel p Instrument Mode Linear Positive Mode Digitizer r Control Mode Automatic onto Manual C Automatic r Voltages Accelerating 20000 Yy Grid 35 0 0 110 0 Guide Wire 0 05 0 000 0 300 Delay Time 70 nsec r Spectrum Acquisition Shots Spectrum 64 Mass Range Da 500 to 2100 M Low Mass Gate Dal 600 Default Extemal File SHUMEREON High Voltage OFF SGUSENZES006 Se Acquisition t Active WellEB 7 Calibration info Figure 6 2 Instrument Control Panel Before Acquiring NOTE If the Instrument Settings control page is not displayed select Instrument Settings from the View menu 2 Inthe Instrument Settings control page click Manual Applied Biosystems Acquiring in Manual Mode from the Instrument Control Panel 3 Specify calibration information e Calibration Matrix Select the matrix used for your application For more information see Matrix influence on page 5 22 e Default or External Select Default or select an external CAL file For more information see Types of calibration on page 6 7 Selecting sample To select sample position and laser intensity do the following position and laser in the Manual Laser Sample Positioning control page see intensity Figure 6 2 on page 6 12 1 If the Manual Laser Sample Positioning co
21. Oscilloscope and LeCroy systems only Wrong Input Bandwidth setting Select Full for Input Bandwidth in Mode Digitizer Options dialog box NOTE Input Bandwidth is not available on Signatec systems Laser not properly aligned Call Applied Biosystems Technical Support High voltage control circuit not functioning properly Call Applied Biosystems Technical Support Flat tops on peaks Signal is saturated laser power is too high Decrease laser setting using the slider controls on the Manual Laser control page until peak tops are sharp 9 10 Applied Biosystems continued Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Round tops on peaks Saturated flat top and unsaturated sharp top scans averaged Decrease laser setting by using the slider controls on the Manual Laser Sample Positioning control page until peak tops are sharp While acquiring laser power changed Averaged scan includes saturated flat top and unsaturated sharp top scans Reacquire using one laser setting that gives sharp peaks NOTE Data acquired using different laser settings may have poor mass accuracy Poor mass accuracy in Delayed Extraction mode external calibration only High voltage power supplies not warmed up Start high voltages by clicking 2 on the toolbar before calibration Mass A
22. See Section A 4 Digitizer Specifications Vacuum system e Dual differential turoomolecular pumping for high vacuum e Multi vacuum gauge capability for independent monitoring of source and analyzer regions lon detection Positive and negative Sample analysis Automated single plate sample loading system sample plates of various formats currently available See B 3 Spare Parts e Manual control using control stick or mouse Sequence control software for automated analysis Dimensions 27 inches 69 cm deep e 25 inches 64 cm wide 65 inches 165 cm high includes cabinet and flight tube A 6 Applied Biosystems Voyager DE PRO Specifications Table A 6 Voyager DE PRO Miscellaneous Specifications Condition Specification Environmental Operating temperature 10 25 C Relative humidity 10 80 non condensing Altitude lt 2 000 m 6 500 ft above sea level e Installation category overvoltage category Il classified as portable equipment e For indoor use only Pollution degree rating 2 and may be installed in an environment that has nonconductive pollutants only Computer Minimum configuration Pentium III 500 MHz with 9 GB hard disk and 128 MB RAM random access memory e 17 inch color monitor Voyager Biospectrometry Workstation User s Guide A 7 Appendix A Specifications A 3 Voyager DE STR Specifications This s
23. Table of Contents 3 4 3 5 Loading Sample Plates in the Mass Spectrometer 3 50 3 4 1 Assigning Plate IDS 0 ce eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaaes 3 50 3 4 2 Using the Mass Accuracy Optimization Option 3 52 3 4 3 Ejecting the Sample Holder 0 ecceeeeeeeeeeeeeeeeteeeeeeeeeeeees 3 54 3 4 4 Loading Sample Plates ceccececeeeeeeeeeaeeaaeeeeeeeetteeeeeeeeeees 3 55 Sample Plate TYPES iernii uier saeesectens dec wies eia a een s 3 61 3 5 1 Sample Plate Types and Applications ccecceeeeeeeeeeeee 3 62 3 5 2 Editable Configuration Plate PLT Types Provided with thes System sesso nn te ments a aa aeaa Daaa athe wares 3 65 3 5 3 Guidelines for Defining Custom Plate Types 3 68 3 5 4 Creating and Editing PLT Files eenen 3 76 3 5 5 Adjusting the Laser Position for a Custom PLT File 3 85 Chapter 4 Voyager Instrument Control Panel Basics vi 4 1 4 2 4 3 4 4 45 Instrument Control Panel sisi 4 2 4 1 1 Parts of the Instrument Control Panel ccsseeeeeeeeeneeeees 4 2 4 1 2 Manual and Automatic Control Modes ccececeeeeneeneeeees 4 6 4 1 3 Accessing the Sequence Control Panel and the Data Explorer Software cceceeceeeeeeeeaeeaeeeneeeeeeeeeeeeeeeeeaes 4 7 Using the Control Pages sn tec cdercendtecucedusensedenehs 4 8 Using the Spectrum Window 4 10 4 3 1
24. e Guide Wire Voltage To obtain suitable mass spectra you may need to adjust these parameters in conjunction with each other See Section H 1 Optimizing a Continuous Extraction Standard Instrument BIC Setting for more information You see the best resolution at laser threshold the minimum laser intensity required to produce a steady detectable signal NOTE High mass compounds may require a higher laser intensity to ionize the sample than low mass compounds You may see the best overall spectrum good resolution and good signal to noise ratio at a setting slightly higher than threshold Voyager Biospectrometry Workstation User s Guide H 11 H Appendix H Continuous Extraction Mode H 2 2 Determining Laser Threshold This section describes H 2 2 1 Overview Definition Overview Adjusting the laser intensity Verifying threshold setting Laser threshold is the minimum laser intensity required to produce a steady detectable signal Laser intensities above threshold generate a dense plume of desorbed neutrals which cause energy loss during acceleration The observed impact of higher laser intensity is a decrease in resolution and mass accuracy and a possible increase in fragmentation Factors affecting Laser threshold is affected by sample preparation The most significant factors affecting threshold are threshold H 12 Applied Biosystems Matrix Absorption coefficient of matrix affects th
25. e Select Graphic Options from the Display menu e Click Use the same settings for all graphs in View Setup to change all traces if desired e Click Graph 1 Setup and set colors as needed When manually setting colors note the following e Selections set to white or line widths set to 0 may not print on certain printers e If you select different trace colors for multiple traces only the color for the active trace is saved Printing 2 Click on the Spectrum window to print 3 To print with the x axis along the longest length of the paper select Print Setup from the File menu and select Landscape orientation 4 18 Applied Biosystems Using the Spectrum Window NOTE If you set Landscape printing orientation within Instrument Control Panel this setting is lost when you close Instrument Control Panel To permanently set the printer see Dedicating a printer to landscape orientation on page 4 20 From the File menu select Print Preview to view the traces before printing NOTE To print without previewing select Print Spectrum from the File menu Click Print NOTE To print a trace that is displayed as Vertical Bars change the Line Width to 1 If Line Width is set to 0 Vertical Bar traces may not print See Section 4 4 Customizing the Instrument Control Panel for information NOTE Line Widths of 0 or 1 or lines set to the color white may not print on certain printers If
26. in Manual Control Mode 8 37 8 5 Acquiring PSD Data with Standard BIC Files in Automatic Control Mode 8 57 8 6 Exploring PSD Mode 8 65 8 7 Viewing PSD Data a se erneer 8 77 Voyager Biospectrometry Workstation User s Guide 8 1 Chapter 8 PSD Analysis 8 1 PSD Quick Start This section gives a quick overview of how to perform a PSD acquisition of angiotensin and an unknown in manual mode For detailed PSD information refer to the sections that follow this PSD Quick Start Before you begin Before starting this PDS Quick Start be familiar with the information in e Section 8 2 1 Post Source Decay Analysis e Section 8 6 1 Observing the Effects of Laser Intensity e Section 5 1 Loading Modifying and Saving Instrument Settings e Section 6 2 Acquiring in Manual Mode from the Instrument Control Panel e Section 6 4 Making Accurate Mass Measurements e Data Explorer User s Guide the sections that describe examining spectra and manual calibration Two types of When you perform PSD analysis you generate two types of calibration in PSD mass calibration analysis e External calibration applied to precursor ion mass Normal mass calibration applied to a reflector mode spectrum to ensure accurate mass of the precursor ion described in Section 6 1 3 Calibrating the Mass Scale e PSD calibration applied to fragment ion masses Special PSD calibration that optimizes
27. on page 2 47 Running OptiPlate to Optimize Mass Accuracy 2 8 Running OptiPlate to Optimize Mass Accuracy This section includes e Overview e Requirements e Preparing to optimize e Running OptiPlate e Evaluating and saving results 2 8 1 Overview What OptiPlate The OptiPlate software compensates for differences in does calibration across a sample plate caused by variations in e Sample plate well depth e Sample plate surface flatness Electric field in the source chamber of the mass spectrometer When you run the OptiPlate software it e Calculates a correction factor called Extraction Correction that minimizes the differences in calibration across a sample plate caused by the factors listed above e Creates a mass accuracy optimization file that contains the Extraction Correction for each optimized position on the plate Voyager Biospectrometry Workstation User s Guide 2 53 Chapter 2 2 54 Using the optimization created by OptiPlate Applied Biosystems Installing the Voyager Biospectrometry Workstations To use the optimization created by OptiPlate during sample analysis select the Use Mass Accuracy Optimization option when you load a sample plate in the Instrument Control Panel The Mass Accuracy Optimization option allows you to use fewer standards and still obtain optimum mass accuracy For more information see e Section 3 2 2 Locating Standards for Optimum Mass Accur
28. 1 2 1 3 1 4 1 5 Workstations Voyager DE and Voyager DE PRO System Overview 1 2 Voyager DE STR System Overview 1 5 MALDI TOF MS Technology Overview 2 1 7 Voyager DE Delayed Extraction Technology 1 11 Parts of the Voyager DE and Voyager DE PRO Systems 1 17 1 5 1 System Components c cceceeeeeeeeeeeeeeeaeeaaeaeeeeeeteeeeeeeeeeees 1 17 1 5 2 Mass Spectrometer cceeeeeeeeeeeeeeeeeeaeaaeaaeeeteeeeeeeeeeeneeees 1 20 1 5 3 Vacuum Syste Me terest a Mier 1 25 1 5 3 1 Voyager DE Vacuum System n 1 26 1 5 3 2 Voyager DE PRO Vacuum System 1 28 1 5 4 Computer Components sssssssssisssisirirrsersssssrstrrtrnrrrnrerresese 1 30 Parts of the Voyager DE STR System 1 31 1 6 1 System Component ccccceceeeeeeeeeeeeeeeaeeaaeeeeeeeeteeseeeeeeeees 1 31 1 6 2 Mass Spectrometer cceeeeeeeeeeeceeeeeeaeaaeaeeeeeeeseeeeeeeeeeeees 1 33 1 6 3 Vacu m Syste Maisan ii e a a e an aie iss 1 37 1 6 4 Front Panel Indicators aione a E 1 39 1 6 5 Computer Components ceceeeeeeeeeeee ee ae eaeeeeeeeeeteeeeeeeeeeeees 1 41 Software OVEVIOW rss Saceeuen cae ated sighs a te a eitetend enr etage 1 42 1 7 1 Control Software Instrument and Sequence Control Panels cceceeeeeeeeeeeeeeeeeaeeaaeeneeneeeee
29. 9 9 H 20 offscale 5 69 saturated 5 69 9 9 H 13 suppression 5 70 with high salt concentration 8 67 Signal intensity see also Intensity signal and accurate mass measurement 6 26 criteria used in automatic laser intensity adjustment 6 43 in PSD mode 8 67 Signal saturated causes signal suppression in other mass regions 5 70 checking 6 17 decreasing laser intensity to correct 5 69 number of counts 5 69 6 17 Index 38 Applied Biosystems Signal to noise ratio and accurate mass measurement 6 26 and laser threshold H 11 and resolution H 8 automatic spectrum evaluation 6 43 calculating during acquisition 6 33 calculating for live data 6 33 criteria used in automatic laser intensity adjustment 6 43 6 44 filtering during acquisition 6 43 6 44 high mass ions 6 5 impact of changing instrument settings parameters 6 25 improving H 18 labels not displayed 6 34 9 20 labels not displayed for all peaks 6 34 mass range used to determine signal 6 43 parameters affecting in Continuous Extraction mode H 11 results 6 34 thin film matrices 6 5 troubleshooting 9 13 9 14 9 15 Signal to noise ratio optimizing Accelerating Voltage 5 87 accumulating spectra 5 85 Guide Wire Voltage 5 88 Low Mass Gate 5 89 overview 5 85 parameters affecting 5 86 Shots Spectrum 5 50 5 89 Signatec digitizer see Digitizer Sinapinic acid chemical structure and molecular weight C 6 concentration 3 7 crystals 3 7 8 45 i
30. Configure for negative ion hardware 1 From the Mode Digitizer dialog box select the Instrument Mode tab 2 Select Negative for Polarity type and click OK Calibration mass shifted up or down by 10 Da Uneven matrix layer causing hot and cold spots Acquire a number of spectra and accumulate scans Prepare new sample spot Resolution labels or signal to noise not displayed Peaks not detected Apply peak detection when acquisition is complete by clicking in the toolbar 9 20 Applied Biosystems Troubleshooting Table 9 2 Instrument Control Panel Troubleshooting Continued Symptom Possible Cause Action Spectrum window is not updated with every laser shot If you are acquiring a large number of data points window is not updated with every shot The exact update rate depends on the Mass Range setting and the Bin Size setting you are using No action Normal occurrence Table 9 3 Sequence Control Panel Troubleshooting Symptom Possible Cause Action All lines in TXT or XLS file you are importing are not imported Any lines following a blank line are ignored and not imported Remove blank lines and import again Table 9 4 PSD Troubleshooting Symptom Possible Cause Action Cannot see low mass ions in PSD mode Guide Wire Voltage too high Adjust See Section 5 4 3 3 O
31. Description Search Pattern File Determines the search pattern used when Use Automated Sample Positioning is enabled See Section 6 6 4 Search Patterns for more information NOTE Before using search pattern files created in version 4 software convert to version 5 format See Section 5 5 Converting Version 4 Methods and Search Pattern Files Number of Positions Displays the number of positions to be collected in the currently selected search pattern file Stop Conditions Enable Stop Conditions if you want acquisition to stop after a specified consecutive number of e Acquisitions fail acceptance criteria e Laser adjustments do not yield acceptable data NOTE You must specify a Stop Condition if you have selected Resolution in Spectrum Acceptance Criteria parameters described on page 6 42 5 38 Applied Biosystems Instrument Settings Parameter Descriptions 5 2 4 Description of Spectrum Accumulation Options This section describes the following Spectrum Accumulation options available in the Automatic Control dialog box described on page 5 33 e Save all spectra e Save all spectra that pass acceptance criteria e Save the first spectrum to pass acceptance criteria e Save the best spectrum e Accumulate all spectra e Accumulate all spectra that pass acceptance criteria Save all spectra in Save All Spectra mode Figure 5 8 All spectra are saved e One spectrum per search pa
32. Experiment1_PSD DAT or Experiment1_PSD_0001 DAT if Autosequence File Names is enabled Selecting and 3 Inthe PSD Acquisition Settings control page select the acquiring a row click the number box in the Segment column that segment corresponds to the segment you want to acquire 4 To start acquiring select Start ha from the Acquisition menu or click 5 Adjust laser intensity to El signal intensity You typically need a higher laser intensity to optimize signal intensity for segments with lower Mirror Ratio settings The laser intensity needed for the first segment the segment with the highest Mirror Ratio setting is typically 150 to 200 counts higher than the laser intensity used to acquire the reflector mode precursor spectrum and increases with each segment Voyager Biospectrometry Workstation User s Guide 8 17 Chapter 8 PSD Analysis Examining and 6 saving the segment 8 18 Selecting and 8 acquiring remaining segments Stopping the 9 experiment Applied Biosystems Examine the spectrum to ensure that fragments are produced If fragments are present click EJ in the toolbar to add the segment to the DAT file After you save the segment the Saved check box in the segment list is checked If the current spectrum does not contain significant fragment ion signal and you do not want to save the spectrum reselect the row and reacquire the spectrum or select a new row CAUTION
33. H 20 Peaks detecting 6 28 do not appear in spectrum 9 7 H 16 flat tops 9 10 integrating 6 29 round tops 9 11 wide H 20 Peptide Fragmentation macro 8 19 Peptides Accelerating Voltage recommendation 5 84 BIC files 5 4 5 5 Guide Wire Voltage recommendation 5 56 Input Bandwidth setting 5 30 matrix for 3 8 C 6 C 7 Starting Mass recommendations 5 90 PerSeptive Biosystems Technical Support see Applied Biosystems Technical Support Physical dimensions A 10 Voyager DE and Voyager DE PRO 2 2 A 3 A 6 Voyager DE STR 2 7 Picolinic acid C 12 Pipette tips recommended 3 37 Plate ID alignment information stored with 2 52 assigning 3 50 description 3 51 plate optimization information stored with 3 52 Voyager Biospectrometry Workstation User s Guide Index 27 Index Plate Maker 3 79 Plate optimization see OptiPlate software Plate view sample position accessing Sample view 4 31 zooming to Sample view 6 13 Plates see Sample plate PLT files see also PLT files creating 100 well plate PLT 3 67 384 well plate PLT 3 67 400 well plate PLT 3 67 64 well disposable plate PLT 3 67 96x2 PLT 3 67 alignment checking 3 73 3 85 contents 3 69 corner positions list of 2 50 default units 3 79 displaying name of PLT file loaded 3 78 editing 3 83 format 3 68 laser adjusting position for custom 3 85 location 3 51 name 3 83 OptiPlate requirements 2 57 position diameter 3 72 provided 3 65 sample plate preparing befo
34. Inthe Instrument Control Panel select Hardware Configuration from the Instrument menu 2 Click the Digitizer tab to display the Digitizer page Figure 2 20 Hardware Configuration xi Vacuum Laser Timed lon Selector High Voltage Instrument Digitizer Type LeCroy V Acquire in Single Shot mode Acquisition will stop automatically after 10 000 shots OK Cancel Apply Figure 2 20 Digitizer Configuration Check the Digitizer type field This is a read only value that displays the digitizer type installed 3 Select Acquire in Single Shot Mode to set the system to save only the last acquired spectrum when you save data instead of averaging spectra 2 44 Applied Biosystems Hardware Configuration You can use single shot mode e As a diagnostic to determine if there is scan to scan variation in signal e To acquire data When single shot mode is enabled a maximum of 10 000 shots are acquired but are not averaged When acquisition is stopped the last single shot acquired is displayed in the Spectrum window A data file contains data for a single scan When single shot mode is disabled the Total Shots specified in the instrument settings are acquired and are averaged as they are acquired Click OK to exit Voyager Biospectrometry Workstation User s Guide 2 45 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 7 Aligning the Sample Plate In
35. M Roskey Smirnov W Stanick M Vestal and K Waddell Oligonucleotide Analysis by MALDI MS Analusis Magazine 1998 28 32 Haff L A and I P Smirnov DNA Sequence Analysis by Matrix assisted Laser Desorption lonization MS Biochemical Society Transactions 1996 901 903 Voyager Biospectrometry Workstation User s Guide Bibliography 5 B j B L j 0 G R A P H Y Bibliography Haff L A and I P Smirnov Multiplex Genotyping of PCR Products with Mass Tag Labeled Primers Nucleic Acids Research 25 1997 18 3749 3750 Haff L A and I P Smirnov Single Nucleotide Polymorphism Identification Assays using a Thermostable DNA Polymerase and Delayed Extraction MALDI TOF Mass Spectrometry Genome Methods 1997 7 378 388 Griffin T J and L M Smith Single nucleotide polymorphism analysis by MADLI TOF mass spectrometry Trends in Biotechnology February 2000 18 77 84 Juhasz P M T Roskey I P Smirnov L A Haff M L Vestal and S A Martin lonization Time of flight Mass Spectrometry to Oligonucleotide Analysis Applications of Delayed Extraction Matrix Assisted Desorption Anal Chem 1996 68 941 946 Roskey M T P Juhasz I P Smirnov E J Takach S A Martin and L A Haff DNA Sequencing by Delayed Extraction MALDI TOF Mass Spectrometry Proc Natl Acad Sci USA 1996 93 4724 4729 Ross P L P A Davis and P Belgrader Analysis of D
36. PLT file is considered sample N Corresponds to Position Numbering parameter in the Create PLT File see Table 3 14 Create PLT File Parameters on page 3 81 Comment Text used to describe the file Optional Start with a semicolon Voyager Biospectrometry Workstation User s Guide 3 71 Chapter 3 Preparing Samples Guidelines for Note the following creating PLT files The diameter of a position on a plate is determined by the plate used e If you are using welled or laser etched plates the position diameter is determined by the well or laser etched position size 2 540 um on a 100 well plate e If you are using plates with polished blank surfaces the position diameter is determined by the size of the physical sample spot delivered by manual application or automated sample preparation device e When creating a PLT file specify absolute X Y coordinates that accommodate the sample diameter but do not allow samples to overlap e After you create a PLT file create an SP file to accommodate the sample diameters defined in the PLT file The DEFAULT SP file is set to analyze 2 540 um diameter positions corresponding to standard sample positions on a 100 well plate NOTE For optimum mass accuracy if you do not use the Optimize Mass Accuracy option do not spot sample on outer rows of the plate 3 72 Applied Biosystems Guidelines for creating SP files Effect of plat
37. X Axis Setup Eg FX Axis Display Range From fo To 2000 Cancel Figure 4 3 X Axis Setup Dialog 4 Set From and To values for the display range m z units then click OK 4 10 Applied Biosystems Using the Spectrum Window Y range 5 From the Display menu select Range 6 Select Y Range to scale the y axis The Y Axis Setup dialog box Figure 4 4 is displayed Y Axis Setup x Scaling Mode Display Relative x m Minimum Absolute Max Y M Use Limit fsa Y Display Range From fe a x To f100 H y OK Cancel Figure 4 4 Y Axis Setup Dialog Box 7 Set the parameters described below Parameter Description Scaling Mode Display Relative Autoscales the trace to the largest peak in the selected range Base Peak Relative Autoscales the trace to the base peak in the entire range notthe selected range Displays a right axis label with the base peak intensity NOTE To turn off the right axis select Graphic Options from the Display menu click the Graph 1 Setup tab then deselect the Show Right Y Axis check box continued Voyager Biospectrometry Workstation User s Guide 4 11 Chapter 4 Voyager Instrument Control Panel Basics Parameter Description Scaling Mode continued Absolute Value Sets the trace to the Y Display Range you enter in the Y Display Range From To boxes Display Min Max Sets the tra
38. and Voyager DE PRO 2 12 mass spectrometer Voyager DE STR 2 23 oscilloscope Voyager DE and Voyager DE PRO 2 19 oscilloscope Voyager DE STR 2 26 2 28 video monitor Voyager DE and Voyager DE PRO 2 21 video monitor Voyager DE STR 2 27 Instrument configuration 2 40 name specifying 2 41 serial number displaying 2 41 status displaying 2 76 type displaying 2 41 Instrument Control Panel basics 4 1 BIC loaded at end of Sequence run 7 28 control buttons 4 24 cursor and grid displaying 4 21 customizing 4 21 Data Explorer accessing from 4 7 high voltage turning on off 4 25 interaction with Sequence Control Panel 1 44 4 33 layout changing 4 8 layout default 4 8 loading sample plates 3 54 Voyager Biospectrometry Workstation User s Guide Index 17 I N D E X Index Instrument Control Panel continued Output window 4 5 overview 1 43 4 2 parts of 4 2 peak detection 6 28 pressures displaying 2 76 Sequence Control Panel accessing from 4 7 software exiting 2 75 software starting 2 74 status bar 4 5 toolbar 4 3 4 24 traces do not print 4 19 traces previewing 4 18 traces printing 4 18 troubleshooting 9 19 zooming 4 13 Instrument mode determining 5 16 displayed in status bar 4 5 setting 5 25 Instrument Settings files BIC see also BIC files see also Instrument Settings parameters Automatic Control mode setting 5 10 6 37 cannot save 5 11
39. cannot run BICs that specify internal and external laser in same sequence 7 8 7 16 7 27 configuring 2 42 custom plates adjusting position for 3 85 description 1 22 1 34 external see Laser external optional nitrogen 1 22 1 34 not aligned with sample position 2 48 9 23 not responding to slider controls 9 20 pulse width 1 22 1 34 rate see Laser rate safety information xxvi single shot mode 2 45 stopping l 7 threshold see Laser threshold troubleshooting 9 20 turning on and off 4 25 type setting 5 26 UV radiation warning xxi 9 3 wavelength pulse width and frequency 1 22 1 34 Laser adjustment buttons description 4 28 increments setting 2 43 Laser attenuator adjusting 2 43 description 1 22 1 34 Index Laser external optional Laser intensity continued cannot run BICs that specify signal to noise adjustment internal and external laser criteria 6 43 6 44 in same sequence 7 8 stopping acquisition before 7 16 7 27 adjusting 6 5 determining if installed 2 43 stored in BIC 6 4 6 19 indicated on Manual Laser Sample strategy if using BIC 5 68 Position control page 5 26 strategy if using default 5 68 resetting 2 71 Laser Intensity Adjustment Criteria specifying in BIC 5 26 how criteria are used during troubleshooting 2 72 9 28 acquisition in Automatic Laser intensity mode 6 58 see also Laser intensity Automatic parameters accessing 5 36 Control mode parameters parameters setting 6 43 adjusting manual
40. file spot the complete sample position layout for which you are creating the PLT file Sample Plate Types First position O00000 OO000 Or Oi OO iC m Last position aces P Figure 3 13 First and Last Positions for a PLT File For more information see Loading samples on page 3 42 Load the sample plate into the system then select BLANK PLT as the Plate Type For information see Section 3 4 Loading Sample Plates in the Mass Spectrometer From the View menu in the Instrument Control Panel select Manual Laser Sample Positioning Voyager Biospectrometry Workstation User s Guide 3 77 Chapter 3 Preparing Samples Figure 3 14 shows the Plate View displaying the logical coordinates for the BLANK PLT file m Manual Laser Intensity _ w Manual Sample Positioning Active Pos 7 Blank plate plt Logical Relative X 23812 500 219 coordinates 21907 Absolute x 1587 500 Y 47307 51 00 Figure 3 14 Logical Coordinates in Plate View in Manual Laser Sample Positioning Control Page 3 78 Applied Biosystems Sample Plate Types Creating a PLT The Create PLT File function creates PLT files that specify file using Create positions PLT File In evenly spaced rows and columns identical Y coordinates for all positions in a row and identical X coordinates for all positions in a column e Sequentially from left to
41. glycosylation phosphorylation and sulfation Pulsed Source ion source in a Delayed Extraction system Precursor fon Used in PSD Analysis software Original ion from which fragments dissociate Precursor lon Selector See Timed lon Selector Product ion Used in PSD Analysis software Fragment ion that dissociates from precursor ion Sensitivity Measure of the amount of sample required to generate an acceptable mass spectrum Usually expressed in terms of strength of signal relative to noise Signal intensity See Intensity Source See lon source Stitch Segment of a PSD composite spectrum Tandem mass spectrometry See MS MS Analysis TC2 Thermocouple vacuum gauge Pressure gauge that operates from 1 millitorr to near atmospheric pressure by measuring the temperature of a heated thermocouple junction As the pressure rises more heat is removed lowering the temperature Timed lon Selector Mechanism for suppressing all but the ion of interest used in PSD analysis Voltage is applied to ion selector plates in the flight tube before and after the ion of interest passes the selector plates Time of flight TOF Mass Analyzer A mass analyzer that measures mass by measuring drift times If a packet of ions leaves the ion source at the same time and with the same energy then their flight time through a field free drift region will depend on their mass Time of flight mass analyz
42. observe the trace in the Spectrum window 1 Examine the number at the top of the right axis in the Current Spectrum trace to determine if signal is saturated Figure 6 4 Signal with counts greater than 64 000 may be saturated Adjust the laser intensity as needed For information see Signal saturation on page 5 69 2 Use the Resolution calculator or the Signal To Noise calculator on the Current trace to determine if data is acceptable For more information see Section 6 5 Evaluating Data in the Instrument Control Panel Hint You can accumulate traces to improve resolution and signal to noise ratio For more information see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Spec 9 TIC 65370964 BP 1297 31 60324 100 2095 0 R392 5 8E 4 90 Check for signal 70 2467 1 saturation Intensity p105 2 0 T T T T 0 1900 2060 2220 2380 2540 2700 Mass m z Figure 6 4 Checking for Signal Saturation Voyager Biospectrometry Workstation User s Guide 6 17 Chapter 6 Acquiring Spectra from the Instrument Control Panel 3 At this point you can e Save the data described in Saving data on page 6 18 e Acquire additional spectra and create an accumulated spectrum described in Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions CAUTION If you do not save data before starting a new acquisition the d
43. parts of 1 21 1 22 overview 1 3 parts of the system 1 18 space required 2 2 specifications A 5 startup and shutdown 2 73 weight 2 3 Voyager DE STR Biospectrometry Workstation Beam guide wire replaced by lon focusing lens on models with serial number 4154 and later 1 33 1 35 digitizer Acqiris 2 26 digitizer oscilloscope 2 26 features 1 6 Guide Wire Voltage not available on STR models with serial number 4154 and later 5 18 mass spectrometer parts of 1 33 overview 1 5 parts of the system 1 31 power requirements 2 7 Voyager DE STR Biospectrometry Workstation continued space required 2 7 A 10 specifications A 8 startup and shutdown 2 73 weight 2 7 W Warnings safety CHCA matrix 2 49 fire hazard and fuse ratings 2 5 high voltage xxii 9 3 removing instrument covers xxii 9 3 sample holder retraction 3 56 3 59 symbols on system xv UV radiation from laser xxi 9 3 Warranty damages claims returns B 5 exceptions B 3 for computers with altered configuration B 1 period B 2 Washing sample cleanup 3 26 Wavelength laser 1 22 1 34 Weight of system Voyager DE 2 3 Voyager DE PRO 2 3 Voyager DE STR 2 7 Well depth 3 70 Windows NT event log see NT Event log version 1 30 1 41 X X axis setting range 4 10 X y Z ions angiotensin F 6 XML files created by OptiPlate software 2 70 Y Y axis scaling 4 11 4 12 Y Offset digitizer signal 5 30 Y axis spectrum 4 11 Z ZipTips sample cleanup 3 31
44. software Angiotensin_PSD BIC is set to acquire 10 segments with the Mirror Ratios listed in Default Mirror Ratio settings on page 8 41 These settings are adequate for many applications You can add or delete segments or change the Mirror Ratio for a segment as needed The PSD software allows complete flexibility in the way you acquire and process PSD data You are not required to acquire a complete set of PSD segments If you are interested in specific fragment masses only you can acquire only the number of segments you are interested in by using Mirror Ratio settings that focus the fragment ion masses of interest Hint The best focusing conditions occur when the fragment ion mass is equal to the Mirror Ratio multiplied by the precursor ion mass equal to the Max Stitch Mass When you type in a Mirror Ratio described in Section 8 4 4 Filling in the Segment List and Saving the BIC File observe the Max Stitch Mass displayed This value reflects the maximum mass of the segment that will be included in the composite spectrum The mass range included in the segment is approximately 15 percent higher than the Max Stitch Mass Acquiring PSD Data with Standard BIC Files in Manual Control Mode 8 4 3 Setting PSD Acquisition Parameters for Manual Mode To set PSD Acquisition parameters e Display the PSD Acquisition control page e Set voltages and external calibration for the precursor spectrum e Set precursor m
45. solution in one sample position by placing the pipette tip in the sample position and slowly expelling while keeping the tip in contact with the sample plate NOTE In general do not touch the pipette tip to the sample plate surface However when you use THF as the solvent in the thick layer method it is necessary to touch the tip to the sample plate to slowly expel the sample onto the sample plate Voyager Biospectrometry Workstation User s Guide 3 21 Chapter 3 Preparing Samples 3 1 4 Preparing Sample In this section This section includes e Sample concentration e Preparing samples for dried droplet application e Preparing samples for thin layer application e Internal standards Sample Prepare sample just before loading the sample plate Refer to concentration Section 3 1 5 Sample Cleanup to determine if your samples should be cleaned up before preparing The ideal sample amount for analysis is Typical Concentration Compound Dried Droplet Application Thin Layer Application Peptides 0 1 to 10 pmol ul lt 0 1 pmol l Proteins 0 1 to 10 pmol ul Oligonucleotides 10 to 100 pmol ul Polymers 100 pmol ul Some proteins particularly glycoproteins yield better results at concentrations up to 10 pmol ul Preparing For peptides and proteins dilute sample at this phase of samples for dried sample preparation to a concentration of 1 to 10 pmol ul For droplet other compoun
46. value in 5 percent increments and observe the spectrum For example if the setting in the BIC file is 80 decrease to 75 then 70 At lower Grid Voltage settings e Lower mass ion resolution degrades e Higher mass ion resolution improves Voyager Biospectrometry Workstation User s Guide 8 75 Chapter 8 PSD Analysis 6 Check the Grid Voltage setting in the standard BIC file provided on your system for angiotensin Angiotensin_Reflector BIC Acquire a spectrum using this setting At this setting you should observe optimum resolution on the high mass peak 8 6 4 Summary The table below summarizes the impact of changing PSD acquisition conditions Condition Impact Laser e Increase higher than normal to induce fragmentation intensity e Setting too high increases baseline noise and eventually decreases signal e Setting too low reduces fragment ion abundance Precursor lon e Turn on to eliminate fragments that do not travel with the Selector selected precursor mass e Decrease the Deflector Gate Width set in Hardware Configuration to narrow the Precursor lon Selector mass window e Extremely small Deflector Gate Widths can weaken fragment ion intensity Grid e Increase or decrease to affect the focus of fragment ions Voltage e Higher Grid Voltage settings shift the optimal focus toward shorter flight times lower masses within a PSD segment Guide Wire e Decreasing the Guide W
47. want to insert another spot then right click Move a spot Edit spot a Place the cursor on the spot you want to move then click and drag the spot to its new location within the sample position area Delete a spot Delete spot Right click the mouse button on the spot you want to delete Delete all Delete all All spots are deleted spots spots 6 56 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Setting x y To set x y coordinates for a spot coordinates 4 Click Menu then select X Y The Set X Y dialog box is displayed Figure 6 21 Set X Y Xx spot fi ok k 373 y fiis0 Cancel Figure 6 21 Set X Y Dialog Box The x y values displayed correspond to the location of the cursor in the sample position area when you select the X Y command not to the spot number displayed in the Spot field 2 Inthe Spot field type a number e If you type a spot number that exists the spot is edited e If you type a new spot number the spot is added 3 Type x and y coordinates for the spot Valid x and y coordinate values correspond to approximately one half the dimensions of the sample position of the currently loaded or selected plate For example the maximum x or y value for a 2540H by 2540W sample position is approximately 1270 4 Click OK Voyager Biospectrometry Workstation User s Guide 6 57 Chapter 6 Acquiring Spectra from the Instrument Control
48. 0 or 1 or lines set to the color white may not print on certain printers If traces do not print change the line width or color 5 Click OK Changing You can change between two line types for the trace line type of trace Lines Displays a continuous trace of connected data points e Vertical Bars Displays a vertical line for each data point where the height of the bar is equal to the intensity of the data point NOTE To print a trace that is displayed as Vertical Bars change the Line Width to 1 If Line Width is set to 0 Vertical Bar traces may not print 4 22 Applied Biosystems Customizing the Instrument Control Panel Graph and Plot Options x View Setup Graph 1 Setup Graph 2 Setup Last Acquired Spec 37 TIC 42851701 BP 2473 23 4249 m Graph Title Setup _ Axis Title Setup Font aia x Color Light Green Y Turn grid Size 10 a cont Aral 7 on and off M Bold l Italics I Underine Size 10 z r Plot Setup F Bold I Italics M Underline T Show Grid e a etup V Show Right Y Axis Turn right axis newid Axis Color Medium Gray x on and off Trace fi Line Width Axis Tick Label Setup IV Show Tick Labels Color Yelow bd Font aval bd Size 10 Size 9 F Bold T Italics Underline Line Style Da z Color Eont anal v Change line type of trace Change color of peak labels
49. 1000000111 H 1 Appendix I Using the Oscilloscope and Control Stick 1 1 Glossary Bibliography Index xii Applied Biosystems Safety and Compliance Information Safety and Compliance Information In this section This section includes e Instrument safety e Safety and EMC standards e Laser safety Instrument Safety In this section This section includes e Notes Hints Cautions and Warnings e Safety symbols e Before operating this instrument e Material Safety Data Sheets MSDSs e General Warnings Notes Hints Notes Hints Cautions and Warnings are used in this Cautions and document as follows Warnings A Note provides important information to the operator For example NOTE If you are prompted to insert the boot diskette into the drive insert it then press any key A Hint provides helpful suggestions not essential to the use of the system For example Hint To avoid complicated file naming use Save First to Pass or Save Best Only modes Voyager Biospectrometry Workstation User s Guide xiii Safety and Compliance Information Remarques recommandations et avertissements Xiv Applied Biosystems A Caution provides information to avoid damage to the system or loss of data For example CAUTION Do not touch the lamp This may damage the lamp A Warning provides specific information essential to the safety of the operator For example WARNING CHEM
50. 1137 596 1165 591 1182 617 DRVYIHPFH x y z Fragment 158 082 132 1025 112 076 L 295 141 269 1614 249 135 HL 442 209 416 2298 396 204 FHL 539 262 513 2825 493 256 PFHL 676 321 650 3414 630 315 HPFHL 789 405 763 4255 743 399 IHPFHL 952 468 926 4888 906 463 YIHPFHL 1051 537 1025 557 1005 531 VYIHPFHL 1207 638 1181 658 1161 632 RVYIHPFHL F 6 Applied Biosystems Observed PSD Fragments in Angiotensin F 4 Observed PSD Fragments in Angiotensin The masses listed below are fragment ions of the M H percursor ion Fragment Mass Fragment Mass Designation Designation monoisotopic average monoisotopic average 70 066 70 1 P R 426 2 426 5 72 081 72 1 V 489 246 489 6 a4 17 86 097 86 1 LL 492 2 492 6 110 072 110 1 H 506 273 506 6 a4 136 076 136 2 Y 513 283 513 6 y4 156 101 156 2 R 517 241 517 6 b4 17 166 062 166 2 H 534 268 534 6 b4 207 125 207 3 HP 28 619 357 619 7 a5 217 134 217 3 PF 28 630 325 630 8 b5 17 223 156 223 3 IH 28 632 331 632 8 IHPFH 230 1 230 3 647 352 647 8 b5 235 120 235 3 HP VY 28 650 341 650 8 y5 235 145 251 151 251 3 IH 739 389 739 9 a6 17 255 109 255 3 b2 17 756 416 756 9 a6 263 140 263 3 VY 767 384 767 9 b6 17 269 161 269 3 y2 784 411 784 9 b6 Voyager Biospectrometry Workstation User s Guide F 7 Appendix F Reference Standard Information Fragment Mass F
51. 14 is determined by the voltages applied to the sample plate Accelerating Voltage and the variable voltage grid Grid Voltage Variable voltage Sample E plate Accelerating grid at of a Voltage at 25 000 V Accelerating Voltage gri i Potential gradient 2 8mm lon source l lonization region Figure 5 14 Sample Plate and Grids Potential Accelerating Voltage Grid Voltage gradient Distance between plate and grid Grid voltage Grid Voltage Accelerating Voltage 5 52 Applied Biosystems Impact of Changing Instrument Settings Parameters For example Figure 5 14 with a 25 000 V Accelerating Voltage and a Grid Voltage of 56 the potential gradient is Potential 25 000 14 000 V gradient 28mm 11 000 V 2 8 mm 3 928 V mm Vary the potential gradient by varying the Grid Voltage and use the recommended Accelerating Voltage for the mass range For more information see Section 5 4 4 2 Setting Accelerating Voltage Maximum allowed The Voyager Instrument Control Panel and electronics allow a potential maximum absolute potential difference of 10 000 V between difference the Accelerating Voltage and the voltage applied to the grid Grid Voltage You can calculate the potential difference using the following equation Accelerating Voltage x 100 Grid Voltage 100 Grid Voltage in conjunction with Delay Time has a direct impact on resolution See Sect
52. 2 Select a file or type in a file name 3 Click Import Information is imported into all columns even if columns are hidden Exporting To export a TXT or XLS file 1 Select Export from the File menu 2 Type ina file name 3 Select TXT or XLS from the Save As Type drop down list 4 Click Save NOTE The Acquisition Status column is not exported The exported Run column contains 0 if Run is unchecked and 1 if Run is checked Voyager Biospectrometry Workstation User s Guide 7 23 Chapter 7 Acquiring Spectra from the Sequence Control Panel Aligning the sample plate High voltage warmup for improved mass accuracy Before acquiring 7 24 Applied Biosystems 7 5 Preparing to Run a Sequence This section describes Aligning the sample plate High voltage warmup for improved mass accuracy e Before acquiring Sample plate alignment is necessary for a sequence run if the laser is not striking the center of the sample position Sample plate alignment may not be necessary on your system particularly if you use 100 well plates and the SPIRAL SP search pattern file provided with the system See Section 2 7 Aligning the Sample Plate to determine if sample plate alignment is necessary If you do need to align the sample plate you must do so for each sample plate you use For maximum mass accuracy allow the high voltage power supplies to warm up for a short period of time before
53. 2 3 Connecting Voyager DE and Voyager DE PRO Workstations then power up 2 6 Applied Biosystems Selecting the Site 2 2 2 Voyager DE STR Workstation In this section This section includes e Required space e Weight e Power voltage requirements Required space The Voyager DE STR system is a floor standing unit that measures e 34 inches 87 cm deep 94 inches 239 cm wide e 46 inches 117 cm high The Voyager DE STR Workstation is constructed on a rolling base For service you must have enough space to pull the workstation forward 24 inches 61 cm to allow access at the rear of the workstation Allow an additional 18 inches 48 cm on the sides for service access Weight The Voyager DE STR system weighs approximately 1 100 pounds 499 kg Power voltage The Voyager DE STR systems can be configured to operate requirements at 110 V voltage is 10 Operating voltage is selected by an internal jumper which must be set by an Applied Biosystems Technical Representative NOTE Workstations are shipped with the internal voltage selector jumper disconnected The Voyager DE STR systems require a single phase 50 60 Hz power source at one of the following voltages e 100V 16A e 120V 16A e 220V 10A e 240V 10A Voyager Biospectrometry Workstation User s Guide 2 7 Chapter 2 Installing the Voyager Biospectrometry Workstations CAUTION Before operation internal jumpers must be set to accom
54. 2 30 2 5 2 Starting the Software ccceeceeeeeeeeeee eee aeaeeeeeeeeeeeeeeeeeeeeeaeaaes 2 32 2 5 3 Exiting the Software ccccccsceeeeeeeeeeeeeeeeeeeeeaaeaaeeneeeeeeeeeeees 2 34 Hardware Configuration 2 35 2 6 1 VACUUM Configuration vsere iaire d nupit uktana E RAKENNA U KREAN An EAER KRS R 2 35 2 6 2 High Voltage Configuration ssssssssesesssnnreresnrrreressrrrrrnnrnnne 2 38 2 6 3 Timed lon Selector Configuration ccccccceeeceeeeeeeeeeeeeeeeees 2 39 2 6 4 Instrument Configuration cceeeeeeeeeeeeeeeeeaeeaaeeaaeaeeeeeeeees 2 40 2 6 5 Laser Configuration ccccceeeeeeeeeeeeeeeeeeeeeeaeaaeaaeeeteeeeeeeeeees 2 42 2 6 6 Digitizer Configuration cece ceeeeeeeeeeeeeeeeeeeeee cee aaaaaeeteeeeeeeees 2 44 Aligning the Sample Plate eeeeeeeeeeeeeeeaes 2 46 Applied Biosystems 2 8 2 9 2 10 2 11 Table of Contents Running OptiPlate to Optimize Mass Accuracy 2 53 2 8 1 OMV VI W ns sr AT te min rate en te enr mre renard des 2 53 2 8 2 R quir ments Sirana a tneennn tanith cabanas eecdesaabendeaes 2 55 2 8 3 Preparing to Optimize 2 0 22 cece ceeeeeeeeeeeeeeeeeeeeeaaeaaeneeeeeeeeeeeees 2 58 2 8 4 Running OptiPlate 0 ccccceeecsceeeeeeeeeeeeeeeeeeeeeaaeaaeeneeeeeeeeeeees 2 63 2 8 5 Evaluating and Saving Results eeen 2 69 Resetting the Optional External Laser 2 71 Startup and Shutdown ss 2
55. 3413 Porcine Trypsin 2211 0968 2211 4322 1 2211 1046 2212 4322 Adrenocortico tropic 2464 1910 2465 71 1 2465 1989 2466 72 hormone ACTH clip 18 39 F 2 Applied Biosystems Calibration Compounds Molecular Weight Protonated Molecular lon Canpouna Charge M H p State Monoisotopic Average Monoisotopic Average Insulin B chain 3493 6435 3495 95 1 3494 6513 3496 96 oxidized Adrenocorticotropic 3656 9216 3659 18 1 3657 9294 3660 19 hormone ACTH clip 7 38 Insulin bovine 5733 58 1 5730 6087 5734 59 2 2865 8083 2867 80 Thioredoxin E coli 11673 47 1 11674 48 oxidized 2 5837 74 Cytochrome C horse 12360 5 1 12361 5 heart 2 6181 25 Myoglobin horse 16951 55 1 16952 56 heart 2 8476 78 Trypsinogen 23980 1 23981 Carbonic anhydrase 29023 1 29024 Enolase Baker s 46671 1 46672 yeast 2 23336 Bovine serum albumin 66430 1 66431 BSA 2 33216 Voyager Biospectrometry Workstation User s Guide F 3 Appendix F Reference Standard Information F 2 Conversion of Mass to Time for Typical Standards The table below includes mass and time values for standards under different acquisition conditions Accelerating Voltage and flight tube length You can use this information when observing the signal on the oscilloscope to determine if the peaks you are seeing are at the correct mass NOTE Th
56. 37 pressure range 9 26 wait time 2 37 TC2 Voyager DE STR description 1 39 max load pressure 2 37 pressure range 9 26 wait time 2 37 Index Technical support contacting 9 7 for computers with altered configuration B 1 Teflon plates see Sample plate Teflon Tektronix oscilloscope see Digitizer see Oscilloscope Temperature operating A 4 A 7 A 10 Templates sample plate 3 61 THAP chemical structure and molecular weight C 11 concentration 3 13 crystals 3 13 3 46 mass spectrum C 5 preparing 3 13 sample concentration 3 13 stability 3 14 when to use 3 3 Thermocouple gauge see TC2 Thin layer sample application technique see Sample loading Thioredoxin BIC file 5 5 molecular weight F 3 Time dispersion correcting 1 24 1 36 Timed lon Selector BIC file to test 5 5 configuring 2 39 8 30 enabling 5 32 function 1 25 1 36 8 20 impact on system performance 2 40 8 29 resolution A 5 A 9 see also Precursor lon Selector setting tracks Precursor lon Selector 5 32 Tips pipette see Pipette tips Tolerance of sample plates 3 73 Voyager Biospectrometry Workstation User s Guide Index 41 Index Toolbars Instrument Control Panel Troubleshooting continued customizing 4 21 laser external 9 28 description 4 3 laser threshold H 19 moving 4 21 Load Eject dimmed 9 19 ToolTips 4 3 mass accuracy 9 11 9 12 9 17 Traces H 23 H 24 f adding 4 14 mass range in Spectrum window does not update with every laser sho
57. 4 882 893 Voyager Biospectrometry Workstation User s Guide 8 23 Chapter 8 PSD Analysis 8 24 Applied Biosystems 8 2 2 Differences From Regular Analysis When operating in PSD mode note the following differences from analysis in non PSD mode Higher laser intensity In non PSD mode you use a laser intensity that yields acceptable performance without fragmentation You need a higher laser intensity to generate PSD fragments Higher Grid Voltage and shorter Delay Time A higher Grid Voltage and shorter Delay Time generates a sharper ion packet at the position of the Precursor lon Selector and improves fragment ion resolution Sample consumed more quickly Higher laser intensities ionize and consume sample more quickly than lower laser intensities used in non PSD mode Different calibration used PSD analysis uses a different calibration function to correctly determine the masses of PSD fragment ions For information see Section 8 2 5 Mass Calculation for Fragment lons Acquisition of multiple spectra at different Mirror Ratio settings to create a single composite spectrum To correctly focus fragments with different mass ranges collect PSD segment spectra with different Mirror Ratio settings The Data Explorer software stitches the segments together to create a composite spectrum For information see the Data Explorer Software User s Guide Chapter 8 Viewing Voyager PSD Data Overview of PS
58. 5 NOTE A saturated spectrum has flat topped peaks If the spectrum goes off the screen adjust the position of the spectrum to see if the top is flat Voyager Biospectrometry Workstation User s Guide H 13 Appendix H Continuous Extraction Mode This File 1 C VOYAGER DA TASKEITHiKO106011 MS Collected 16 95 8 42 AM 1290 98 70000 60000 50000 540000 30000 20000 10000 500 1000 1500 2000 2500 Mass miz Figure H 5 Saturated Spectrum 3 Check the spectrum for the peak of interest Zoom in on the spectrum as needed See Section 4 3 Using the Spectrum Window H 4 Zoomin on the appropriate mass range to check for the peak of interest If peak of interest is 5 Ifthe peak of interest is present the current laser intensity present is the high setting for the sample class matrix you are analyzing Decrease the laser intensity by 10 percent and acquire another 10 spectra Continue decreasing the laser intensity by 10 percent until the signal disappears When signal disappears the current laser intensity is the low setting for the sample class matrix you are analyzing If peak of interest is 6 If the peak of interest is not present the current laser not present intensity is the low setting for the sample class matrix you are analyzing Increase the laser intensity by 10 percent and acquire another 10 spectra Continue increas
59. 5 4 4 2 Setting Accelerating Voltage and Section 5 4 3 5 Optimizing Grid Voltage High mass ions fragmenting when you are using a cyano matrix Internal energy of ions causing fragmentation Use Sinapinic acid matrix which yields ions with a lower internal energy than a cyano and therefore causes less fragmentation of high mass ions Masses off by 22 or 38 Da Sample ionized to M Na or M K instead of M H No action Data accurate Voyager Biospectrometry Workstation User s Guide continued 9 17 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Observed mass not equal to monoisotopic mass Observed mass is average mass except when resolving isotopes No action Data accurate Large tail on the high mass side of peak Unresolved salt or buffer adducts due to sample contamination Clean up sample See Section 3 1 5 Sample Cleanup 9 18 Applied Biosystems Troubleshooting 9 2 2 Software Troubleshooting This section includes e Instrument Control Panel troubleshooting e Sequence Control Panel troubleshooting e PSD troubleshooting e Checking the Windows NT Event Log Table 9 2 Instrument Control Panel Troubleshooting Symptom Possible Cause Action Load Eject button or Communication problem Select Reinitialize from command dimmed b
60. 5 format See Section 5 5 Converting Version 4 Methods and Search Pattern Files Select Stop Conditions if you want to stop the acquisition when a specified number of e Consecutive acquisitions fail acceptance criteria e Laser adjustments within a search pattern position do not yield acceptable data when using manual sample positioning For more information see Stop Conditions on page 5 38 Click OK For more information on Automatic Control parameters see Section 5 2 3 Automatic Control Dialog Box Voyager Biospectrometry Workstation User s Guide 6 41 Chapter 6 Acquiring Spectra from the Instrument Control Panel Setting spectrum acceptance and laser adjustment 4 6 42 criteria Applied Biosystems If you selected an accumulation condition that uses acceptance criteria see step 9 on page 6 40 In the Automatic Control dialog box see Figure 6 15 on page 6 38 click the Spectrum Acceptance Criteria button The Spectrum Acceptance Criteria dialog box Figure 6 16 is displayed Acceptance Adjustment Criteria x m Acceptance Criteria T Minimum Signal Intensity 0 local base peak T Maximum Signal Intensity 7 local base peak I Signal to Noise F local base peak I Resolution Ep local base peak FWHM Note If no criteria selected all spectra will pass Used for spectrum acceptance and automated laser adjustment Used for spectrum acceptance only r C
61. 50 Input Bandwidth 5 30 Number of Data Points Digitized 5 29 Vertical Offset 5 30 Vertical Scale 5 30 Digitizer Signatec connecting 2 13 optimized laser rate supported 5 26 A 12 Digitizer Tektronix oscilloscope connecting to Voyager DE and Voyager DE PRO 2 19 connecting to Voyager DE STR 2 26 optimized laser rate supported 5 26 A 12 Dihydroxybenzoic acid mixture see DHBs Dihydroxybenzoic acid see DHB Dimensions Voyager DE STR 2 7 Voyager DE and Voyager DE PRO 2 2 A 3 A 6 Voyager DE STR A 10 Dimers caused by 6 21 H 8 troubleshooting 9 15 Directory for data file Instrument Control Panel 6 14 Sequence Control Panel 7 14 Disk space check in Automatic Control mode 6 58 check in Sequence Control 7 25 clearing 9 6 required to run software 2 29 9 6 swap file 9 6 Display range scaling 4 11 X range expanding 4 10 y range expanding 4 10 Disposable plate see Sample plate types of Dithranol chemical structure and molecular weight C 11 concentration 3 19 crystals 3 19 Index Dithranol continued mass spectrum C 5 preparing 3 19 sample concentration 3 19 when to use 3 3 Docked control pages 4 9 Dried droplet sample application see Sample loading Drift time and molecular weight 1 8 1 10 equation for multiply charged ions 1 10 equation for singly charged ions 1 10 fragments 8 71 Drying sample plates consequence of loading wet plate 3 43 3 50 time needed 3 43 Dynamic range adjusting 5 60 E Ed
62. 50 of the laser intensity that yields signal intensity between 20 000 and 60 000 counts see below Determining the Due to variations in sample preparation and crystallization it laser intensity may be necessary to determine the laser intensity each time you run OptiPlate To determine the laser intensity that yields a signal intensity between 20 000 and 60 000 counts 1 Inthe Instrument Control Panel load the OptiPlate_Linear BIC or OptiPlate Reflector BIC file provided on your system 2 Inthe Instrument Settings page change Control Mode from Automatic to Manual 3 Select a position spotted with standard mixture and matrix from the sample plate you spotted in Preparing the plate to optimize on page 2 58 4 Start acquisition move around on the sample position until you observe an acceptable signal then adjust the laser intensity until signal intensity is between 20 000 and 60 000 counts 5 Move around to several other locations on the sample position to verify that the selected laser intensity yields signal in the correct range 6 Stop acquisition Setting Minimum _ To set the Minimum and Maximum Laser Intensity settings in and Maximum Automatic Control Laser Intensity 1 Inthe Instrument Settings page change Control Mode from Manual to Automatic 2 Click Automatic Control Voyager Biospectrometry Workstation User s Guide 2 61 Chapter 2 2 62 Applied Biosystems 3 Installing t
63. 60 Applied Biosystems Impact of Changing Instrument Settings Parameters Decrease if signal is weak Fr Increase if Vertical signal exceeds scale Scale E ES E S Figure 5 18 Effect of Vertical Scale in Digitizer Settings Suggested settings Use the settings listed below as a starting point for Vertical Scale Adjust as needed to bring the signal into the proper range Mass Range Da Vertical Scale mV 0 10 000 1 000 10 000 50 000 200 gt 50 000 50 Not available on the Signatec digitizer Voyager Biospectrometry Workstation User s Guide 5 61 Chapter 5 Optimizing Instrument Settings When to increase Increase the Vertical Scale if signal goes offscale Figure 5 18 The Vertical Scale setting is typically between 50 mV and 1 000 mV If you set it at 1 000 mV the maximum Vertical Scale setting and signal is offscale decrease the laser intensity to decrease the signal intensity The offscale signal exceeds 64 000 counts see the right hand axis When to decrease Decrease the Vertical Scale if signal intensity of tallest peak is not greater than approximately 20 percent of the vertical maximum Figure 5 18 If you are at the minimum Vertical Scale setting and signal is not greater than approximately 20 percent of the vertical maximum follow the guidelines in Section 5 4 4 Optimizing Signal to Noise Ratio to increase signal intensity Verti
64. 60 columns Grid Offset Grid 1 2 3 P maximum of 30 12345 maximum of 60 16 00000000000 16 00 00 00000 2 DO 0 0 0 0 0 0 60 0 0 2600000000000 3010 0 060 000000 410D0000000000 36 060 0000 060000 5000000000000 v v maximum of 30 maximum of 15 Nee Figure 2 26 Grid or Offset Grid Pattern in PLT File NOTE All PLT files provided with the system contain fewer than 900 positions and specify a grid see PLT files provided on page 3 67 New PLT files created using Create PLT File specify a grid see Creating a PLT file using Create PLT File on page 3 79 If you use a PLT file that does not meet these requirements the position location and spacing displayed in the OptiPlate dialog box do not accurately reflect the position location and spacing specified by the PLT file However you can still obtain valid Extraction Corrections for the positions If you use a PLT file that contains more than 900 positions an error message is displayed and you cannot run OptiPlate Voyager Biospectrometry Workstation User s Guide 2 57 2 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 8 3 Preparing to Optimize Preparing the plate to optimize 2 58 Applied Biosystems This section includes e Preparing the plate to optimize e Preparing the system e Verifying laser intensity WARNING CHEMICAL HAZARD Angiotensin ACTH and Insulin may cause an allergic skin and
65. 7 28 7 6 4 Stopping a Sequence ee eeceecceeeeeeeeeee cee ceaaeeeeeteeteeeeeeeeeeeaeaaas 7 29 7 6 5 Checking Sequence Status ccccecceeeeeeeeeeeeeeeeeeeeeeeeeeeaeaaes 7 29 Automatic Calibration During a Sequence Run 7 32 7 7 1 Calibration Options in a Sequence cccseeeeeeeeeeeeeeeeeaeeaes 7 32 7 7 2 External Calibration Standard Requirements 7 33 7 7 3 Performing Close External Calibration c cseeeeteeeeees 7 34 7 7 4 Internal Standard Calibration Considerations 7 39 Customizing the Sequence Control Panel 7 43 7 8 1 Customizing the Sequence Display cccssseeceeeeanseeeeeees 7 43 7 8 2 Setting Sequence Control Panel Preferences 7 45 Chapter 8 PSD Analysis x 8 1 8 2 PSD Quick SAT En Wee ees CE CA aoe EE 8 2 8 1 1 PSD Analysis of Angiotensin ccceeeeeeeeeeeeeeeaeeaeeeeeeteeeees 8 7 8 1 2 PSD Analysis of an UNKNOWN 0 cccceeeeeeeeeeee eee aeeaeeteeeeeeeees 8 15 Overview of PSD Analysis 8 20 8 2 1 Post Source Decay Analysis ceccccecseeeeeeeeeeeeeeeeeeeeeeeees 8 20 8 2 2 Differences From Regular Analysis esessessesesesrreerrresnnne 8 24 8 2 3 Segments and Composite Spectra ceseeeeeeeeeeeeeeeeeees 8 25 8 2 4 PSD Data Files a te ae ner tease caatiuiaees 8 27 8 2 5 Mass Calculation for Fragment
66. 7 7 BIC file loaded in Instrument Control Panel 7 28 cannot run BICs that specify internal and external laser in same sequence 7 16 creating 7 13 general sequence parameters setting 7 13 loading 7 24 parameters 7 13 parts of 7 13 pausing and resuming 7 28 run list see Sequence run list saving 7 20 starting 7 25 status 7 29 stopping 7 29 Sequence Control Panel see also Acquiring data Sequence Control Panel see also Sequence accessing from Instrument Control Panel 4 7 basics 4 32 checking disk space 7 25 data file name 7 16 data file name including position number in 7 16 7 46 directory for data files 7 14 interaction with Instrument Control Panel 1 44 4 33 layout organizing 4 35 list of data files acquired 7 14 log file 7 14 organizing 4 35 overview 1 44 4 32 7 2 parts of 4 32 peak detection if no SET file specified 7 17 peak detection specifying 7 17 Run column 7 20 run list see Sequence run list sample plate aligning 7 24 sample position 7 16 selecting rows to run 7 20 software exiting 2 34 software starting 2 33 toolbar 4 32 troubleshooting 9 21 Sequence Control Panel BIC file optimizing for 7 7 requirements 7 8 7 27 selecting 7 16 Sequence Control Panel CAL file applied to multiple spectra in a data file 7 4 creating 7 11 how it is used 7 3 7 4 selecting 7 19 Index Sequence Control Panel calibration applied to multiple spectra in a data file 7 4 external overview 7 5 7 6
67. A m Glossary 3 lt DBNWOrYH G L O S S A R Y Glossary Matrix Low mass UV absorbing substance used in MALDI technology to enable sample ionization Common matrices include sinapinic acid dihydroxy benzoic acid and a cyano 4 hydroxy cinnamic acid See Appendix C Matrixes for more information Metastable ion analysis See MS MS analysis Mirror A single plate at high voltage Commonly used to refer to the reflector in a TOF instrument Mirror voltage ratio Ratio of the mirror high voltage supply to the acceleration high voltage supply Mirror Voltage ratio x accelerating voltage MS MS analysis Selection of a precursor ion for fragmentation and the collection of the fragment ions MS MS analysis can yield sequencing information for a peptide By acquiring data using different mirror voltages you can focus on different segments of the fragment population After acquisition you can piece together the segments to examine a composite PSD spectrum Multiply charged ions See M 2H 2 and M 3H M Z Mass to charge ratio Parent ion See Precursor ion Glossary 4 Applied Biosystems Post Source Decay PSD Fragmentation of an ion during flight after it leaves the ion source region Post translational modification Non amino acid modifications made to a peptide or protein by chemical or enzymatic means Examples include deamidation acetylation oxidation
68. All display network and printer drivers Follow the instructions provided with the software listed above to install it This section describes e Installing the Voyager software e Starting the software e Exiting the software Voyager Biospectrometry Workstation User s Guide 2 29 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 5 1 Installing the Voyager Software Installing To install the Voyager software 1 Insert the Voyager CD into the CD drive in the computer The installation routine automatically starts and the Welcome dialog box appears NOTE If the installation routine does not automatically start click Start on the Windows NT desktop click Run type D VOYAGER SETUP or the drive designation for your CD drive and click OK 2 Click Next The Select Components dialog box appears with all options selected by default e Voyager software Includes the Instrument Control Panel and Sequence Control Panel and all standard calibration reference REF plate type PLT default settings SET files and search pattern SP files provided e Data Explorer software lIncludes the Data Explorer software and default settings SET files e Example files Includes standard instrument settings BIC files and installs them in C VOYAGER DATA NSTALLATION NOTE The standard BIC files are installed in a different directory than the BIC files that have been optimize
69. Calibration During a Sequence Run NOTE This method is useful for plates with larger sample positions If you are using a 400 position or 92x2 position plate follow the procedure in Sample and standard in separate sample positions on page 7 34 NOTE Use plates without laser etched sample positions or wells if you spot standard and sample as shown below m Manual Laser Intensity KIKI Manual Sample Positioning Active Pos fat X 100 well plate plt Standard Sample Logical coordinates microns Figure 7 9 Location of Sample and External Standard for Maximum Mass Accuracy Without Using the Optimize Mass Accuracy Option Voyager Biospectrometry Workstation User s Guide 7 37 Chapter 7 Acquiring Spectra from the Sequence Control Panel To acquire sample and standard create two search pattern files to analyze the sample and standard spots See Creating search pattern files for close external calibration on separate spots on page 7 38 for more information Create two instruments settings BIC files that contain the same settings but specify the sample search pattern file and the standard search pattern file To run samples spotted as illustrated in Figure 7 9 assuming that subsequent positions are spotted in the same way set up the run list as shown in the following table Position pase re Instrument Settings File Calibration Type
70. Da Guidelines for Acceptable resolution is determined by the mass range you acceptable are analyzing Refer to the following tables for the typical resolution esolution at a given signal to noise ratio for different mass ranges ST Signal to Noise Mass Range Typical Resolution in Linear Mode Ratio 1 000 2 000 2 000 or greater 50 1 or greater 2 000 20 000 Determined by width of isotopic envelope 50 1 or greater gt 20 000 Sample dependent 20 1 or greater On Voyager DE STR models resolution is approximately 30 percent higher than the values listed Required signal to noise ratio to obtain the resolution stated Mass Range Typical Resolution in Reflector Mode ee 500 1 500 5 000 or greater 50 1 or greater 1 500 6 000 7 000 or greater 50 1 or greater 6 000 15 000 Determined by width of isotopic envelope 20 1 or greater gt 15 000 Sample dependent 20 1 or greater On Voyager DE STR models resolution is approximately 30 percent higher than the values listed Required signal to noise ratio to obtain the resolution stated Voyager Biospectrometry Workstation User s Guide 5 75 Chapter 5 Optimizing Instrument Settings If you cannot obtain the resolution listed for the mass range of interest consider the following possible causes e Laser intensity is too high e Sample oxidized fresh sample needed e Sample does not ionize well in the matrix e
71. Data Explorer e If Data Explorer is already running and displaying a data file the data file is automatically closed when you access Data Explorer from the Instrument Control Panel Voyager Biospectrometry Workstation User s Guide 4 7 Chapter 4 Voyager Instrument Control Panel Basics 4 2 Using the Control Pages 4 8 Displaying control pages Applied Biosystems The Instrument Control Panel allows you to display organize and rearrange one or more control pages This section describes Displaying control pages Types of page control You can display control pages in several ways Select individual control pages from the View menu Select the View menu then select Default Layout to display the Spectrum window Data Storage and Manual Laser Sample Positioning control pages NOTE Depending on the size of your computer screen some control pages may be only partially displayed To display the entire control page click drag the page borders to resize Hint toolbar button is available for selecting Default Layout See the Data Explorer Software User s Guide Customizing toolbars on page 1 21 for information on customizing the toolbar The M button is located in the Window category Using the Control Pages Types of page You can select between two types of page control for the control control pages Docked Pages are attached or anchored to other pages or the edge
72. Edit existing PLT files with a text editor m Comment RS Create PLT Close Figure 3 15 Create PLT File Dialog Box 3 80 Applied Biosystems Sample Plate Types 4 Set the parameters described below Table 3 14 Create PLT File Parameters Parameter Description Number of columns Limited by plate size Total number of positions columns x rows cannot exceed 32 766 NOTE The OptiPlate application can use PLT files with a maximum of 30 columns in a grid pattern or 60 columns in an offset grid pattern Number of rows Maximum of 52 Total number of positions columns x rows cannot exceed 32 766 NOTE The OptiPlate application can use PLT files with a maximum of 30 rows NOTE The OptiPlate application can use PLT files with a maximum of 30 rows in a grid pattern or 15 rows in an offset grid pattern Position Width and Width and height of positions Height um Corresponds to WellWidth and WellHeight parameters in the PLT file see Table 3 13 PLT File Parameters on page 3 69 Position Depth mm Depth of indentation of a non flat plate for example a welled plate from the plate surface to the bottom of the indentation A setting of 0 is equal to a flat plate Measure the depth of a position on the plate with a micrometer to determine the value to enter This parameter corrects for the distance between the sample in a welled plate and the variable volt
73. Figure 8 8 Timed lon Selector Tab in Hardware Configuration Dialog Box Set the Deflector Gate Width as needed A lower setting increases specificity but may decrease sensitivity CAUTION Do not change the Flight Length to Deflector parameter This parameter is optimized for your system 8 3 Enhancing Fragmentation with CID Enhancing Fragmentation with CID Overview Collision induced dissociation CID is a technology that enhances fragmentation in Post Source Decay PSD analysis CID is available as an option on the Voyager DE PRO and Voyager DE STR workstations The CID option includes e A0 5cc cylindrical cell collision cell in the ion source region connected to the grounded aperture e External CID box with valves to control CID gas flow Figure 8 9 shows the plumbing of the CID option To Fore pump Purge line Flight tube Collision cell 3 way Valve Turbo S l pump 1 ___ _ _ Gas inlet Metering line valve CID Grids Main box in source source Wenn External gas source or atmosphere Figure 8 9 CID Option Plumbing When supplied with gas the collision cell has a higher pressure than the source As ions leave the source they pass through the collision cell and interact with the collision gas molecules Energy is transferred to the ions and fragmentation is enhanced Voyager Biospectrometry Workstation User s Guide 8 31 Chap
74. If the Data Storage control page Figure 6 3 is not displayed select Data Storage from the View menu Data Storage Directory E Woyager Data El Filename NW Autosequence Filenames Sample Description Comment Figure 6 3 Data Storage Control Page 2 Click E to select a directory in which all data files will be stored 6 14 Applied Biosystems Acquiring in Manual Mode from the Instrument Control Panel Type a filename Select Autosequence Filenames if you want the software to determine the next available sequential filename NOTE If you deselect Autosequence Filenames the software uses the name in the Filename field and will overwrite an existing data file If Autosequence is enabled a 4 digit suffix starting at 0001 is automatically appended to the filename specified For example SAMPLE_0001 Before acquiring the software checks the directory specified in the Data Storage page for root file name then appends the next available suffix The software does not overwrite existing file names or reset the suffix to 0001 If you want to reset the suffix to 0001 delete or move existing files from the directory or use a different file name Enter comments that will be saved with the data file This step is optional Voyager Biospectrometry Workstation User s Guide 6 15 Chapter 6 Acquiring Spectra from the Instrument Control Panel Starting To start acquiring select St
75. In the Run list type the starting sample position to fill down NOTE If you want to fill down all positions on the plate you must start at row 1 Select the rows to fill down NOTE To fill down all positions on the plate click on the Sample Position column header to select all rows From the Edit menu select Fill Down Selected rows are filled with the incremented sample position number You can import and export the following file types for use in the Sequence Control Panel Text files TXT tab or comma delimited Excel Worksheet files XLS A file that you import into the run list must meet the following conditions Must contain 11 columns of information to correspond to all columns in the grid except the Acquisition Status column If you do not have information for a column leave a blank in that column Columns must be in the same order as the columns in the run list see Table 7 1 Run List Parameters on page 16 The Run column must include a 1 equal to the Run field being checked or 0 equal to the Run field being unchecked BIC SET or CAL file columns must specify a full path for the file Creating a Sequence NOTE If you do not specify a path the software assumes a path of C VOYAGER e Does not contain blank lines Any lines following a blank line are ignored and not imported Importing To import a TXT or XLS file 1 Select Import from the File menu
76. Instrument Settings Control page is used to determine the t value precursor ion flight time needed for the PSD calibration equation described on page 8 28 The t value is determined using the standard calibration equation t in the standard equation described on page 6 9 You will also specify an external PSD calibration below which is used to determine the values for a 6 andy needed for the PSD calibration equation To generate the calibration 1 Open the precursor spectrum data file in the Data Explorer software 2 Create a single point calibration using the precursor ion mass For more information see the Data Explorer Software User s Guide Chapter 5 3 2 Manually Calibrating 3 Save the calibration file CAL by exporting the calibration constants from the data file For more information see the Data Explorer Software User s Guide Exporting BIC MSM and CAL files on page 1 36 Voyager Biospectrometry Workstation User s Guide 8 39 Chapter 8 PSD Analysis 8 4 2 Determining the Number of Segments to Acquire for a Complete Composite Spectrum Resolution and 8 40 the number of segments Applied Biosystems This section includes e Resolution and the number of segments e Decrement ratio Default Mirror Ratio settings e Size of segments Default number of segments e Acquiring only select segments If you are interested in a complete composite spectrum the numbe
77. Instrument Settings Parameters Relationship to Delay Time and Grid Voltage are interactive parameters For Grid Voltage each Grid Voltage there is an optimum Delay Time and for each Delay Time there is an optimum Grid Voltage The best approach for optimizing Delay Time is to leave the Grid Voltage at a fixed value and optimize Delay Time until you obtain optimum resolution For more information see Section 5 4 3 4 Optimizing Delay Time Adjusting In general e Higher masses require a longer Delay Time e Delay Time is affected by matrix For more information on Delay Time see e Section 5 4 3 4 Optimizing Delay Time e Delay Time nsec on page 5 18 Voyager Biospectrometry Workstation User s Guide 5 55 Chapter 5 Optimizing Instrument Settings 5 3 4 Understanding Guide Wire Voltage NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later By applying voltage to the beam guide wire Figure 5 16 you can overcome the dispersion effect from the source and refocus ions on the detector WWOOHOOOOHOOOOAT Guide Wire Voltage applied ions focused on detector 1019912 Figure 5 16 Beam Guide Wire All models in general e Increase Guide Wire Voltage to increase sensitivity e Decrease Guide Wire Voltage to increase resolution A higher Beam Guide Wire voltage can negatively impact resolution because it is focusing ions that would n
78. Juhasz P M Vestal and S A Martin On the Initial Velocity of lons Generated by Matrix Assisted Laser Desorption lonization and Its Effect on the Calibration of Delayed Extraction Time Of Flight Mass Spectra J Am Soc Mass Spectrom 1997 8 209 217 2 Juhasz P M L Vestal and S A Martin Novel Method for the Measurement of the Initial Velocity of lons Generated by MALDI Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics May 12 16 1996 Portland OR p 730 Voyager Biospectrometry Workstation User s Guide 5 21 Chapter 5 Optimizing Instrument Settings Matrix influence 5 22 Applied Biosystems The initial velocity is the average speed at which matrix ions desorb The initial velocity of matrix contributes to the higher order terms in the calibration equation see Figure 6 1 on page 6 9 The software allows you to correct the calibration equation for matrix initial velocity by selecting a matrix in instrument settings see page 5 20 Table 5 5 Matrix Initial Velocity Settings Initial Velocity Matrix m sec 2 a cyano 4 hydroxycinnamic acid 300 Sinapinic acid 350 DHB 500 3 hydroxypicolinic acid 550 1 Juhasz P M Vestal and S A Martin On the Initial Velocity of lons Generated by Matrix Assisted Laser Desorption lonization and Its Effect on the Calibration of Delayed Extraction Time Of Flight Mass Spectra J Am S
79. Mode High laser power causing the following This File 2 CAVOYAGERADATSAIKEITHIKO106002 MS Collected 16 95 8 12 AM 20 Matrix peaks seen with Low Mass Gate On 100004 Sample mass slightly higher than expected due to collision of ions with excess neutrals generated by the laser a Broad peak Poor resolution less than 100 4000 No separation between molecular ion Dimer and matrix adduct ion o 5000 10000 15000 20000 25000 30000 35000 Mass mie Excess chemical noise 2000 Figure H 1 Example of Poor Mass Spectrum for Myoglobin Using Sinapinic Acid H Laser power near threshold producing This File 1 CAVOYAGERADATAKEITHISMOOTHMS Collected 16 95 8 17 AM Savitsky Golay Order 2 Points 19 162351 5 50004 4000 8 Sharp narrow peak Good resolution greater than 350 30004 Cont Dimer minimized 2000 Good separation between molecular ion 1000 and matrix adduct ion 15000 Mass mis 5000 10000 No matrix peaks seen Minimal noise Figure H 2 Example of Good Mass Spectrum for Myoglobin Using Sinapinic Acid Voyager Biospectrometry Workstation User s Guide H 9 Appendix H Continuous Extraction Mode Matrix and sample peaks saturated flat topped This File 1 CAVOYAGER DATA KEITHYKO106011 MS High laser power causing the following Collected 16 95 8 42 AM 1299 98 3
80. Panel 6 6 6 Process that Occurs During Acquisition in Automatic Mode NOTE This process occurs when you acquire using a BIC file that has Use Automated Laser Intensity Adjustment enabled Checking disk Before starting a run that uses automated data evaluation space described on Setting spectrum acceptance and laser adjustment criteria on page 6 42 the system checks that at least 100 MB of disk space is available NOTE If the run requires more than 100 MB for data file storage a message is displayed during the run when free disk space is no longer available Prescan mode Prescan mode allows the system to determine the appropriate laser setting for a sample position before acquiring spectra This feature minimizes sample loss Prescan mode does not consider signal to noise ratio or resolution when determining laser setting You can select one of the following Prescan mode choices e At First Search Pattern Position Only e At Each Search Pattern Position NOTE If Prescan is not selected the system operates in Acquisition mode See Acquisition mode on page 6 61 Shots Spectrum in Each time the system begins acquiring in Prescan mode it Prescan mode acquires five Shots Spectrum or the number of Shots Spectrum specified in the Instrument Settings control page if less than five 6 58 Applied Biosystems During Prescan In Prescan mode the system does the following 1 Acquiring in Autom
81. Sample is too dilute or too concentrated e Impurities are suppressing ionization of sample There are heterogeneous components in the peak 5 4 3 3 Optimizing Guide Wire Voltage NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later Start with the BIC file in which you optimized Delay Time or Grid Voltage Use the following Guide Wire settings to optimize resolution Mode Guide Wire Voltage Setting Linear Peptides lt 10 000 Da 0 05 Proteins 0 1 or higher for best sensitivity Reflector 0 0 005 or as low as possible NOTE Decreasing Guide Wire to these suggested values may decrease signal to noise ratio For more For more information see information Section 5 4 4 3 Setting Guide Wire Voltage e Section 5 3 4 Understanding Guide Wire Voltage 5 76 Applied Biosystems Optimizing Instrument Settings Parameters 5 4 3 4 Optimizing Delay Time This section includes e Overview Inherent Delay Time offset e Optimizing Delay Time e Setting Delay Time to resolve isotopes across a broad mass range Overview Use this procedure when operating in Linear mode or Reflector mode Start with a standard BIC file optimize the Delay Time and leave the Grid Voltage unchanged Before optimizing read Setting Delay Time to resolve isotopes across a broad mass range on page 5 81 NOTE You can alternatively fo
82. Save the current segment if the data is acceptable before starting to acquire the next segment If you do not you will lose the data for the current segment Repeat step 3 through step 7 to collect remaining segments NOTE Segments are listed in the Data Explorer software in the order in which they are acquired If segments with duplicate Mirror Ratios are contained in the file the software uses the last acquired segment when it generates the composite spectrum After you acquire all necessary segments select Stop Experiment from the Acquisition menu The PSD data file is closed You cannot view the PSD data file in the Data Explorer software until you stop the experiment CAUTION If you stop an experiment without saving any segments no DAT file is created PSD Quick Start Confirming or To confirm or investigate fragment ion identity you can use the investigating following tools fragment ion Ifthe peptide sequence is known Use the lon identity Fragmentation calculator in the Data Explorer software to apply fragment labels to the unknown composite spectrum For information see the Data Explorer Software User s Guide Section 8 2 Applying Fragment Labels e If the peptide sequence is not known Use the Peptide Fragmentation macro provided with the Data Explorer software to investigate the sequence in the unknown composite spectrum For information see the Data Explorer Software User s Guid
83. Select a value Voyager Biospectrometry Workstation User s Guide 8 49 Chapter 8 PSD Analysis Using the Fill To use the Fill Down function Down function 4 if you want the Fill Down function to calculate values for Mirror Ratio and Guide Wire instead of copying the value from the first row set the following in the Mirror Ratio Guide Wire Fill Down section e Decrement Ratio To calculate the needed Mirror Ratios For more information see Decrement ratio on page 8 41 Increase this value to create smaller segments Decrease this value to create larger segments e Guide Wire Tracks Mirror Ratio not available on Voyager DE STR models with serial number 4154 and later If you want to automatically adjust the Guide Wire settings according to the Mirror Ratio setting NOTE Do not select Guide Wire Tracks Mirror Ratio option on Voyager DE PRO or Voyager DE STR systems This parameter is for use with older systems only 2 Type in values in any row that you want to fill down into selected rows 3 Click on the Segment number to select the row containing the values to fill down 4 Click LA The following occurs e The Mirror Ratio is calculated and entered in all rows below If no Decrement Ratio is specified the value from the first cell is copied to selected cells below NOTE If you selected Guide Wire Tracks Mirror Ratio Guide Wire is also calculated and filled in 8 50 Applie
84. The PLT file and alignment information associated with the Plate ID are automatically loaded Alternatively you can specify a new Plate ID and select a PLT file For more information see Assigning Plate IDs on page 3 50 e Click OK 2 50 Applied Biosystems Aligning the Sample Plate Aligning To align the loaded plate 1 Tape the transparency to the video monitor 2 Select Align Sample Plate from the Sample Plate menu The Sample Plate Alignment wizard Figure 2 25 is displayed Sample Plate Alignment x The Sample Plate Alignment Wizard allows you to determine the laser position in the sample well and adjust the sample well XY position if necessary You then calibrate the sample well XY position using four sample well positions Use the Sample Plate Alignment Wizard to configure the well positions when you are running in Automatic Control mode and the laser beam is not striking the center of the sample well Click Next to begin the Sample Plate Alignment process Nest gt Cancel Figure 2 25 Sample Plate Alignment Wizard 3 Click Next The sample plate moves to the first alignment position on the sample plate See Table 2 1 Four Corner Positions on Sample Plates on page 2 50 to determine your plate positions 4 Start the laser using the control stick NOTE When aligning the sample plate starting the laser does not start acquisition Voyager Biospectrometry
85. Three point tp andA Calculates by linear least squares fit of the data points from standard mass Acquiring Use standards that are above and below the mass range of calibration interest standards To ensure that sample and standard peaks are similar in size and shape use the same acquisition conditions for sample and standard in particular e Instrument mode linear or reflector e Signal intensity e Laser intensity e Voltages Accelerating Grid and Guide Wire e Low Mass Gate To obtain accurate mass values in the calibration you need well shaped symmetrical peaks You may need to smooth or noise filter peaks before calibrating See the Data Explorer Software User s Guide for more information 6 10 Applied Biosystems Acquiring in Manual Mode from the Instrument Control Panel 6 2 Acquiring in Manual Mode from the Instrument Control Panel This section includes e Manually acquiring evaluating and saving spectra e Manually accumulating spectra from multiple acquisitions Before acquiring spectra become familiar with the information in Section 6 1 Before You Begin 6 2 1 Manually Acquiring Evaluating and Saving Spectra This section describes the following manual mode procedures e Selecting instrument settings e Selecting sample position and laser intensity e Setting Data Storage e Starting acquisition e During acquisition e Evaluating data e Saving data e Saving the instrume
86. Threshold Troubleshooting Symptom Action Signal fades very quickly Increase the laser intensity by 1 to 2 percent while acquisition is occurring If signal does not increase move to a new point in the sample well while acquisition is occurring If signal does not increase increase the laser setting further 2 to 3 percent at a time Signal increases quickly then saturates Stop acquisition Decrease the laser setting by one step use the slider controls on the Manual Laser page Acquire again Hysteretic signal behavior lag in response For example you observe signal at a laser intensity of 300 signal disappears when you decrease to 290 signal does not reappear when you increase intensity back to 300 Move to new spot in sample well if this problem prevents you from generating a signal that is at least 10 percent of full scale If the signal is below 10 percent of full scale sample may be consumed Voyager Biospectrometry Workstation User s Guide H 19 Appendix H Continuous Extraction Mode Table H 7 Laser Threshold Troubleshooting Continued Symptom Action Signal is flat Laser setting may be too low increase Sample may be consumed move to a new position in sample well Sample may not be present try new position Sample may not ionize well use different matrix Cannot see ions in Reflector Check that you can see ions in Linear mode mode e If
87. User s Guide 3 19 Chapter 3 Preparing Samples Table 3 9 Synthetic Polymer Matrix Information Matrix concentration 0 1 M 1071 M Final sample concentration 0 1 mM 10 4 M Solvents Sample and matrix dependent Choose solvents in which polymer and matrix are soluble Preparation Follow the procedure in Preparing matrix on page 3 5 and combine 1 1 sample matrix See page 3 21 for application techniques Apply vacuum drying for non volatile solvents water DMF Acetone and THF dry instantaneously Crystals No crystals visible If sample position looks glassy or shiny it may indicate sample concentration is too high Analyze polymer samples within one hour of loading on the sample plate Many polymer matrix mixtures are not stable once they are loaded Areas in which you see hot and cold spots are much smaller with polar compounds than with non polar compounds Stability Prepare weekly Store THF and acetone solutions in tightly sealed vials 3 20 Applied Biosystems Preparing Samples You can load polymer samples on sample plates in two ways Thin layer polymer method yields even response but provides adequate sample response for only 10 laser shots Load 0 3 ul sample matrix solution in one sample position e Thick layer polymer method yields less even response but provides adequate sample response for 50 to 100 laser shots Load 2 to 3 ul sample matrix
88. View in Manual Laser Sample Positioning Control Page on page 3 78 for information on displaying logical coordinates NOTE On a standard 100 well plate position 91 is located at X 1587 5 Y 1587 5 and corresponds to the lower left of the sample plate For optimum mass accuracy do not specify coordinates on the outer edges of a plate continued 3 70 Applied Biosystems Sample Plate Types Table 3 13 PLT File Parameters Continued Parameter Description X and Y continued The following coordinates correspond to the corner positions that define the area of the sample plate that provides optimum mass accuracy Absolute X 6667 5 Absolute Y 42227 5 Top left corresponds to position 1 e Absolute X 42227 5 Absolute Y 42227 5 Top right e Absolute X 6667 5 Absolute Y 6667 5 Bottom left e Absolute X 42227 5 Absolute Y 6667 5 Bottom right NOTE Sample plate alignment for custom plate types PLT files that you create requires sample spots on any four corner positions You can use any four corner positions but for maximum mass accuracy do not use positions outside the area defined by the coordinates above Position name optional Displayed as the Active Position name in the Sample View see Figure 4 8 on page 4 27 NOTE If you do not specify a position name in the file the position number is displayed The first sample in the PLT file is considered sample 1 The Nth sample in the
89. Voyager DE and Voyager DE PRO Voyager DE STR Voyager DE and To load sample plates Voyager DE PRO Eject the sample holder as described in Section 3 4 3 Ejecting the Sample Holder 2 Hold the sample plate with the bottom of the numbers facing toward the analyzer for standard 100 well plate and with the slanted underside of the plate facing to the left 3 Slide the sample plate into the holder from the right side until it snaps into place Figure 3 5 The ball bearings on the holder snap into the plate CAUTION If the sample plate does not snap into place it may be inserted into the holder the wrong way and it may jam inside the instrument Remove the plate slide it into the holder with the slanted underside of the plate facing to the left and toward the back of the instrument and snap it into place Voyager Biospectrometry Workstation User s Guide 3 55 Chapter 3 Preparing Samples ss Figure 3 5 Loading the Sample Plate in a Voyager DE or Voyager DE PRO WARNING PHYSICAL INJURY HAZARD Fingers can get caught in the sample holder To avoid injury do not click Load to retract the sample holder when your fingers are near the sample holder 4 From the Sample Plate menu select Load to retract the sample plate and insert it into the main source chamber The Load Eject Sample Plate dialog box Figure 3 6 is displayed 3 56 Applied Biosystems Loadin
90. Workstation User s Guide 2 51 Chapter 2 Alignment information saved 2 52 for future use Applied Biosystems Installing the Voyager Biospectrometry Workstations 5 Mark the laser position on the transparency Do not mark the sample position Be sure the laser is centered on the video monitor and does not appear as shown in Figure 2 24 on page 2 48 6 Center the sample position under the laser using the control stick 7 When the position is centered click Next The system advances to the next corner position for the PLT type 8 Repeat step 3 through step 7 for each of the four corners of the sample plate The software calculates the alignment and uses the settings to ensure all sample positions are centered under the laser If the alignment is successful a message is displayed 9 Do one of the following Click To Finish Save the alignment for the specified Plate ID Cancel End the Sample Plate procedure without saving the alignment for the plate Back Repeat the alignment procedure NOTE A message is displayed if the alignment is outside the preferred tolerance Repeat the alignment procedure by clicking Back If the alignment is still outside of tolerance call Applied Biosystems Technical Support The alignment information is saved with the Plate ID and applied each time you load this Plate ID into the system For more information see How the system aligns a plate
91. Zooming sample position 6 13 spectrum trace 4 13 Voyager Biospectrometry Workstation User s Guide Index Index 45 Index Index 46 Applied Biosystems
92. accumulating spectra 6 19 acquisition PSD starting 8 38 acquisition starting 4 25 6 16 acquisition stopping 4 25 6 16 data saving 6 18 Data Storage setting 6 14 evaluating data 6 17 Index Manual Control mode continued Mass accuracy optimization instrument settings selecting 6 11 continued laser intensity adjusting 4 28 using an optimized plate 3 39 overview 4 6 6 2 6 11 using an unoptimized plate 3 40 process that occurs during 3 53 acquisition 6 16 Mass assignment precision 6 26 sample position selecting 6 13 saving BIC after acquisition to store laser intensity 6 19 Mass range acquisition 5 19 displaying full range on Manual Laser Sample Positioning oscilloscope l 3 control page does not update with every laser displaying 4 27 shot 6 16 9 21 laser position 4 31 in Instrument Settings 5 19 location 4 27 PSD composite spectrum 8 25 parameters 4 27 PSD segments 8 54 8 64 shape of positions 3 69 3 82 Voyager DE 1 4 using 4 27 Voyager DE PRO 1 4 Mass accuracy Voyager DE STR 1 6 and location of standard 3 38 H 24 Mass resolution see Resolution mass calibration 6 7 6 10 Mass spectrometer effect of charges on 9 17 maintenance 9 3 lec ob Na and Kon 9 17 parts of Voyager DE 1 20 1 22 actors av ecung 6 26 parts of Voyager DE PRO 1 21 improving 1 14 2 56 3 38 6 4 1 22 6 5 7 24 H 23 H 24 parts of Voyager DE STR 1 33 improving by deisotoping before rear panel Voyager DE STR 2 23 c
93. acquisition This reduces variability in accelerating voltages and yields more reproducible ion flight times To turn on the high voltage power supplies click in the toolbar NOTE The high voltage power supplies are automatically turned off after 60 minutes if the mass spectrometer is not used To change the Idle Time see High Voltage Configuration on page 2 38 Before acquiring a sequence 1 Load a sequence by doing one of the following e Create a new sequence See Section 7 3 Before Creating a Sequence Open an existing sequence by clicking in the toolbar and selecting an SEQ file 2 Check system status See Section 2 11 Checking System Status and Pressures Running a Sequence 7 6 Running a Sequence This section includes e Starting a sequence e What the system checks when you start a sequence e During and after acquiring a sequence e Stopping a sequence e Checking sequence status 7 6 1 Starting a Sequence CAUTION After you start a sequence do not change instrument settings or any other parameters in the sequence Changing settings while a sequence is running may cause the sequence to stop Checking disk Before starting a sequence run the system checks that at space least 150 MB of disk space is available if you have enabled the Low Disk space message in Preferences described in Section 7 8 2 Setting Sequence Control Panel Preferences NOTE If the run requires more th
94. and display the Sample View Note the relative x and y coordinates for a minimum of three positions on the standard spot and a minimum of six positions on the sample spot Create the search pattern file using Windows Notepad as described Creating and Editing SP Files Using the Search Pattern Editor on page 6 50 Figure 7 10 shows an example search pattern SP file for internal calibration using separate spots sinternal calibration using two spots 5 25 00 std spots 1048 635 857 635 762 635 Sample spots 635 635 730 635 825 635 635 730 730 730 825 730 Figure 7 10 Example SP File for Internal Calibration Using Separate Spots Voyager Biospectrometry Workstation User s Guide 7 41 Chapter 7 Acquiring Spectra from the Sequence Control Panel 7 42 Performing internal standard calibration with an external For applications in which the concentration of internal standard to sample varies you can set the sequence to perform an external and an internal calibration This provides a certain level of mass accuracy even if the internal standard peak is suppressed and the internal calibration fails An calibration example of this application is analysis of proteolytic digests backup which use autolytic enzyme peaks as internal standards Set up the run list as shown in the following table Position Ru nn lle Calibration Type eee 91 STD1 STD BIC INTERNA
95. and matrix for matrix solutions With the 1 1 dilution used in this strategy the typical concentration of organic may not be high enough to keep the matrix in solution Load 0 5 to 1 ul of sample on the plate of the appropriate concentration to yield the necessary final sample concentration followed by 0 5 to 1 ul of matrix See Matrix Information on page 3 6 for final sample concentrations A homogeneous mixture is critical for good crystallization Applying sample before matrix prevents matrix from drying before it mixes with sample Loading Samples on Sample Plates 3 2 Loading Samples on Sample Plates In this section This section describes e Overview e Locating standards for optimum mass accuracy e Loading samples dried droplet application e Loading samples thin layer application e Examining crystals on sample plates 3 2 1 Overview In this section This section describes e Types of applications e Types of sample plates e Handling sample plates e Guidelines for good crystallization Recommended pipette tips Types of Two techniques are available for sample application applications Dried droplet Simple application technique suitable for most applications Can use any type of sample plate e Thin layer Use for increased sensitivity when analyzing peptides with a concentration lt 0 1 pmol ul Requires polished blank sample plate no etched positions Voyager Biospectrometry Work
96. are acquired under the same conditions All spectra that you save are placed in one DAT file no DAT file is created until you save a segment When you save the first PSD segment the software creates a PSD DAT file During a PSD acquisition the PSD DAT file remains open and you can acquire multiple segment traces accumulate or discard traces and save traces Each time you save a trace it is appended to the currently open PSD DAT file At the end of the acquisition you stop the experiment which closes the PSD DAT file and makes it available for viewing in the Data Explorer software After an experiment is closed and the DAT file is closed you cannot reopen the DAT file to append additional segments 8 52 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Manual Control Mode NOTE In the Data Explorer software you cannot view the PSD DAT file you are currently acquiring until you stop the experiment The PSD DAT file is open and not available for viewing while the experiment is running NOTE If you stop an experiment without saving any segments no DAT file is created NOTE All instrument settings except Shots Spectrum are disabled as soon as you start acquisition in PSD mode until you stop the experiment Make sure instrument settings are correct before starting acquisition Acquiring PSD To acquire PSD segments segments 4 inthe Manual Laser Intensity Sample Positi
97. displayed in the Instrument Control Panel if you open a BIC file set to PSD mode Voyager Biospectrometry Workstation User s Guide 8 7 Chapter 8 PSD Analysis PSD acquisition settings fur Vorager instrument Control Panel BIC C VOYAGER Data nstallation angiotensin_psd bic SH erel Ele Edt View Instument Acquistion SamplePlate Display Tools Applications EES r Data Storage Filename O F Autosequence Filenames Sample Description Comment r Manual Laser Intensity 1 4 4 gt Manual Sample Positioning Active Pos 91 z 100 well plate pit oooooooood Directory C Woyager Data FE Intensity r Mirror ratio Guide wire auto fill Decrement ratio 0 75 Guide wire tracks minor ratio Precursor mass 1296 7 I Precursor ion selector 1005 905 804 704 604 504 404 304 204 moence Eeee rure Current Spectrum 0 shots XX 0 Saved 460 800 T T 0 1200 1600 2000 Mass m z 1 000 0 020 200 of x geje B Instrument Mode PSD Positive Mode Digitizer r Control Mode Manual C Automatic 20 r Voltages Accelerating 20000 v Grid 80 50 0 99 9 Guide Wire 0 02 0 000 0 300 Delay Time 100 nsec p Spectrum Acquisition Shots Spectum 50 Mass Range Da 324175 to 1626 58 m essa p Calibration Matriz
98. e PSD For PSD Analysis allows you to create composite spectra from fragment ion spectra Uses the reflector detector See Chapter 8 PSD Analysis for more information NOTE Reflector and PSD modes are not available on the Voyager DE system If you have a Voyager DE system buttons for these two modes are not displayed Extraction Type Specifies the type of extraction to use e Delayed wUse for normal operation Described in Section 1 4 Voyager DE Delayed Extraction Technology e Continuous Use for diagnostic purposes For more information see Appendix H Continuous Extraction Mode Polarity Type Specifies the polarity of spectra to acquire e Positive e Negative continued Voyager Biospectrometry Workstation User s Guide 5 25 Chapter 5 Optimizing Instrument Settings Table 5 6 Instrument Mode Parameters Continued Parameter Description Laser Type Specifies the type of installed laser e Internal e External optional NOTE When you switch from the internal laser to the external laser you must manually reset the external laser before acquiring data See Section 2 9 Resetting the Optional External Laser NOTE When you save or load a BIC file with the external laser specified a message prompts you to reset the external laser the Manual Laser Sample Position control page see Figure 4 8 on page 4 27 displays External and the laser intensity range is
99. eae Name File 91 STD1 STD BIC specifies STD SP INTERNAL UPDATE STD CAL 91 SAMP1 SAMP BIC specifies SAMP SP EXTERNAL STD CAL 92 STD2 STD BIC specifies STD SP INTERNAL UPDATE STD CAL 92 SAMP2 SAMP BIC specifies SAMP SP EXTERNAL STD CAL 93 STD3 STD BIC specifies STD SP INTERNAL UPDATE STD CAL 93 SAMP3 SAMP BIC specifies SAMP SP EXTERNAL STD CAL 7 38 Creating search calibration on To create search pattern files for close external calibration on pattern files for separate spots close external 1 Spot sample and standard as close to each other as possible within 1 mm see Figure 7 9 on page 7 37 separate spots NOTE For optimum mass accuracy calibration standards must be as close to unknowns as possible An internal standard spot within 1 mm of a sample spot can yield mass accuracy close to the expected mass accuracy for internal standard calibration on your system Applied Biosystems Automatic Calibration During a Sequence Run NOTE Use plates without laser etched sample positions if you spot standard and sample as shown in Figure 7 9 Load the sample plate select the position containing sample and standard and display the Sample View Note the logical x and y coordinates for a minimum of three positions on the standard spot and a minimum of six positions on the sample spot Create the two search pattern files using Windows Notepad as described in Cr
100. improve external calibration mass accuracy see Appendix A Specifications for external calibration specifications for your system perform plate optimization before acquiring data For more information see Section 3 4 2 Using the Mass Accuracy Optimization Option e Mass calibrate on the same sample plate you will use to analyze samples If you do not use the Optimize Mass Accuracy option note the following For rapid screening in which high mass accuracy is not needed one calibration standard located in the center of the plate is adequate e For applications requiring higher mass accuracy more calibration standards are needed You must experiment to determine the number and location of standards required to provide the mass accuracy you require e For optimum mass accuracy place samples in sample positions adjacent to standards Voyager Biospectrometry Workstation User s Guide 7 33 Chapter 7 Acquiring Spectra from the Sequence Control Panel e For maximum mass accuracy spot sample and standard as close to each other as possible within 1 mm This is referred to as close external calibration For more information see Section 7 7 3 Performing Close External Calibration 7 7 3 Performing Close External Calibration NOTE The Mass Accuracy Optimization feature provides mass accuracy comparable to close external calibration and requires fewer calibration standards For information see Section 3 4 2 Using
101. in PSD calibration when you generate the composite spectrum Even if prompt and PSD fragments are identical in chemical structure they will travel down the flight tube at different times Figure 8 18 Prompt fragments Accelerated based on their initial fragment mass and reach the reflector sooner than their corresponding PSD fragments PSD fragments Accelerated based on the mass of the molecular ion from which they form and reach the reflector later than the prompt fragments Because of this difference in flight times the Precursor lon Selector can screen out prompt fragments while allowing PSD fragments to pass into the reflector Voyager Biospectrometry Workstation User s Guide 8 71 Chapter 8 PSD Analysis Path of prompt fragment ion Path of molecular ion Prompt fragment PE Path of PSD fragment ion 1er lon Source Reflector Prompt fragment PSD fragment formed formed lt Reflector Ta lon Source Precursor lon Selector on Prompt fragment deflected Figure 8 18 Flight Path of Prompt and PSD Fragments 8 72 Applied Biosystems Exploring PSD Mode 8 6 3 Observing the Effects of Grid Voltage Adjusting Grid Voltage affects resolution In this section In this section you will e Understand the function of Grid Voltage in focusing ions e Observe the impact of Grid Voltage on higher and lower mass ions
102. in for fragment ion mass accuracy step 4 You use this multi point external Angio_PSD CAL calibration in PSD analysis to obtain maximum mass accuracy for the fragment ions Voyager Biospectrometry Workstation User s Guide 8 3 Chapter 8 PSD Analysis Table 8 1 Steps to Perform PSD Analysis of Angiotensin Continued See Step Result page 6 To verify that the Angio_PSD CAL yields 8 14 acceptable mass accuracy Angiotensin_PSD BIC with Add the PSD calibration generated in Angio CAL step 5 to PSD Acquisition parameters Angio_PSD CAL Open Angiotensin_PSD BIC select External PSD Calibration File then select Angio_PSD CAL for fragments Acquire PSD segments precursor and Recalibrated angiotensin PSD 8 14 fragment spectra with PSD calibration Composite spectrum with optimum fragment ion mass accuracy oP 8 4 Applied Biosystems PSD Quick Start Table 8 2 Steps to Perform PSD Analysis of an Unknown See Step Result page Generate a PSD calibration on angiotensin by PSD calibration 8 3 performing step 1 through step 6 in Steps to for fragment ion mass accuracy Perform PSD Analysis of Angiotensin on Angio_PSD CAL page 8 3 Generate a precursor spectrum on the Unknown Reflector spectrum 8 7 unknown in Reflector mode use Precursor PSD_Precursor BIC provided 2 NOTE This step assumes th
103. in the run list described in Section 7 4 2 Creating a Run List 7 4 1 Setting General Sequence Parameters Set parameters as needed 1 From the View menu select General Sequence Parameters The General Sequence Parameters dialog box is displayed Figure 7 5 General Sequence Parameters x Data Files E Woyager Datas An Directory E Author MVreeland Comments fin Run Log File Name fests OK Cancel Figure 7 5 General Sequence Parameters Dialog Box Voyager Biospectrometry Workstation User s Guide 7 13 Chapter 7 Acquiring Spectra from the Sequence Control Panel 2 Type or select the Directory name in which to store the data files 3 Enter text as needed for Author and Comments This information is stored with the sequence SEQ file 4 Specify the Run Log file name the default name is taken from the sequence name If you want to store the run log in a directory other that the Directory specified above click E and select a directory for the file The run log file contains the list of lines from the run list that were executed during the sequence and any errors that occurred If a line in the run list generated more than one data file the log lines are duplicated and file names are incremented accordingly 5 Click OK 7 4 2 Creating a Run List This section includes e Sample order in the run list e Creating a run list e Modifying and customizing the run list Filling down
104. inches 102 cm to the right side of the mass spectrometer for Video monitor for sample chamber e Computer monitor control stick and keyboard e Printer e Optional oscilloscope or external digitizer Weight The Voyager DE system weighs approximately 250 pounds 113 kg The Voyager DE PRO system weighs approximately 350 pounds 159 kg Power voltage The Voyager DE and Voyager DE PRO systems require requirements Location Power Voltage Required US 120 V 20 A single phase dedicated circuit 60 Hz power source European 230 V 10 A single phase 50 60 Hz power source You must be able to quickly disconnect the main power supply to the instrument if necessary In addition you need grounded outlets for the e Computer CPU e Computer monitor e External digitizer or oscilloscope if your system includes e Printer optional Voyager Biospectrometry Workstation User s Guide 2 3 Chapter 2 2 4 Selecting input voltage Applied Biosystems Installing the Voyager Biospectrometry Workstations If the system is set for a voltage that does not accommodate your power source select a different input voltage setting on the Voyager system If you select a different input voltage setting install fuses of the proper rating Extra fuses for different voltage settings are supplied with the system Perform this procedure before plugging in and powering up the system for the first
105. internal overview 7 5 7 6 internal update overview 7 5 7 6 type selecting 7 18 Sequence Control Panel macro creating 7 8 how it is used 7 3 selecting 7 17 7 20 Sequence Control Panel sequence loading 7 24 pausing and resuming 7 28 starting 7 25 stopping 7 29 Sequence Control Panel SET file creating 7 12 defaults used if none specified 7 17 description 7 3 how it is used 7 3 selecting 7 17 Sequence run list cannot run BICs that specify internal and external laser in same sequence 7 16 creating 7 14 customizing 7 21 importing and exporting 7 22 importing blank lines not supported 7 23 9 21 importing lines not imported 7 23 9 21 modifying during run 7 28 sample order 7 14 Serial number displaying 2 41 Service contract 9 3 SET file Sequence Control Panel creating 7 12 defaults used if none specified 7 17 description 7 3 how it is used 7 3 selecting 7 17 Voyager Biospectrometry Workstation User s Guide Index 37 I N D E X Index Shots Spectrum description 5 19 does not match number of times laser fires 5 19 for maximum mass accuracy 6 6 impact on signal to noise 5 50 improving signal to noise ratio 5 89 incorrect number 9 20 maximum number 5 19 5 89 maximum number overriding with manual accumulation 5 89 saving a single shot 2 45 troubleshooting 9 20 Shutting down computer 2 75 mass spectrometer 2 75 Side panel Voyager DE and Voyager DE PRO mass spectrometer 2 9 Signal flat 9 7
106. lon Selector in PSD mode the mass specified for the Timed lon Selector in Reflector mode also changes Setting voltages 3 For optimum mass accuracy change the Accelerating and external Voltage and Grid Voltage on the Instrument Settings calibration for the control page to the values you used to acquire the precursor precursor spectrum and to generate the calibration spectrum NOTE If you use different voltage settings Accelerating Voltage Grid Voltage or Delay Time to acquire the precursor spectrum generate the calibration and acquire the fragment spectra the software compensates for the different settings However using different voltage settings may not yield optimum mass accuracy 8 44 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Manual Control Mode 4 Setting precursor 5 mass and PSD calibration for fragment spectra In the Calibration section of the Instrument Settings control page select the matrix you are using and the CAL file you created in Generating an external calibration for the precursor ion on page 8 39 NOTE The calibration you specify on the Instrument Settings control page is used to determine the t value precursor ion flight time needed for the PSD calibration equation described on page 8 28 The t value is determined using the standard calibration equation t in the standard equation described on page 6 9 You will also specify an ex
107. malfunction Technical Support Beam guide wire Call Applied Biosystems malfunction Technical Support Internal detector Call Applied Biosystems power supply setting Technical Support too high Grounded grid inion Call Applied Biosystems source damaged Technical Support continued 9 14 Applied Biosystems Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor signal to noise ratio on sample peaks Matrix peaks causing excessive noise in detector Turn on Low Mass Gate in BIC file to suppress matrix peaks Dimer in spectrum Laser intensity too high causing signal Adjust laser by using the slider controls on the Manual saturation Laser Sample Positioning control page Dimers trimers and Sample Prepare sample matrix with a tetramers in spectrum concentration too high final sample concentration appropriate for sample and matrix See Section 3 1 3 Matrix Information Mass calibration on Spectrum window not accurate Wrong Flight Length entered in Hardware Configuration Call Applied Biosystems Technical Support Voyager Biospectrometry Workstation User s Guide continued 9 15 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Peaks not symmetrical Laser intensity too high Decrease laser intensity by
108. monitor PB100844 To Video on side panel Power cord of mass spectrometer Figure 2 12 Rear Panel of Video Monitor NOTE In the US only you can plug the video monitor into a grounded wall outlet or into the receptacle on the mass spectrometer Voyager Biospectrometry Workstation User s Guide 2 27 Chapter 2 2 28 Installing the Oscilloscope video monitor and computer monitor Applied Biosystems Voyager Biospectrometry Workstations Make the following connections on the rear panel of the Voyager DE STR system Voyager DE STR Figure 2 8 on Connect to Cable page 2 23 Video Video input of BNC with video video monitor adapter Figure 2 12 VGA Computer monitor 9 pin connector GPIB Oscilloscope IEEE 488 Figure 2 10 on page 2 26 NOTE Thread cables for CH1 CH2 and CH3 Aux1 input through the circular hole in the front panel of the workstation Installing Software 2 5 Installing Software All necessary software is installed on your Voyager workstation when it is shipped to you Use these instructions to reinstall software or install a new version of software The Voyager software requires a total of 100 MB of free disk space plus additional space for data files Before installing the Voyager software make sure the following software is installed on your computer e Microsoft Windows NT based software e
109. necessarily take 10 seconds with a Bin Size of 1 nsec Changes in mass in the low mass range has a greater impact on laser firing rate than changes in mass in the high mass range For additional information on laser firing rates and digitizers see Figure A 1 on page A 12 continued Voyager Biospectrometry Workstation User s Guide 5 27 Chapter 5 Optimizing Instrument Settings Linear Reflector Click the Linear or Reflector Digitizer tab to display the Digitizer Linear Digitizer or Reflector Digitizer page Figure 5 5 parameters NOTE The Reflector Digitizer tab is not displayed on the Voyager DE system Mode Digitizer x Refectar Draiteer Advanced Instrument Mode Linear Digitizer Bin Size nsec e 7 Number of Data Points 25000 Vertical Scale mY full scale 1000 x Vertical Offset full scale 0 0 LA Input Bandwidth MHz Full be Figure 5 5 Mode Digitizer Settings Dialog Box with Linear Digitizer Tab Displayed Linear and Reflector Digitizer parameters are described in Table 5 7 5 28 Applied Biosystems Instrument Settings Parameter Descriptions Table 5 7 Linear and Reflector Digitizer Parameters Parameter Description Bin Size nsec Determines the time interval nanoseconds between subsequent acquired data points see Figure 5 17 on page 5 59 Use this setting to optimize resolution Bin Size and Number of Data Points Digitized are dependent value
110. noise NOTE To maintain signal to noise ratio use a lower Input Bandwidth setting Mass Range Input Bandwidth Da Linear Mode Reflector Mode PSD Mode 0 500 Full Full 200 250 MHz 500 6 000 200 250 MHz Full OT AO MD on PSD 6 000 50 000 200 250 MHz 200 250 MHz segments below 0 3 gt 50 000 20 25 MHz 20 25 MHz Mirror Ratio NOTE Settings below 200 in Linear mode may not allow resolution of isotopes Voyager Biospectrometry Workstation User s Guide 5 63 Chapter 5 Optimizing Instrument Settings 5 4 Optimizing Instrument Settings Parameters In this section This section describes e Optimization strategy e Determining the laser setting e Optimizing resolution e Optimizing signal to noise ratio Before you begin Before optimizing instrument settings parameters be familiar with the information in e Data Explorer Software User s Guide Appendix B Overview of Isotopes e Section 5 1 2 Standard Instrument Settings BIC Files Provided e Section 5 1 3 Opening and Viewing Instrument Settings e Section 5 1 5 Saving and Printing Instrument Settings e Section 6 1 Before You Begin e Section 6 3 Obtaining Good Spectra in Delayed Extraction Mode 5 64 Applied Biosystems Optimizing Instrument Settings Parameters 5 4 1 Optimization Strategy This section gives a suggested approach for optimizing instrument settings For details on changi
111. not described in the Voyager Biospectrometry Workstation User s Guide Reference The reference documents shipped with your system are documentation Voyager Biospectrometry Workstation User s Guide Printer documentation depends on the printer you purchase Use this documentation to set up and service your printer XXIX How to Use This Guide XXX Send us your comments Applied Biosystems Microsoft Windows NT User s Guide and related documents Use this guide to learn detailed information about the Microsoft Windows NT user interface e Varian Turbopump Instruction Manual e Varian Turbocontroller Instruction Manual e Varian Multigauge Controller Manual e Mechanical Pump Operating Instructions e GAST Compressor Operating and Maintenance Instructions You also receive the appropriate manual for the digitizer included with your system We welcome your comments and suggestions for improving our manuals You can send us your comments in two ways e Use the Technical Publications Customer Survey at www pbio com techsup survey htm e Send e mail to TechPubs pbio com 1 Introducing the Chapter Voyager 1 Biospectrometry Workstations This chapter contains the following sections 1 1 Voyager DE and Voyager DE PRO System Overview 1 2 1 2 Voyager DE STR System Overview 1 5 1 3 MALDI TOF MS Technology Overview 1 7 1 4 Voyager DE Delayed Ex
112. obvious crystals if vacuum dried or if solvent is a fast drying organic Stability Prepare weekly 3 18 Applied Biosystems Synthetic polymer matrixes Preparing Samples WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Tetrahydrofuran is a flammable liquid and vapor It may be harmful if swallowed Exposure may cause eye and respiratory tract irritation central nervous system depression and liver and kidney damage N N Dimethylformamide DMF is harmful if inhaled It is a combustible liquid and vapor Exposure may cause eye skin and respiratory tract irritation and damage to the liver kidneys and heart It is a suspect cancer hazard Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on Choice of matrix depends on the sample you are analyzing Aromatic for example polystyrene Dithranol 25 mg ml and 1 mg ml silver trifluoroacetate Ag TFA dissolved in tetrahydrofuran THF e Polar DHB 10 mg ml in deionized water e Non polar Indole acetic acid 10 mg ml or DHB in THF dimethylformamide DMF or acetone Voyager Biospectrometry Workstation
113. of spectra to acquire 5 35 process that occurs during 6 65 Accumulation spectra manual clearing trace 6 20 overriding maximum number of Shots Spectrum 5 89 procedure 4 26 6 19 PSD 8 55 resolution improving 5 71 signal to noise ratio improving 5 85 Accuracy see Mass accuracy Accurate mass measurements 6 26 Acajiris digitizer see Digitizer Acquiring data Instrument Control Panel see also Automatic Control mode Manual Control mode PSD acquisition PSD mode accumulating spectra 6 19 8 55 Automatic Control mode 6 36 8 57 data saving 6 18 Data Storage setting 6 14 evaluating data automatically 6 42 evaluating data manually 6 17 guidelines 6 4 laser intensity setting automatically 6 39 laser intensity setting manually 6 14 Manual Control mode 6 11 moving position on sample spot 6 5 multiple spectra in one data file 5 35 5 39 Index 2 Applied Biosystems Acquiring data Instrument Control Panel continued multiple spectra recommended maximum in one data file 5 35 obtaining maximum mass accuracy 6 6 options 6 2 overview 4 6 6 2 PSD automatic mode 8 57 PSD manual mode 8 37 resolution calculating during 6 30 sample position selecting automatically 6 41 sample position selecting manually 6 13 saving BIC after acquisition to store laser intensity 6 19 signal to noise ratio calculating during 6 33 single shot mode 2 45 starting 4 25 6 16 status 2 78 4 5 4 25 stopping 4 25 6 16 stopping before
114. of the Instrument Control Panel You can do the following with a docked page e Deselect it from the View menu to close it e Double click it to change it to a floating page described below This automatically maximizes the page Double click it again to dock the page e Right click on it and deselect Allow Docking to change it to a floating page This automatically maximizes the page Double click on the page to dock the page e Click drag the page borders to resize it Floating Pages are not docked to other pages or the edge of the Instrument Control Panel You can do the following with a floating page Deselect it from the View menu or click X in the top right corner of the page to close it e Click drag the page borders to resize it e Click drag it to a different location on the screen e Double click to change it to docked page Voyager Biospectrometry Workstation User s Guide 4 9 Chapter 4 Voyager Instrument Control Panel Basics 4 3 Using the Spectrum Window This section includes e Adjusting the display range e Zooming on traces e Adding traces to a window e Annotating traces e Previewing and printing traces 4 3 1 Adjusting the Display Range To set the display range in Spectrum window 1 Click the Spectrum window to activate it 2 From the Display menu select Range X range 3 Select X Range to set the x axis range The X Axis Setup dialog box Figure 4 3 is displayed
115. of the product or the software not authorized by Applied Biosystems THE ABOVE PROVISIONS SET FORTH APPLIED BIOSYSTEMS SOLE AND EXCLUSIVE REPRESENTATIONS WARRANTIES AND OBLIGATIONS WITH RESPECT TO ITS PRODUCTS AND APPLIED BIOSYSTEMS MAKES NO OTHER WARRANTY OF ANY KIND WHATSOEVER EXPRESSED OR IMPLIED INCLUDING WITHOUT LIMITATION WARRANTIES OF Voyager Biospectrometry Workstation User s Guide B 3 Appendix B Warranty Service Information B 4 Warranty limitations Applied Biosystems MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE ALL OF WHICH ARE EXPRESSLY DISCLAIMED THE REMEDIES PROVIDED HEREIN ARE BUYER S SOLE AND EXCLUSIVE REMEDIES WITHOUT LIMITING THE GENERALITY OF THE FOREGOING IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT OR ON ANY OTHER BASIS FOR DIRECT INDIRECT PUNITIVE INCIDENTAL MULTIPLE CONSEQUENTIAL OR SPECIAL DAMAGES SUSTAINED BY THE BUYER OR ANY OTHER PERSON WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGE INCLUDING WITHOUT LIMITATION DAMAGE ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE OPERATION OF ANY EQUIPMENT OR SO
116. page 2 11 shows the location of the two boards that can be installed in your computer depending on the digitizer option selected for your system NOTE The computer layout may change without notice Boards may be located in slots that differ from those shown in Figure 2 9 and Figure 2 4 Voyager Biospectrometry Workstation User s Guide 2 25 Chapter 2 Installing the Voyager Biospectrometry Workstations Oscilloscope Figure 2 10 shows the front panel of the oscilloscope The CH1 CH2 and CH3 or Aux 1 input cables thread through the center hole in the front panel of the Voyager STR workstation These cables are connected internally to the detector and control board To CH3 from internal control board 600 gt 0 Ca from internal from internal Linear detector Reflector detector output output Figure 2 10 Oscilloscope Connections Acgiris 2 GHZ Figure 2 11 shows the connectors on the Acqiris 2 GHz digitizer digitizer board The input cables from these connectors INPUTA INPUTB and EXTERNAL connect internally to the mass spectrometer linear detector reflector detector and control board respectively EXTERNAL PB100831 To linear To reflector To internal detector detector control board Figure 2 11 Acqiris 2 GHz Digitizer Board Connections 2 26 Applied Biosystems Connecting the Voyager DE STR Workstation Video monitor Figure 2 12 shows the rear panel of the video
117. precursor spectrum 2 From the View menu select Data Storage Set parameters as needed See Setting Data Storage parameters on page 6 14 for information Hint Include a_PSD suffix when you name PSD data files to help you distinguish them from non PSD data files For example if you type in Experiment1_PSD as the file name the complete data file name will be Experiment1_PSD DAT or Experiment1_PSD_0001 DAT if Autosequence File Names is enabled Selecting and 3 Inthe PSD Acquisition Settings control page press and acquiring a hold the Control key and click all rows that correspond to segment the segments you want to acquire NOTE All instrument settings are disabled as soon as you start acquisition in PSD mode until you stop the experiment Make sure instrument settings are correct before starting acquisition Voyager Biospectrometry Workstation User s Guide 8 63 Chapter 8 PSD Analysis CAUTION Select all necessary segments before continuing You cannot acquire additional segments into the current data file after you start acquisition When acquisition is complete the data file is automatically closed If you want to acquire additional segments as part of the same data file you must repeat the acquisition with all necessary segments selected 4 To start acquiring select Start Acquisition from the Acquisition menu or click Pal During The following occurs for each segment spec
118. process of accumulating only acceptable spectra allows you to improve the resolution of the final spectrum you save For more information on manually accumulating spectra see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Voyager Biospectrometry Workstation User s Guide 5 71 Chapter 5 Optimizing Instrument Settings 5 4 3 1 Overview This section includes e Parameters to adjust e DE parameters e Relationship between DE parameters e Readjusting the laser after optimization Parameters to You can set the following parameters to optimize resolution adjust Figure 5 24 e Guide Wire Voltage e Delay Time e Grid Voltage The following sections give guidelines for setting these parameters To Optimize Adjust Resolution Guide Wire Voltage Delay Time Grid Voltage Figure 5 24 Optimizing Resolution 5 72 Applied Biosystems Optimizing Instrument Settings Parameters DE parameters Improved resolution in Delayed Extraction technology is achieved by velocity focusing ions See Velocity focusing on page 1 15 for more information Two instrument settings parameters affect the velocity focusing of ions in Delayed Extraction e Delay Time Time in nanoseconds after the laser ionizes the sample at which full Accelerating Voltage is applied creating the potential gradient that accelerates ions Grid Voltage Voltage applied to the vari
119. provided control page with the software This is a PSD mode BIC file set to Automatic Control mode with the following settings Parameter Setting Automated Laser Intensity Enabled Minimum Maximum Laser Step and Prescan settings not used Number of Spectra to Acquire 1 one spectrum saved for each PSD segment all segments saved in one DAT file Spectrum Accumulation Save All Spectra no Acceptance Criteria applied Automated Sample Positioning Enabled with Random Uniform as the default however if you change the setting your change is retained 8 58 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Automatic Control Mode NOTE If you start with a BIC file that is not set as indicated in the table above and you change to Automatic Control mode and PSD Operation mode the software automatically changes the settings in the BIC file to those in the table above These changes are not stored permanently in the BIC file unless you save it 2 If Control mode is not set to Automatic click Automatic 3 Ifthe PSD Acquisition Settings control page Figure 8 13 is not displayed select PSD Acquisition from the View menu Mirror resio Guide wiro auto ft Precursor mess 129656 F Precursor ion gelactor Loser horement used during auto contol only 4 4 x Decrement ratio hs o Guide wine tracks mirror rato r PSD Calbenton F Deraur Baana PSD
120. respiratory reaction Exposure may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Alpha cyano 4 hydroxycinnamic acid CHCA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Running OptiPlate to Optimize Mass Accuracy To prepare the plate 1 Spot the sample plate with the standard mixture and matrix prepared in the previous section Follow the procedure in Section 3 2 3 Loading Samples Dried Droplet Application Spot a minimum of 15 positions on a plate For best results spot the number of positions indicated below for the plate type you are optimizing Plate Number of Positions to Spot 100 well 100 positions 400 well 100 positions every fourth row and all perimeter positions 96 x 2 well 96 Spot either A or B rows and all perimeter positions Spot one position as the reference position with the standard mixture and matrix For best results select a position in the center of the plate for example position 55 or 56 on a 100 well plate NOTE During the optimization procedure the system automatically reanalyzes this position to maintain an accurate calibration A position in the middle of the plat
121. saves the data file for the position e Reacquires the reference spectrum every 3 minutes and updates the calibration to adjust for instrument drift over time Voyager Biospectrometry Workstation User s Guide 2 67 Chapter 2 If acquisition fails or peaks are not found Installing the Voyager Biospectrometry Workstations The OptiPlate software may analyze a position more than one time as described below If The software Acquisition fails in Reference or non reference position Continues acquiring until the number of consecutive failing acquisitions set in Stop Conditions is met see page 2 60 Expected masses not found in reference position Reacquires the reference position until a second successful acquisition occurs then checks for presence of expected peaks If no peaks are found the OptiPlate run stops Expected masses not found in non reference position Does not calculate Extraction Correction Marks position with and continues the OptiPlate run Pausing and resuming the plate optimization 2 68 Applied Biosystems To stop the OptiPlate run for any reason for example you notice that many positions display a black edge indicating they have failed optimization or you realize that you did not save changes to the BIC file 1 Click Stop Acquisition 2 Wait for acquisition to stop in the Instrument Control Panel 3 If necessary eject the sa
122. see also PSD acquisition practicing 8 37 8 65 Accelerating Voltage setting 8 44 Precursor lon Selector see 8 59 Precursor lon Selector accumulating spectra 8 55 precursor see PSD precursor autofill list 8 50 _ Spectrum BIC files 5 6 resolution improving 8 29 8 40 CID option 8 31 8 73 segments see PSD segments setting 5 25 signal intensity different from regular analysis 8 67 comparison to Reflector mode 8 24 constants 8 28 data file not available for viewing until experiment closed 8 52 standard instrument settings H 4 default values 8 48 summary of parameters definition 8 20 affecting 8 76 effect of Grid Voltage 5 51 troubleshooting 8 29 9 21 enabling 5 25 8 43 Vertical Scale 8 48 exploring 8 65 PSD precursor spectrum fill down list 8 50 see also PSD mode fragment ion yield poor 8 29 acquiring 8 38 fragmentation enhancing 8 31 BIC file 5 6 Grid Voltage observing effects CAL generating from 8 39 of 8 74 calibration 8 39 Grid Voltage setting 8 44 8 59 mass determining in Reflector Guide Wire Voltage setting 8 48 mode 8 38 Guide Wire Tracks Mirror mass entering in PSD mode 8 45 Ratio 8 50 8 60 Voyager Biospectrometry Workstation User s Guide Index 31 I N D E X Index PSD segments see also PSD fragments accumulating 8 55 acquiring in any order 8 55 acquiring selected 8 42 appended to DAT file 8 52 list autofilling 8 50 mass range 8 54 8 64 number of and composite spect
123. select one of the Spectrum Accumulation conditions described below For information on the Automatic Laser Sample Positioning Spectrum Accumulation function see e Section 5 2 3 Automatic Control Dialog Box e Section 6 6 Acquiring in Automatic Mode from the Instrument Control Panel For a description of a search pattern file see Section 6 6 4 Search Patterns Two options are available for accumulating spectra from multiple search pattern positions Accumulate all Averages the acquired spectra regardless of their quality A single spectrum is created See Section 5 2 3 Automatic Control Dialog Box for more information e Accumulate all passing Evaluates the acquired spectra against the Acceptance Criteria specified then averages only passing spectra See Section 5 2 3 Automatic Control Dialog Box for more information NOTE If no Acceptance Criteria are specified all spectra are averaged Voyager Biospectrometry Workstation User s Guide 6 65 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 6 7 1 Process that Occurs when Accumulating All Spectra 6 66 Applied Biosystems When accumulating all spectra Accumulation mode is determined by the Spectrum Accumulation conditions described in Section 5 2 8 Automatic Control Dialog Box the system does the following during acquisition 1 Sets the laser intensity as described in Section 6 6 6 Process that Occurs During Acq
124. settings 5 61 Voyager Biospectrometry Workstation User s Guide Index 43 Index Video camera magnification Voyager DE and Voyager DE PRO 1 22 magnification Voyager DE STR 1 35 not aligned with sample position 2 48 9 23 Video monitor connecting Voyager DE and Voyager DE PRO 2 21 2 27 connecting Voyager DE STR 2 28 Views sample plate see Plate view see Sample view Voltage see also Accelerating Voltage see also Grid Voltage see also Guide Wire Voltage see also High voltage input selecting 2 4 setting operating Voyager DE STR 2 7 Voltage difference see Potential gradient Voyager control software see also Instrument Control Panel see Sequence Control Panel disk space required 2 29 Voyager Mass Standards Kit B 6 Voyager processing software disk space required 2 29 starting 2 32 Voyager DE Biospectrometry Workstation digitizer Acqiris 2 17 digitizer LeCroy 2 15 digitizer oscilloscope 2 19 digitizer Signatec 2 13 features 1 4 input voltage selecting 2 4 Index 44 Applied Biosystems Voyager DE Biospectrometry Workstation continued mass spectrometer parts of 1 20 1 22 overview 1 2 parts of the system 1 17 space required 2 2 specifications A 2 startup and shutdown 2 73 weight 2 3 Voyager DE PRO Biospectrometry Workstation digitizer Acqiris 2 17 digitizer LeCroy 2 15 digitizer oscilloscope 2 19 digitizer Signatec 2 13 features 1 4 input voltage selecting 2 4 mass spectrometer
125. signal to noise 5 50 Fill Down Sequence Control Panel 7 21 Filtering peaks monoisotopic 7 3 7 12 7 17 Firing rate laser 5 26 Flight Length to Detector impact on system performance 2 40 setting 2 40 Flight tube description 1 23 1 35 height Voyager DE and Voyager DE PRO 2 2 A 3 A 6 length Voyager DE 1 4 length Voyager DE PRO 1 4 length Voyager DE STR 1 6 Floating control pages 4 9 Focusing velocity see Velocity focusing Fonts changing Instrument Control Panel 4 21 Fore pump Voyager DE function 1 27 vacuum gauge 1 27 Fore pump Voyager DE PRO function 1 29 vacuum gauge 1 29 Fore pump Voyager DE STR function 1 37 vacuum gauge 1 39 Foreline valve location 1 26 1 28 1 38 Fragment ions see also PSD fragments PSD segments Prompt fragments and Grid Voltage 8 73 Fragment ions continued and laser intensity 8 68 and Precursor lon Selector 8 69 calibration 8 28 fast see Prompt fragments investigating identity 8 19 kinetic energy 8 22 optimum resolution observed near Max Stitch Mass 8 23 8 42 poor yield 8 29 prompt 6 24 8 70 8 71 PSD 6 24 8 21 8 70 8 71 PSD kinetic energy 8 21 Fragmentation and Delay Time 5 49 and Grid Voltage 5 50 5 53 and laser intensity 5 50 causing with higher laser intensity 6 24 enhancing with CID 8 31 fragment types 6 24 Front panel description 1 39 9 25 Fuse Voyager DE and Voyager DE PRO changing 2 4 9 4 rating 2 4 2 6 9 6 FWHM 6 30 G Gas CID 8 33 Gau
126. spectra H 8 automatic mode 8 59 description 1 12 1 13 PSD Acquisition settings manual Grid Voltage setting H 6 mode 8 44 Guide Wire Voltage setting H 6 System Status 2 76 instrument setting parameters that Control stick affect H 11 connecting Voyager DE and instrument settings changing H 2 Voyager DE PRO 2 22 Instrument settings optimized connecting Voyager DE STR 2 25 for H 4 diagram l 7 instrument settings optimized moving position on sample spot I 7 for H 3 starting acquisition 1 7 laser intensity H 6 H 12 stopping acquisition l 7 Voyager Biospectrometry Workstation User s Guide Index 9 I N D E X Index Converting version 4 methods MNU and search pattern SP files before converting 5 95 Data Processing parameters not supported 5 95 external calibration set to default 5 95 HV Tune Ratio replaced by new parameter 5 95 information that is converted 5 93 manual control mode set 5 95 new parameter 5 95 overview 5 92 path for SP not retained 5 95 procedure 5 96 Coordinates determining x y for PLT file positions 3 83 Counts absolute see Absolute counts Create PLT File parameters 3 81 using 3 79 Crystallization 3 HPA 3 46 alpha cyano 3 45 desired pattern 3 45 DHB 3 46 DHBs 3 46 examining 3 45 guidelines for 3 37 sinapinic acid 3 45 THAP 3 46 troubleshooting 9 9 uneven recommended search pattern for 6 49 Current Spectrum trace definition 4 14 during acquisition 6 16 evaluating 6 17 Index 1
127. store important data for retrieval at a later time and remove the original data 9 6 Applied Biosystems Troubleshooting 9 2 Troubleshooting This section includes e Spectrum troubleshooting e Software troubleshooting e Hardware troubleshooting Troubleshooting information is organized according to likelinood of possible cause from most likely to least likely possible cause If you are unable to solve your problem using the information in the following tables call Applied Biosystems Technical Support To reach Applied Biosystems Technical Support refer to the list of offices on the back cover of this book 9 2 1 Spectrum Troubleshooting Table 9 1 Spectrum Troubleshooting sample region matrix peaks seen Symptom Possible Cause Action Flat signal on oscilloscope Laser intensity too Adjust laser by using the or in spectrum window for low slider controls on the Manual continued on next page Laser Sample Positioning control page Analyzing cold spot Analyze different position in sample position Sample consumed at Analyze different position in location in wellwhere sample position laser is hitting Mass range setting Adjust See Section 5 3 5 incorrect Understanding Digitizer Settings continued Voyager Biospectrometry Workstation User s Guide 9 7 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Sym
128. system type To perform internal calibration you include an internal standard in your sample You then display the data file in the Data Explorer software and calibrate using the internal standard Voyager Biospectrometry Workstation User s Guide 6 7 Chapter 6 Acquiring Spectra from the Instrument Control Panel When to calibrate 6 8 Applied Biosystems e Sequence Control calibration Provides external internal and internal with automatic updating calibration options during acquisition from the Sequence Control Panel For more information see Section 7 7 1 Calibration Options in a Sequence For the mass accuracy specifications for your instrument refer to Appendix A Specifications NOTE Mass accuracy for sample positions in the outer rows of a sample plate may be poorer than the mass accuracy of the sample positions in the inner rows Calibrate e Atleast once a day You may want to include calibration standards on each sample plate If the ambient temperature in your lab is fluctuating more than 5 C check calibration and recalibrate if necessary The calibration software compensates for changes in instrument settings However if you require optimum mass accuracy generate a calibration file with identical instrument settings or use internal calibration if possible Before You Begin Calibration The general equation that the Voyager software uses for equations calibration with a CAL fil
129. the refrigerator overnight Filter then wash the precipitate with cold water This procedure is not optimized for yield NOTE The organic concentration above assumes you are premixing sample solution matrix solution in a 1 10 dilution If you are mixing sample solution matrix solution on the plate in a 1 1 dilution increase the proportion of acetonitrile to 60 percent to prevent the matrix from precipitating Adjust the other components accordingly NOTE A higher concentration of TFA up to 3 may improve sample solubility Crystals Rounded see Figure 3 1 on page 3 45 Stability Prepare weekly 1 Shevchenko A M Wilm O Vorm M Mann Anal Chem 1996 68 850 858 3 10 Applied Biosystems CHCA for thin layer application Preparing Samples Use for peptides lt 3 000 Da WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Alpha cyano 4 hydroxycinnamic acid CHCA may cause eye skin and respiratory tract irritation Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on Isopropanol is a flammable liquid and vapor It may cause eye skin and upper respirato
130. the Mass Accuracy Optimization Option Overview You have two options for close external calibration e Sample and standard in separate sample positions e Sample and standard in the same sample position NOTE The closer the locations of sample and standard the better the mass accuracy Sample and if you are acquiring samples and standards from different standard in sample positions enter the standards in the run list preceding separate sample the unknowns that use the calibration positions For optimum mass accuracy place samples in sample positions adjacent to standards See the example in Figure 7 8 7 34 Applied Biosystems Automatic Calibration During a Sequence Run 33 34 35 36 37 O82220 43 44 45 46 47 O2 20 53 54 55 56 57 O02220 100 well plate 400 position plate Standard G Sample C Empty 22 FODOUOO 96 x 2 position plate Figure 7 8 Sample and Standard Locations for Optimum Mass Accuracy Without Using the Optimize Mass Accuracy Option To run samples spotted on a 100 well plate as illustrated in Figure 7 8 set up the run list as shown in the following table 100 well Base File Instrument Calibration Type Calibration Position Name Settings File File 45 STD1 STD BIC INTERNAL UPDATE STD CAL 34 SAMP1 SAMP BIC EXTERNAL STD CAL 35 SAMP2 STD BIC EXTERNAL STD CAL 36 SAMP3 SAMP BIC EXTERNAL STD CAL 44 SAMP4 STD BIC
131. the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified number of spectra to acquire or the number of search pattern positions in the SP file For more information see Section 6 6 7 Process that Occurs when Accumulating Spectra from Multiple Search Pattern Positions 5 48 Applied Biosystems Impact of Changing Instrument Settings Parameters 5 3 Impact of Changing Instrument Settings Parameters This section includes e Summary of parameters e Understanding Grid Voltage e Understanding Delay Time e Understanding Guide Wire Voltage e Understanding Digitizer settings 5 3 1 Summary of Parameters Optimizing Changing instrument settings parameters can impact the parameters in a sensitivity resolution or signal to noise ratio in different ways specific order For optimum performance optimize parameters in the order listed in Section 5 4 3 Optimizing Resolution e Section 5 4 4 Optimizing Signal to Noise Ratio List of parameters The following table lists the impact of changing these parameters Parameter Mode Impact Delay Time Linear Impact dependent on Grid Voltage nanoseconds Reflector Performance drops off if set too high or too low for corresponding Grid Voltage May vary with matrix Critical parameter with optimum values for maximum resolution continued
132. the coordinates to enter in the PLT file display the Sample View see Figure 4 9 on page 4 31 and record the Absolute X Absolute Y logical coordinates pairs for the first and last position See Logical Coordinates in Plate View in Manual Laser Sample Positioning Control Page on page 3 78 for information on displaying logical coordinates Comment Comment stored with PLT file Optional 5 Click next to the Plate File PLT field Specify the directory in which to store the PLT file type the name for the file then click Save 6 Click Create PLT 7 Check the sample plate alignment See Section 3 5 5 Adjusting the Laser Position for a Custom PLT File 3 82 Applied Biosystems Sample Plate Types Creating or NOTE Display the Sample View when you create or edit a editing a PLT file PLT file with Notepad editor Move the sample plate to the using Notepad positions you want analyzed and note the Absolute X editor Absolute Y coordinates to enter in the PLT file To create or edit a PLT file using Notepad editor 1 Perform the procedure in Preparing the sample plate on page 3 76 2 Open the Microsoft Windows NT Notepad text editor See the Microsoft Windows NT User s Guide if you need help using Notepad 3 Open the PLT file to edit 4 To enter a comment on the first line type a semicolon then type a description of the sample plate for example 96 position r
133. the first position analyzed the second entry is the second position analyzed and so on 0 1270 Y 1270 0 99 1270 0 0 1270 Figure 6 17 Location of Coordinates in a Sample Position on a 100 Position Plate Voyager Biospectrometry Workstation User s Guide 6 47 Units in search pattern files Default search pattern file Chapter 6 Acquiring Spectra from the Instrument Control Panel For Voyager software version 5 0 and later the default units for X Y coordinates in search pattern files is microns For pre 5 0 Voyager software the default units for X Y coordinates is steps Before using search pattern files created in version 4 software convert to version 5 format See Section 5 5 Converting Version 4 Methods and Search Pattern Files The default search pattern file for a 100 position plate DEFAULT SP causes a serpentine crossing of the sample position determined by the following 20 X Y coordinates X coordinate um Y coordinate um X coordinate um Y coordinate um 1 952 5 158 75 11 635 635 2 635 317 5 12 238 125 396 875 3 317 5 476 25 13 158 75 238 125 4 JO 635 14 476 25 0 5 317 5 793 75 15 873 125 238 125 6 635 635 16 1031 875 158 75 7 317 5 396 875 17 635 396 875 8 O 158 75 18 317 5 555 625 9 396 875 79 375 19 10 714 375 10 873 125 317 5 2
134. the toolbar to turn the high voltage on and off You can also control high voltage by selecting Turn On Off High Voltage from the Instrument menu NOTE High voltage is automatically turned on when an acquisition is started High voltage is automatically turned off when exiting the Instrument Control Panel or ejecting a sample plate Click in the toolbar to load or eject the sample plate You can also load or eject the sample plate by selecting Load or Eject from the Sample Plate menu x Click in the toolbar to select the active sample position You can also select from the Manual Laser Sample Position control page described on page 4 27 Click in the toolbar to turn acquisition laser on and off You can also control acquisition by selecting commands from the Instrument menu When the system is acquiring e The button is depressed e The Acquisition field in the status bar at the bottom of the Instrument Control Panel is green and displays Acquisition ON e High voltage is on Click H in the toolbar to manually save the selected current or accumulated trace You can also save data by selecting Save Spectrum from the Acquisition menu For additional information see Saving data on page 6 18 Voyager Biospectrometry Workstation User s Guide 4 25 Chapter 4 Voyager Instrument Control Panel Basics Accumulating spectra Clearing an accumulated spectrum 4 26 Applied Biosystems Click in the too
135. this section This section describes e Overview e When to align e PLT files and multiple alignments e How the system aligns a plate e Overview of video monitor display e What you need e Corner positions in PLT files Using the control stick e Before aligning e Aligning Alignment information saved for future use Overview The Sample Plate Alignment function allows you to align the center of sample positions under the laser beam When to align For most standard welled plates and PLT files provided alignment is not frequently required Alignment is more typically required e When you are using a custom PLT file and are spotting sample plates using an automated device e When you are using Teflon plates because there can be some variation in the laser etched indentations on the plate Align the Sample Plate if the laser beam is not centered on a sample position When you advance to a sample position using the Manual Sample Position control page e Before you acquire data from the Instrument Control Panel and use automated sample positioning e Before you acquire data from the Sequence Control Panel 2 46 Applied Biosystems Aligning the Sample Plate PLT files and A PLT file defines the sample positions on a sample plate For multiple alignments example 100 PLT may define a 100 well plate If you have more than one 100 well plate you may need to align each plate The Voyager software al
136. time This procedure is required for Voyager DE and Voyager DE PRO systems only To select input voltage 1 Remove the power cord from the mass spectrometer 2 Carefully remove the voltage selector fuse holder from the system Figure 2 1 using a small flat blade screw driver 3 Carefully remove the voltage selector from the holder and insert the selector with the proper voltage displayed in the window of the holder CAUTION The plastic tabs that hold the voltage selector in place are fragile Do not exert force when removing this piece Selecting the Site Fuses Voltage selector Window Holder PB100507 Figure 2 1 Selecting Input Voltage and Installing Fuses WARNING FIRE HAZARD Using a fuse of the wrong type or rating can cause a fire Replace fuses with those of the same type and rating Voyager Biospectrometry Workstation User s Guide 2 5 Chapter 2 Installing the Voyager Biospectrometry Workstations 4 Insert two fuses of the proper rating for the selected voltage Electrical Rating Volts Amps EF Fuse 5 x 20 mm 100 V 10A T10A 250V 120 V 10A T10A 250V 220 V 6 3A T6 3A 250V 240 V 5A T5A 250V 5 Insert the voltage selector fuse holder into the receptacle 6 Plug in the mass spectrometer Connect devices to the mass spectrometer as described in Section
137. to selected location of Figure 4 9 on on a position the sample position page 4 31 4 30 Applied Biosystems Controlling the Workstation Hint If you right click on a position you can change between Sample View and Plate View m Manual Laser Intensity 0 al gt Manual Sample Positioning Active Pos fn b 100 well plate Laser position Scroll bars gt Coordinates Relative x 1587 50000 Y 1587 50000 Fie of Active Absolute x o o00000 Y 0 000000 es Position Figure 4 9 Manual Laser Sample Position Control Page Sample View Adjusting sample Adjust the sample position in the Sample view display by position in the doing any of the following Sample view Click the up down and left right scroll bars e Click on the sample position to move to that X Y position e Type coordinates microns in X and Y fields and click GO NOTE The motor controlling plate movement moves in step units Coordinate values entered are rounded to the nearest step value 1 step 3 175 microns e Use the control stick to move to a sample position For more information see Section 1 3 Using the Control Stick Voyager Biospectrometry Workstation User s Guide 4 31 Chapter 4 Voyager Instrument Control Panel Basics 4 6 Sequence Control Panel Sequence Control The Voyager Sequence Control Panel Figure 4 10 allows Panel you to collect data for m
138. using the slider controls on the Manual Laser Sample Positioning control page Sample contains more than one component Purify sample before analyzing On Voyager DE PRO and Voyager DE STR systems isotopes are only partially separated because you are using a setting very close to laser threshold Increase laser intensity by using the slider controls on the Manual Laser control page to improve symmetry but data is accurate as is NOTE Increasing the laser intensity may decrease resolution Signal is saturating detector Decrease laser intensity by using the slider controls on the Manual Laser control page 9 16 Applied Biosystems continued Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action On Voyager DE PRO and Voyager DE STR systems cannot see high mass ions in Reflector mode Refer to Flat signal on oscilloscope or in spectrum window for sample region matrix peaks seen symptom on page 9 7 Refer to Flat signal on oscilloscope or in spectrum window for sample region matrix peaks seen action on page 9 7 lons not reaching detector Accelerating Voltage too low lons fragmenting before reaching the detector Accelerating Voltage too high or Grid Voltage too low Verify that you can see ions in Linear mode then adjust voltages in Reflector mode and rerun See Section
139. will also specify an external PSD calibration below which is used to determine the values fora B and y needed for the PSD calibration equation NOTE If default calibration yields acceptable mass accuracy for your application an external calibration is not required In the PSD Acquisition Settings control page type the Precursor mass NOTE Precursor mass is used for PSD calibration Type in an accurate value with appropriate precision for example type 1296 68 not 1297 Select Precursor lon Selector if it is not already selected To set PSD calibration select the default calibration or select a previously generated external PSD CAL file For more information see the Data Explorer User s Guide Chapter 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files Acquiring PSD Data with Standard BIC Files in Automatic Control Mode NOTE The calibration you specify on the PSD Acquisition Settings control page is used to determine the values fora B and y needed for the PSD calibration equation described on page 8 28 The calibration you specify on the Instrument Settings control page in step 4 is used to determine the value for t precursor ion flight time needed for the PSD calibration equation NOTE If default PSD calibration yields acceptable mass accuracy for your application an external calibration is not required NOTE PSD CAL files are named with the
140. with the plate you are using and the last optimization created will be used Note that all items listed above yield mass accuracy better than the accuracy you obtain if you do not use the Mass Accuracy Optimization option Voyager Biospectrometry Workstation User s Guide 3 53 Chapter 3 Preparing Samples 3 4 3 Ejecting the Sample Holder 3 54 Applied Biosystems To eject the sample holder 1 In the Voyager Instrument Control Panel select Eject from the Sample Plate menu The Load Eject dialog box is displayed Load Eject Sample Plate x Plate ID ne Plate Type al Last Aligned Optimization ey Created F UseHe Eject Cancel Figure 3 4 Load Eject Sample Plate Dialog Box 2 Click Eject The following occurs A Load Status dialog box displays hardware status during the ejection sequence High voltage is turned off The mass spectrometer video monitor displays the sample plate moving The sample holder moves out of the main source chamber out of the sample loading chamber then is ejected from the instrument Loading Sample Plates in the Mass Spectrometer NOTE Do not leave the sample holder in the Eject position for more than a few minutes Minimize the time the instrument is exposed to the atmosphere to reduce the time needed to reach high vacuum after you insert a new plate 3 4 4 Loading Sample Plates This section describes loading sample plates in _
141. you are converting an autosampler MNU file open the converted BIC file and e On the Instrument Settings page set the Control mode to Automatic e Inthe Automatic Control mode dialog box respecify the SP file to ensure that the correct path is used for the SP file NOTE No path is retained for the SP file during conversion If the SP file specified is located in the Voyager program directory the software can use the file during acquisition If the SP file is not in the Voyager program directory an error message is displayed when the BIC file is run It is good practice to move SP files to a specific directory before converting and to respecify the SP file in the BIC after converting Converting Version 4 Methods and Search Pattern Files Voyager 4 x File Converter x m Method Files MNU m Search Pattern Files SP Add Converted method files have a BIC extension For example angio mnu becomes angio bic Converted search pattem files are overwritten Cancel If converted BIC will be used for automated control set Control mode to Automatic and specify the complete search pattern path before using Figure 5 27 Voyager 4 x File Converter Dialog Box If you are converting an MNU file that specifies an external calibration calibration mode is reset to Default in the BIC file To specify an external calibration for the converted BIC file creat
142. 0 317 5 952 5 6 48 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Spiral search The SPIRAL SP file provided is a 20 point search pattern that pattern file begins searching at the center of the sample position and spirals outward This is the best search pattern for uneven matrix crystals Search pattern for if you create a custom plate type for a plate without custom plates laser etched sample position or wells with position diameter larger or smaller than 2 540 microns create an SP file for analysis of the plate The DEFAULT SP file is set to analyze 2 540 micron diameter positions corresponding to standard sample positions For more information see Guidelines for creating SP files on page 3 73 Voyager Biospectrometry Workstation User s Guide 6 49 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 6 5 Creating and Editing SP Files Using the Search Pattern Editor This section includes e Creating or editing an SP file e Using the Search Pattern Generator e Drawing a search pattern e Setting x y coordinates Creating or To create or edit an SP file editing an SP file 4 _ in the instrument Control Panel e Select Select Sample Plate from the Sample Plate menu e Select the Plate ID associated with the PLT file for which you are creating the SP file then click OK 2 From the Tools menu select Search Pattern Editor The Se
143. 0 05 400 800 1200 1600 2000 Mass m z Figure 6 9 Example of Good Mass Spectrum for Angiotensin Voyager Biospectrometry Workstation User s Guide 6 23 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 3 2 Laser Intensity Overview Laser intensity does not have a major impact on resolution or Fragment ions generated at higher laser intensity signal to noise ratio You need to find the laser setting that gives you an acceptable signal to noise ratio and acceptable resolution optimum is not necessary and then fine tune If adjusting Grid Voltage and Delay Time causes the signal to saturate you may need to make additional laser adjustments For more information on adjusting the laser see Section 5 4 2 Determining the Laser Setting Continue acquiring and decreasing the laser intensity until you observed a moderate resolution and a signal to noise ratio of approximately 50 1 Higher laser power can cause two types of fragment ions to form Prompt fast fragments Form in the ion source before ion acceleration detected in Linear and Reflector mode PSD slow fragments Form in the flight tube after ion acceleration detected in Reflector mode Figure 6 10 shows a partial spectrum of Angiotensin obtained at high laser intensity in Reflector mode The high laser intensity has generated fragments 100 90 80 704 60 50 40 30 20 10 0 Intensity Mixture o
144. 0 5000 Mass m z Figure 5 23 Signal Acceptable at Lower Laser Intensity To optimize laser intensity continue acquiring and adjusting the laser intensity if necessary until you observe e Acceptable peaks no saturation e Moderate resolution e Signal to noise ratio of approximately 50 1 NOTE By default the right axis is set to Display Relative mode which means it updates to reflect the intensity of the most intense peak in the currently displayed region of the trace You can set the right axis to display an absolute value if desired For more information see Accessing graphic options on page 4 22 5 70 Applied Biosystems Optimizing Instrument Settings Parameters 5 4 3 Optimizing Resolution This section includes e Overview e Acceptable resolution in Delayed Extraction Mode e Optimizing Guide Wire Voltage e Optimizing Delay Time e Optimizing Grid Voltage For information on measuring resolution see Section 6 5 2 Calculating Mass Resolution and Data Explorer Software User s Guide Section 6 3 Using the Mass Resolution Calculator Manually The manual accumulation feature of the Voyager system accumulating allows you to acquire a spectrum examine the spectrum for spectra to signal quality visually or using the Resolution or P Signal to Noise Ratio calculator and then accumulate the improve A spectrum with other previously examined spectra or discard resolution the spectrum This
145. 0 Applied Biosystems Cursor Instrument Control Panel enabling 4 21 oscilloscope moving l 5 Customizing Instrument Control Panel 4 21 toolbars 4 21 Cytochrome c mass to time conversion F 5 molecular weight F 3 D Damage reporting B 5 DAT format extracting information from 1 44 opening BIC from 5 7 overview 1 44 Data acceptable spectra in Continuous Extraction mode H 8 acceptable spectra in Delayed Extraction mode 6 21 Data Explorer SET file see SET file Sequence Control Panel Data Explorer software accessing from Instrument Control Panel 4 7 overview 1 45 Data file directory setting 6 14 including position number in name 7 16 7 46 multiple spectra recommended maximum 5 35 multiple spectra saving 5 35 naming 6 14 opening BIC from 5 7 PSD contains multiple spectra 8 27 Data file continued PSD not available for viewing until experiment closed 8 52 saving 6 18 saving automatically 6 40 saving multiple spectra in 5 39 Sequence Control Panel 7 16 size parameter affecting 5 58 Data points decreasing number collected 5 59 determining number in peak 4 22 impact on laser firing rate 5 27 increasing number collected 5 59 5 88 number digitized 5 29 Data Storage information displayed in Output window during acquisition 6 18 maximum number of lines displayed in Output window 4 6 parameters 6 14 setting 6 14 status 2 78 Daughter ion see PSD precursor spectrum DE technology see Delayed Extracti
146. 012 Substance P mix Timed lon Selector 1 348 1 388 Table H 3 Continuous Extraction PSD Mode Standard Instrument Settings Files BIC File Sample Test Mass Range Da P1000 to Low mass peptide mix Mirror ratio varies for PSD PSD ions for precursor 1009 or angiotensin alone analysis mass 1 297 H 4 Applied Biosystems Optimizing a Continuous Extraction Standard Instrument BIC Setting Optimization When optimizing a Continuous Extraction instrument settings strategy file you e Start with a standard instrument settings file e Fine tune laser setting for major improvement in performance e Optionally adjust Grid Voltage and Guide Wire Voltage for slight improvement in performance Optimizing 1 Openastandard instrument settings file for the mass range you are analyzing If an instrument settings file for the mass range you are analyzing does not exist open a standard instrument settings file with the closest higher mass 2 Save the instrument settings file under a new name Standard instrument settings files are read only and cannot be saved 3 Select a calibration CAL file If you are screening samples use the default calibration 4 Ifthe mass range needs adjusting change the digitizer setting See Section 5 3 5 Understanding Digitizer Settings 5 To include matrix peaks in the spectrum for calibration deselect the Low Mass Gate field or set the Starting Mass to a mass below the matrix pea
147. 0510 DHB dried under vacuum Milky amorphous PB100511 DHB or DHBs air dried Irregular crystals in ring Figure 3 3 Microscopic View of Sample Plate with DHB or DHBs Matrix Applied Biosystems Cleaning Sample Plates 3 3 Cleaning Sample Plates In this section This section describes procedures for cleaning Teflon plates e Gold and stainless steel plates Cleaning Teflon NOTE Avoid using strong organic solvents such as plates acetone Use acetonitrile if a solvent is necessary Avoid ultrasonic cleaning devices WARNING CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NOTE Do not use metal brushes abrasive surfaces or solid detergents to clean Teflon sample plates they are fragile Avoid rubbing the surface of the plate If necessary wipe gently with lint free lab tissues To clean Teflon plates 1 Rinse the plate with luke warm water 2 If necessary wipe gently with a lint free lab tissue to remove matrix 3 Wash the plate with a 1 percent solution of a liquid detergent 4 Rinse with deionized water Voyager Biospectrometry Workstation User s Guide 3 47 Chapter 3 Preparing Samples 5 I
148. 06 plate PLT staggered wells A P x 1 24 400 well Reusable with 400 20 x 20 990 60 1 760 22 x 1 709 42 plate PLT wells A T x 1 20 96 well x 2 Reusable with 192 8 x 24 1 500 1 100 x 1 900 plate PLT staggered wells A H x 1_a 12 a and 1_b 12_b Voyager Biospectrometry Workstation User s Guide 3 67 Chapter 3 Preparing Samples 3 5 3 Guidelines for Defining Custom Plate Types PLT file format Optional values coordinates PLT file parameters 3 68 Applied Biosystems You can define custom plate types of unlimited positions by creating your own PLT files This section describes e PLT file format e PLT file parameters e Guidelines for creating PLT files e Guidelines for creating search pattern SP files For information on creating new and editing existing PLT files see Section 3 5 4 Creating and Editing PLT Files A PLT file is an ASCII text file in which each line of text represents an individual position on the plate Figure 3 11 Table 3 13 describes the parameters in a PLT file WellUnits 2 WellShape 1 WellWidth 1016 WellHeight 1016 WellDepth 0 5 6667 5 42227 5 12 Position 42227 5 42227 5 19 name 6667 5 6667 5 82 42227 5 6667 5 89 Position Figure 3 11 Example PLT File The parameters in a PLT file are described in Table 3 13 Sample Plate Types Table 3 13 PLT File Parameters Parameter Description WellUni
149. 071 page C 5 polymers to minimize Na and e 211 076 MW 226 06 Da Color of crystals K adduct formation solution on o OH Yellow 2 4 6 trihydroxy Applications Make 9 1 dilution of e 169 050 acetophenone Small matrix diammonium THAP Oligonucleotides Citrate see mass Color of crystals Matrix 10 mg ml in spectrum on solution 50 50 water page C 5 White acetonitrile MW 168 04 Da Diammonium citrate 50 mg ml in COCH water HO H PB100261 OH Voyager Biospectrometry Workstation User s Guide C 11 Appendix C Matrixes Table C 1 Matrix Information Matrix Solution Characteristic Matrix Applications Color Matrix lons Concentration monoisotopic trans 3 Applications 10 M in solvent e 187 063 indoleacrylic acid Non polar appropriate for sample e 188 071 IAA see mass polymers e 170 061 spectrum on Color of crystals 144 081 page C 6 Bs e 130 066 solution MW 187 2 e 375 134 White 329 120 284 131 7 02H HC c g N N PB100491 Picolinic acid Tang K N I Taranenko S L Allman C H Chen L Y Chang and K B Jacobson Picolinic Acid as a Matrix for Laser Mass Spectrometry of Nucleic Acids and Proteins Rapid Commun Mass Spectrom 1994 8 673 677 Nicotinic acid Ehring H M M Karas F Hillenkamp Org Mass Spectrom 1992 27 472 480 C 12 Applied Biosystems D Log Sheets This appendix includes log
150. 1 Process that Occurs when Accumulating All Specta a mil ln E le eel 6 66 6 6 7 2 Process that Occurs when Accumulating Passing Spectra cies daveb 6 67 Chapter 7 Acquiring Spectra from the Sequence Control 7 1 7 2 7 3 Panel OVER SRE RS eet eis OTA CR es cee ors ee eee Oe 7 2 Understanding Settings Macros and Calibration 7 3 Before Creating a Sequence 7 7 7 3 1 Optimizing Instrument Settings BIC Files for a SEQUENCE RUN Aktien idan drame aie 7 7 7 3 2 Gr ating Macros a ae aap em et nki inania 7 8 7 3 3 Creating Calibration CAL Files ccccseeeeeeeeeeeeeeeeeeeeeees 7 11 7 3 4 Creating Processing Settings SET Files 7 12 Voyager Biospectrometry Workstation User s Guide ix Table of Contents 7 4 7 5 7 6 7 7 7 8 Creating a Sequence 4 4 issus 7 13 7 4 1 Setting General Sequence Parameters 0 cceeeeeeeeeeees 7 13 7 4 2 Creating a Run List sper maaa a e a Ta E Raai 7 14 Preparing to Run a Sequence eee eeeee eee 7 24 RUMNING a S QUENC a e a a nana ea a a EEE EG at 7 25 7 6 1 Starting a Sequence eee ceeceeceeeeeeeeeee eee aeaa eee eetteeeeeeeeeeeeeaeaaes 7 25 7 6 2 What the System Checks When You Start a Sequence 7 26 7 6 3 During and After Acquiring a Sequence
151. 10 20 25 MHz gigasample sec LeCroy LSA1000 2 Gs S 750 MHz 0 5 1 2 4 10 20 25 and 200 MHz Signatec DA500A 500 Ms S 500 MHz 2 4 10 20 None Tektronix Scope 500 Ms S 500 MHz 2 4 10 20 20 and 100 MHz Tektronix Scope 1 Gs S 500 MHz 1 2 4 10 20 20 and 250 MHz Tektronix Scope 2 Gs S 500 MHz 0 5 1 2 4 10 20 20 and 250 MHz Tektronix Scope 4 Gs S 1 GHz 0 25 1 2 4 10 20 20 and 250 MHz Samples second is equivalent to a sampling rate in hertz Specifications supported by the Voyager software for Tektronix Scopes Different models of Tektronix Scopes are available Refer to the Tektronix User s Manual for specifications on your particular model Voyager Biospectrometry Workstation User s Guide A 11 Appendix A Specifications Laser firing rates Figure A 1 shows the estimated laser firing rates for different digitizer models nN A a hi o a o o a on a 0 0 EE EEEEEEEESES Number of Data Points Estimated Laser Firing Rate Hz Signatec Digitizer a LeCroy Digitizer a Tektronix Scope Acquiris Digitizer Figure A 1 Estimated Laser Firing Rates Supported by Different Digitizer Models A 12 Applied Biosystems B Warranty Service Information This appendix contains the following sections B 1 Limited Product Warranty B 2 B 2 Damages Claims Returns B 5 Bi Spare P
152. 2 Da STR models or 0 3 Da PRO models of the expected masses generate a PSD calibration and reacquire the PSD segments with the PSD calibration Generating a PSD This section gives a brief description of how to generate a calibration PSD calibration For more information see the Data Explorer User s Guide Section 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files NOTE This is a PSD calibration that affects fragment ion masses 1 Inthe Data Explorer software open the PSD data file acquired in the previous section if it is not already open 2 From the Process menu select Mass Calibration then select PSD Calibration 3 Select the Angiotensin_Fragments REF file provided with the software Voyager Biospectrometry Workstation User s Guide 8 13 Chapter 8 PSD Analysis Adding PSD calibration Acquiring PSD segments with PSD calibration 8 14 Applied Biosystems 3 Select the peaks listed in Table 8 3 as reference masses for calibration by right click dragging on a peak then select the correct mass for the peak in the Reference Peak dialog box For best mass accuracy e Select a minimum of seven reference masses e Select peaks from different regions of the spectrum to ensure that high and low Mirror Ratios are represented Click Solve and Plot to generate the calibration then click Apply Calibration to save the calibration constants in the data file C
153. 4 4 Optimizing Signal to Noise Ratio aeee 5 85 5 5 Converting Version 4 Methods and Search Pattern Files 5 92 Voyager Biospectrometry Workstation User s Guide 5 1 Chapter 5 Optimizing Instrument Settings 5 1 Loading Modifying and Saving Instrument Settings This section includes e Using instrument settings BIC files e Standard instrument settings BIC files provided e Opening and viewing instrument settings e Modifying an instrument settings BIC file e Saving and printing instrument settings e Setting instrument settings files to read only status 5 1 1 Using Instrument Settings BIC Files 5 2 Instrument settings file definition Applied Biosystems NOTE Instrument settings and BIC files replace methods and MNU files used in previous versions of Voyager systems Instrument settings control operation of the Voyager mass spectrometer and are stored in BIC files Instrument settings include e Mode digitizer settings e Control mode manual or automatic e Voltages e Spectrum acquisition settings e Laser intensity settings e Calibration settings e PSD acquisition settings For more information see Section 5 2 Instrument Settings Parameter Descriptions and Chapter 8 PSD Analysis You can create instrument settings files with different settings and save each file for future use When you analyze samples you can select an instrument settings file with th
154. 4 6 Sequence Control Panel 4 32 4 7 How the Instrument and Sequence Control Panels Interact 4 33 Voyager Biospectrometry Workstation User s Guide 4 1 Chapter 4 Voyager Instrument Control Panel Basics 4 1 Instrument Control Panel The Voyager Instrument Control Panel allows you to directly control the Voyager mass spectrometer to acquire and inspect mass spectra in Manual or Automatic Control mode NOTE The Voyager Sequence Control Panel allows you to collect data for multiple samples using different conditions For more information see Section 4 6 Sequence Control Panel and Section 4 7 How the Instrument and Sequence Control Panels Interact This section includes e Parts of the Instrument Control Panel e Manual and Automatic Control modes e Accessing the Sequence Control Panel and the Data Explorer Software 4 1 1 Parts of the Instrument Control Panel The Instrument Control Panel includes Toolbar e Control pages e Spectrum window e Status bar e Output window When you start the Voyager Instrument Control software the Instrument Control Panel Figure 4 1 is displayed NOTE If the Instrument Control Panel is not displayed as shown in Figure 4 1 select Default Layout from the View menu 4 2 Applied Biosystems Instrument Control Panel Instrument settings file name Su Varager Instrument Control Panel SPEC Default Insu
155. 5 88 3 66E 05 5 79E 05 5 32E 05 7 27E 05 8 91E 05 Cytochrome C 12361 5 6 58E 05 1 04E 04 9 57E 05 1 31E 04 1 60E 04 horse heart 6181 25 4 65E 05 7 36E 05 6 76E 05 9 24E 05 1 13E 04 4121 17 3 80E 05 6 01E 05 5 52E 05 7 55E 05 9 24E 05 3091 12 3 29E 05 5 20E 05 4 78E 05 6 54E 05 8 00E 05 Myoglobin 16952 5 7 71E 05 1 22E 04 1 12E 04 1 53E 04 1 87E 04 horse heart 8476 75 5 45E 05 8 62E 05 7 92E 05 1 08E 04 1 33E 04 5651 5 4 45E 05 7 03E 05 6 47E 05 8 84E 05 1 08E 04 4238 76 3 85E 05 6 09E 05 5 60E 05 7 65E 05 9 37E 05 Bovine Serum 66431 1 53E 04 2 41E 04 2 22E 04 3 03E 04 3 71E 04 Albumin BSA 33216 1 08E 04 1 71E 04 1 57E 04 2 14E 04 2 62E 04 22144 3 8 81E 05 1 39E 04 1 28E 04 1 75E 04 2 14E 04 16608 5 7 63E 05 1 21E 04 1 11E 04 1 51E 04 1 86E 04 Voyager Biospectrometry Workstation User s Guide F 5 Appendix F Reference Standard Information F 3 Theoretical Cleavages for Angiotensin Monoisotopic masses Da for the theoretical cleavages of angiotensin are listed below as calculated for the positive ion mode Monoisotopic M H for the sequence DRVYIHPFHL is 1296 685 a b c Fragment 88 040 116 035 133 061 D 244 141 272 136 289 162 DR 343 209 371 204 388 231 DRV 506 273 534 268 551 294 DRVY 619 357 647 352 664 378 DRVYI 756 416 784 411 801 437 DRVYIH 853 468 881 463 898 490 DRVYIHP 1000 537 1028 532 1045 558 DRVYIHPF
156. 500 Mass m z Figure C 1 Sinapinic Acid Matrix Spectrum Spec 1 BP 172 0 56919 100 172 0 5 7E 4 379 1 a E 294 1 380 1 0 0 100 180 260 340 420 500 Mass m z Figure C 2 cyano 4 hydroxycinnamic acid CHCA Matrix Spectrum C 2 Applied Biosystems Matrixes Spec 1 BP 154 1 40516 Intensity 0 1 0 100 180 260 340 420 500 Mass m z Figure C 3 2 5 dihydroxybenzoic acid 2 5 DHB Matrix Spectrum Spec 1 BP 137 1 59419 m 137 1 5 9E 4 90 1511 273 1 168 1 287 2 Intensity 742 288 2 om 0 260 340 420 500 Mass m z Figure C 4 Mixture of 2 5 dihydroxybenzoic acid and 5 methoxysalicylic acid DHBs Matrix Spectrum Voyager Biospectrometry Workstation User s Guide C 3 Appendix C Matrixes Spec 1 BP 243 1 28598 100 243 1 2 9E 4 Intensity 7 7 0 340 420 500 Mass m z Figure C 5 2 4 hydroxy phenylazo benzoic acid HABA Matrix Spectrum Spec 1 BP 235 1 47437 100 edo 4 7E 4 Intensity 50 140 230 320 7 0 410 500 Mass m z Figure C 6 3 hydroxypicolinic acid 3 HPA Matrix Spectrum C 4 Applied Biosystems Matrixes Spec 1 BP 227 1 27080 1005 221 2 7E 4 90 s0 2 70 3 6 26 1 5 so 40 228 1 30 211 1 0 0 100 180 260 340 420 500 Mass m z Figure C 7 Dithranol Matrix Spectrum Spec 1 BP 169 1 4977
157. 50004 300004 250004 200004 Counts 150004 100004 50004 5 1000 Excess chemical noise Sample mass slightly higher than expected due to collision of ions with excess neutrals generated by the laser Broad peak Poor resolution 1500 2000 500 Mass miz Minor components or fragments of major components ionized Figure H 3 Example of Poor Mass Spectrum for Angiotensin Well resolved matrix peaks at 172 190 and 379 Da This File 3 CAYOYAGERIDATASKEITHYKO106010 MS Laser power near threshold producing Collected 16 95 8 39 AM Peaks not saturated 15000 10000 8 8 No minor components 5000 Minimal noise 500 1000 Correct mass of sample peak Sharp narrow peak Good resolution greater than 400 1500 2000 2500 Figure H 4 Example of Good Mass Spectrum for Angiotensin H 10 Applied Biosystems Parameters affecting resolution and signal to noise ratio Laser threshold Obtaining Good Spectra in Continuous Extraction Mode These parameters have a primary impact on resolution and signal to noise ratio in Continuous Extraction mode e Laser position on the sample hot or cold spot e Laser intensity These parameters have a secondary impact on resolution and signal to noise ratio in Continuous Extraction mode e Digitizer setting e Accelerating Voltage e Grid Voltage
158. 6 1005 169 1 _5 0E 4 2 2 a 0 4 0 100 180 260 340 420 500 Mass m z Figure C 8 2 4 6 trihydroxyacetophenone THAP Spectrum Voyager Biospectrometry Workstation User s Guide C 5 Appendix C Matrixes Spec 1 BP 170 1 54855 100 5 5E 4 904 80 gt 70 S 60 S 50 40 30 20 375 1 437 1 10 ot 0 100 420 500 Mass m z Figure C 9 trans 3 indoleacrylic acid IAA Matrix Spectrum Table C 1 Matrix Information h se ne Matrix Solution a Se Matrix Applications Color Matrix lons Concentration 5 monoisotopic Sinapinic acid Applications e 10 mg ml in 70 30 e 225 076 see mass Peptides water acetonitrile 224 068 spectrum on Proteins 0 1 TFA final e 207 066 z conc page 0 2 Color of crystals 431 134 MW 224 07 Da solution e 10 mg ml in 50 50 water acetonitrile ot White 0 1 TFA final A NOTE Matrix conc if sample powder may also contaminated a a contain orange CH OCH oH ws crystals Do not use crystals when preparing solutions C 6 Applied Biosystems Table C 1 Matrix Information Matrixes Matrix Solution Characteristic Matrix Applications Color n Matrix lons Concentration monoisotopic Alpha cyano Applications 10 mg ml in 50 50 e 164 047 4 hydroxycinnamic e Peptides water acetonitrile e 195 050 acid aCHCA lt p 0 1 TFA final conc e 172 040 roteins 379 093 see mass Color
159. 73 Checking System Status and Pressures 2 76 Chapter 3 Preparing Samples 3 1 3 2 3 3 Preparing Samples ss ssisuinsssnrenanmme sen deevecds ert eeteaviee animer 3 2 3 1 1 Selecting a Matrix ccccccceeceeeeeeeeeeeeee cee aeeaaeeaeteeeeeeeeeeeeeeaeeaea 3 3 3 1 2 Preparing Matrix oueon een en rnar a na r iea AEEA 3 4 3 1 3 Matrix Informations eurent AD 3 6 3 1 4 Preparing Sample ieni nysiiiriiotii manei padio 3 22 3 1 5 Sampl Clean p nisitan saan inan dents aaa aaraa 3 25 3 1 5 1 WAS RING ee a E A A ENEA URTEA ARNE 3 26 3 1 5 2 Drop Dialysis Floating Membrane 3 27 3 1 5 3 Cation Exchange cccceceeeeeeeeeeeceeaeeeneeeeeeeeeeees 3 29 gSA ZipTIPS sin Bia a ee ee ee 3 31 3 1 6 Mixing Sample and Matrix Dried Droplet Application 3 33 Loading Samples on Sample Plates 3 35 3 2 1 OVE EW Lisez tran ah tie alec rm de dunia Hane are RIDE 3 35 3 2 2 Locating Standards for Optimum Mass Accuracy 3 38 3 2 3 Loading Samples Dried Droplet Application 3 41 3 2 4 Loading Samples Thin Layer Application 3 44 3 2 5 Examining Crystals on Sample Plates ssseeeeeeeeeeees 3 45 Cleaning Sample Plates eceeeeeeeee eee ee neces teen ee eeeeeeeeeeeeees 3 47 Voyager Biospectrometry Workstation User s Guide v
160. 8 90 92 94 96 Grid Voltage E 2 Applied Biosystems F Reference Standard Information This appendix contains the following sections F 1 Calibration Compounds F 2 F 2 Conversion of Mass to Time for Typical Standards F 4 F3 Theoretical Cleavages for Angiotensin F 6 F 4 Observed PSD Fragments in Angiotensin F 7 Voyager Biospectrometry Workstation User s Guide F 1 Appendix F Reference Standard Information 4 F 1 Calibration Compounds The table below includes masses for common calibration compounds NOTE You can display reference mass information in the Data Explorer software by viewing the VOYAGER REF file in the mass calibration function Molecular Weight Protonated Molecular lon Compound oe M H Monoisotopic Average Monoisotopic Average Leucine Enkephalin 555 2693 555 63 1 556 2771 556 64 des Arg Bradykinin 903 4603 904 04 1 904 4681 905 05 Bradykinin 1059 5614 1060 23 1 1060 5692 1061 24 2 eo ori 531 1 Angiotensin 1295 6775 1296 50 1 1296 6853 1297 51 Substance P amide 1346 7281 1347 65 1 1347 7360 1348 66 Glu Fibrino peptide B 1569 6696 1570 60 1 1570 6774 1571 61 Neurotensin 1671 9097 1672 95 1 1672 9175 1673 96 Adrenocorticotropic 2092 0789 2093 45 1 2093 0867 2094 46 hormone ACTH clip 1 17 Bovine Trypsin 2162 0492 2163 3413 1 2163 0574 2164
161. 8 Applied Biosystems Optimizing Instrument Settings Parameters Ifthe spectrum you obtain is not acceptable increase or decrease the laser again in 50 to 100 step increments or decrements and reacquire If you pass the optimum laser setting increase or decrease using a setting that is midway between the previous two step increments or decrements Signal saturation lf the laser intensity is too high the signal may be saturated Figure 5 22 A saturated peak can be determined by Signal is saturated if it Display exceeds Spectrum window 64 000 counts Oscilloscope in 1 V full 5 screen grid line scale range divisions in the 200 mV mode 10 screen grid line divisions in the 100 mV mode Spec 1 BP 2120 2 65279 ian 1318 17 2120 172493 23 6 5E 4 3693 23 Intensity 8701 66 Bi 9 963996 27 Mass m z Figure 5 22 Signal Saturated at High Laser Intensity Voyager Biospectrometry Workstation User s Guide 5 69 Chapter 5 Optimizing Instrument Settings NOTE Saturated signal in any region of the spectrum may suppress peaks in the Mass Range of interest Decreasing the laser should optimize the signal Figure 5 23 Spec 1 BP 1318 6 62479 100 1296 6 2E 4 90 80 2467 z 70 2 60 2120 85 amp 50 40 3660 30 a 665 78 l 10 1340 94 2136 0 699 68 gi dl 1 ro mane 2516 83 PEE RO aes lp 200 1160 2120 3080 404
162. 9 73 B j B L I 0 G R A P H Y Voyager Biospectrometry Workstation User s Guide Bibliography 3 B j B L j 0 G R A P H Y Bibliography Peptide Protein Applications Bieman K Mass Spectrometry of Peptides and Proteins Annu Rev Biochem 1992 61 977 1010 Bieman K Sequencing of Peptides by Tandem Mass Spectrometry and High Energy Collision Induced Dissociation Meth Enzymol 1990 193 455 Billeci T M J T Stults Tryptic Mapping of Recombinant Proteins by Matrix Assisted Laser Desorption lonization Mass Spectrometry Anal Chem 1993 65 1709 1716 Binz P M Muller D Walther W V Bienvenut R Gras C Hoogland G Bouchet E Gasteiger R Fabbretti S Gay P Palagi M R Wilkins V Rouge L Tonella S Paesano G Rossellat A Karmime A Bairoch J Sanchez R D Appel and D F Hochstrasser A Molecular Scanner To Automate Proteomic Research and To Display Proteome Images Anal Chem 1999 71 4981 4988 Chait B T R Wang R C Beavis S B H Kent Protein Ladder Sequencing Science 1993 262 89 92 Chaurand P and F Luetzenkirchen Peptide and Protein Identification by Matrix Assisted Laser Desorption lonization MALDI and MALDI Post Source Decay Time of Flight Mass Spectrometry J Am Soc Mass Spectrom 1999 10 91 103 Henzel W J T M Billeci J T Stults S C Wong C Grimley and C Watanabe Identify
163. 90 95 20 000 100 000 400 1 000 90 95 gt 100 000 1 000 2 000 90 95 Table 5 13 Voyager DE PRO Reflector Mode Delay Time and Grid Voltage Values Reflector Mode Mass Range Da Delay Time Grid nsec Voltage 500 2 000 50 200 72 78 Voyager Biospectrometry Workstation User s Guide 5 79 Chapter 5 Optimizing Instrument Settings 5 80 Applied Biosystems Table 5 13 Voyager DE PRO Reflector Mode Delay Time and Grid Voltage Values Continued Reflector Mode Mass Range Da Delay Time Grid nsec Voltage 2 000 10 000 100 500 72 78 10 000 100 000 300 600 72 78 gt 100 000 No data No data available available Table 5 14 Voyager DE STR Reflector Mode Delay Time and Grid Voltage Values Reflector Mode Mass Range Da Delay Time Grid nsec Voltage 500 2 000 50 100 62 72 2 000 10 000 50 500 62 72 10 000 20 000 200 700 62 72 20 000 100 000 500 1 000 62 72 gt 100 000 No data No data available available NOTE Changing the Delay Time in increments smaller than 20 nsec may have no significant impact on resolution For information see Inherent Delay Time offset on page 5 77 Optimizing Instrument Settings Parameters 4 Continue increasing or decreasing the Delay Time in 100 nsec for Linear mode or 50 nsec for Reflector mode increments or decrements until optimum resoluti
164. A1 Max Operating Pressure Torr fi 5e 006 Wait Time sec fea Sample Loading Chamber TC Max Load Pressure Torr 0 08 Wait Time sec 120 Figure 2 15 Vacuum Configuration 3 Check the following values as needed e Source Chamber BA1 Max Operating Pressure Pressure Torr above which the high voltage power supplies are automatically turned off to prevent damage to the instrument Valid range is 10 to 10 9 Default is 9x10 If the Source Chamber pressure is above the Max Operating Pressure an error message is displayed and the high voltage cannot be turned on The software writes an error in the Windows NT Event log For more information see Checking the Windows NT Event Log on page 9 22 2 36 Applied Biosystems Hardware Configuration e Source Chamber BA1 Wait Time Time seconds that the software waits for the instrument to reach the Source Chamber Maximum Operating Pressure after the sample plate is loaded If the wait time is exceeded an error message is displayed which gives you the option of an additional wait time or ejecting the plate Valid range is 0 to 300 seconds Default is 120 seconds e Sample Loading Chamber TC2 Max Load Pressure Pressure Torr which must be reached before the system moves the sample plate from the load chamber to the source chamber during the load cycle Valid range is 8x107 to 107 Default is 8x10 2 If Max Load Pressure is not reached sa
165. ACCUIACY moeren nae eaa TEESE ten 2 53 2 9 Resetting the Optional External Laser 2 71 2 10 Startup and Shutdown 2 73 2 11 Checking System Status and Pressures 2 76 Voyager Biospectrometry Workstation User s Guide 2 1 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 1 Installing the System Your Voyager Biospectrometry Workstation is initially installed by an Applied Biosystems Technical Representative Do not use the Voyager system before it is properly installed Use the information in this chapter if you move the Voyager system after initial installation 2 2 Selecting the Site This section includes Voyager DE and Voyager DE PRO Workstations e Voyager DE STR Workstation 2 2 1 Voyager DE and Voyager DE PRO Workstations In this section This section includes e Required space e Weight e Power voltage requirements e Selecting input voltage Required space The Voyager DE and Voyager DE PRO mass spectrometers measure 27 inches 69 cm deep e 25 inches 64 cm wide 65 inches 165 cm high includes cabinet and flight tube Allow an additional 10 inches 26 cm above the top of the flight tube for service Allow 4 inches 10 cm at the back of the mass spectrometer for fans and cables 2 2 Applied Biosystems Selecting the Site Allow 4 inches 10 cm on the right side of the mass spectrometer for cables Allow an additional 40
166. AT file after calibration The specified CAL file is updated or created and contains new calibration constants after calibration For more information see Section 7 7 3 Performing Close External Calibration For information on CAL files see e Section 7 2 Understanding Settings Macros and Calibration e Creating Calibration CAL Files on page 7 11 NOTE The calibration specified in the Sequence Control Panel is performed after the calibration performed by the BIC file used to acquire the data file and the data file is updated with the calibration performed in the Sequence Control Panel continued 7 18 Applied Biosystems Creating a Sequence Table 7 1 Run List Parameters Continued Field Description Internal External Calibration continued NOTE If the DAT file contains multiple spectra generated using the Save All or the Save All Passing option in Automatic Control the calibration is applied to all spectra in the DAT file Calibration File required entry for External and Internal Update calibration optional entry for Internal calibration CAL file to use to calibrate the data file acquired in the row If you specify External or Internal Update a CAL file name is required even if a corresponding CAL file with constants does not exist see below for further explanation You can specify an existing CAL file or the name of a CAL file that will be cre
167. Adjusting the Display Range cccceeeeeeeeeneeeeeeeeeaaeeeeeeeeeas 4 10 4 3 2 ZOOMING on Traces oirin reier irene Traean EE RENS 4 13 4 3 3 Adding Traces to a Window 4 13 4 3 4 Annotating TraCeS sanoio ar ieii edea ia 4 16 4 3 5 Previewing and Printing Traces ssssssssrrrsesssrrirrssrrreeeernna 4 18 Customizing the Instrument Control Panel cccceeeeeeeeeeeeeeeees 4 21 Controlling the Workstation esssssseessssesesrsesernrrnnrrrrennrrrrennen 4 24 4 5 1 Using Toolbar Buttons and Instrument Menu Commands 4 24 4 5 2 Adjusting Laser Intensity and Selecting Sample Position 4 27 Applied Biosystems 4 6 4 7 Table of Contents Sequence Control Panel ss 4 32 How the Instrument and Sequence Control Panels Interact 4 33 Chapter 5 Optimizing Instrument Settings 5 1 5 2 5 3 5 4 Loading Modifying and Saving Instrument Settings 5 2 5 1 1 Using Instrument Settings BIC Files cceeeeeeeeeeeees 5 2 5 1 2 Standard Instrument Settings BIC Files Provided 5 3 5 1 3 Opening and Viewing Instrument Settings 5 7 5 1 4 Modifying an Instrument Settings File BIC 5 8 5 1 5 Saving and Printing Instrument Settings 5 11 5 1 6 Setting Instrument Settings Files to Read Only Status 5 13 In
168. B N Pamanik Anal Chem 1995 67 675 679 B j B L j 0 G R A P H Y Voyager Biospectrometry Workstation User s Guide Bibliography 1 B I B L j 0 G R A P H Y Bibliography Karas M U Bahr F Hillenkamp nt J Mass Spectrom lon Proc 1989 92 231 242 Nordhoff E et al Rapid Commun Mass Spectrom 1992 6 771 776 Papac D I A Wong A J S Jones Anal Chem 1996 68 3215 3223 Pieles U W Zurcher M Schar and H E Moser Nucl Acids Res 1993 21 3191 3196 Russel D J Am Soc Mass Spectrom 1996 7 995 1001 Shevchenko A M Wilm O Vorm M Mann Anal Chem 1996 68 850 858 Strupat K M Karas F Hillenkamp Int J Mass Spectrom lon Proc 1991 111 89 102 Wu K J A Steding C H Becker Rapid Commun Mass Spectrom 1993 7 142 146 Delayed Extraction Brown R S and J J Lennon Anal Chem 1995 67 1998 2003 Brown R S J J Lennon and D Christie Desorption 94 Mass Spectrometry of Large Organic lons by Particle and Photon Induced Desorption March 27 81 1994 Sunriver OR USA p 63 Colby S M T B King and J P Reilly Rapid Commun Mass Spectrom 1994 8 865 868 Juhasz P M Vestal and S A Martin On the Initial Velocity of lons Generated by Matrix Assisted Laser Desorption lonization and Its Effect on the Calibration of Delayed Extraction Time Of Flight Mass Spectra J Am Soc Mass Spectro
169. Biosystems Sample plate continued Biacore Chip 3 63 cleaning 3 48 3 49 consequence of loading wet plate 3 43 corner position numbers list of 2 50 crystallization examining 3 45 crystallization poor 3 25 custom plate types guidelines for defining 3 68 drying time 3 43 ejecting 3 54 4 25 handling 3 37 load no plate 3 57 3 59 loading 4 25 maximum number of spots on disposable 3 74 moving position under laser l 7 name of plate loaded 3 78 optimization see OptiPlate software ordering B 7 proper orientation 3 55 3 58 provided editable 3 65 relative coordinates 3 78 search pattern files for custom plate types 3 72 6 49 selecting type 3 56 3 59 spotting sample and standard location 3 38 H 24 templates 3 61 tolerance positional 3 74 types of physical plates 3 36 user defined 3 61 using outer rows 3 38 H 24 volume of sample to load 3 43 well size 6 47 wet consequence of loading 3 50 Sample plate aligning corner position numbers list of 2 50 examples of good and bad alignment 2 48 how the system aligns 2 47 if alignment fails 2 52 multiple alignments supported 2 47 PLT file selecting before starting 2 50 procedure 2 50 Sequence run 7 24 what you need 2 49 when to align 2 46 Sample plate loading dried droplet application 3 41 in mass spectrometer 3 50 overview 3 35 techniques 3 35 thin layer application 3 44 Sample plate types of 96x2 applications 3 64 96x2 PLT file
170. By using the Mass Accuracy Optimization feature in the Voyager software you can obtain optimum mass accuracy using fewer calibration standards For information see Section 3 4 2 Using the Mass Accuracy Optimization Option and Section 2 8 Running OptiPlate to Optimize Mass Accuracy 6 36 Applied Biosystems 6 6 2 Setting Instrument Settings for Automatic Control Mode Acquiring in Automatic Mode from the Instrument Control Panel This section includes e Specifying Automatic Control settings e Setting spectrum acceptance and laser adjustment criteria e Saving the instrument settings BIC file Specifying After you optimize an instrument settings file for Manual Automatic Control mode and ensure that parameters are adjusted for Control settings optimum performance you can adjust the instrument settings for Automatic Control mode if desired For information on optimizing for Manual Control mode see Chapter 5 Optimizing Instrument Settings You can set parameters to control the following in Automatic Control mode e Laser intensity adjustment e Spectrum accumulation and saving e Sample positioning e Stop conditions To set instrument settings for Automatic Control mode 1 Inthe Instrument Control Panel open or create an instrument settings BIC file with the appropriate parameters For more information see Section 5 4 Optimizing Instrument Settings Parameters NOTE Make sure the instrument setting
171. CAL HAZARD To prevent eye injury always wear eye protection when working with solvents AVERTISSEMENT RISQUE CHIMIQUE Pour viter les blessures aux yeux porter toujours des protections pour les yeux lorsque vous manipulez des solvants WARNING PHYSICAL INJURY HAZARD Use the Voyager Biospectrometry Workstation only as specified in this document Using this system in a manner not specified may result in injury or damage to the system Safety and Compliance Information AVERTISSEMENT DANGER DE BLESSURES CORPORELLES Veuillez suivre avec attention les indications figurant dans ce document lorsque vous utilisez la Station de Travail de Biosptectrom trie Voyager Un usage diff rent de la station pourrait causer un accident ou endommager le syst me Voyager Biospectrometry Workstation User s Guide xxiii Safety and Compliance Information Safety and EMC Electromagnetic Compliance Standards xxiv US Safety and EMC Standards Applied Biosystems Safety This instrument has been tested to and complies with standard ANSI UL 3101 1 Electrical Equipment for Laboratory Use Part 1 General Requirements 1st Edition It is an ETL Testing Laboratories listed product EMC This device complies with Part 15 of the FCC Rules Operation is subject to the following two conditions 1 This device may not cause harmful interference and 2 this device must accept any i
172. Celbenton Fie ooo Figure 8 13 PSD Acquisition Settings Control Page Automatic Mode Setting voltages 4 and external calibration for the precursor spectrum For optimum mass accuracy change the Accelerating Voltage and Grid Voltage on the Instrument Settings control page to the values you used to acquire the precursor spectrum and to generate the calibration NOTE If you use different voltage settings Accelerating Voltage Grid Voltage or Delay Time to acquire the precursor spectrum generate the calibration and acquire the fragment spectra the software compensates for the different settings However using different voltage settings may not yield optimum mass accuracy Voyager Biospectrometry Workstation User s Guide 8 59 Chapter 8 PSD Analysis Setting precursor mass and PSD calibration for fragment spectra 8 60 Applied Biosystems 6 In the Calibration section of the Instrument Settings control page select the matrix you are using and the CAL file you created in Generating an external calibration for the precursor ion on page 8 39 NOTE The calibration you specify on the Instrument Settings control page is used to determine the t value precursor ion flight time needed for the PSD calibration equation described on page 8 28 The t value is determined using the standard calibration equation t in the standard equation described on page 6 9 You
173. Control Panel Control Panel Applied Biosystems To start the Voyager Instrument Control Panel from the Windows NT desktop double click the Voyager Control Panel icon on the desktop The Instrument Control Panel is displayed Figure 2 13 NOTE If the Instrument Control Panel is not displayed as shown in Figure 2 13 select Instrument Page Control from the View menu then select Default Layout for control mode y Vopager Instrument Control Panel ISPEC Delault Instrument Settings Ul Eie Edt View Instrument Acquistion SamplePlate Display Tools Applications jesuo we eo le z E Be LS ie S ce Dat Steg s p Instrument Mode zf jiii Linear Positive Mode Digtizer Directoy E WoyagenData GL Control Mode IF Autosequence Filenames Manual C Automatic Aroro Gord Votages Accelerating 20000 Grid iad 00 1100 GudeWre 005 Sample Descrption Comment 0 000 0 300 p Manual Laser Intensity Delay Time joo nsec 4 gt Manual Sample Positioning ActivePos st 100 well plate pit bosoocococo D 200000 Spectrum Acquistion Shots Spectrum 50 Intensity Mass Range Da 500 to F Low Mats Gate Da D p Calibration Mattie 2 Cyano 4 hydroxycinnamic aci id o 400 800 1200 1600 2000 Mass m z ee Automated Corirol _ Data Storage 7 i fiomOFF C i in Looooooo bLoooovocoo O00000000 boocococoococo Lbooooooocoo
174. Control e Single sample Section 6 2 Acquiring in mode in e Manual control of laser Manual Mode from the Instrument intensity sample position Instrument Control Panel Control Panel data accumulation data storage Manual data evaluation Default or external calibration continued 6 2 Applied Biosystems Before You Begin Table 6 1 Voyager Acquisition Options Continued Acquisition nn For more Description Option information see Automatic e Single sample Section 6 6 Acquiring in Control mode in e Automatic manual control of Automatic Mode from the Instrument Control Panel laser intensity or sample position Automatic control of data accumulation and data storage Automatic data evaluation based on acceptance criteria you specify Default or external calibration Instrument Control Panel Batch mode in Sequence Control Panel Multiple samples Uses instrument settings previously optimized for Automatic Control in Instrument Control Panel Default external internal and internal update calibration Optional macro functions that can be applied before and after calibration Chapter 7 Acquiring Spectra from the Sequence Control Panel Voyager Biospectrometry Workstation User s Guide 6 3 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 1 2 Guidelines for Acquiring This section includes e High voltage warmup for
175. D Analysis 8 2 3 Segments and Composite Spectra Overview To obtain the most information about an ion collect fragment ion spectra across a molecular weight range from the mass of the original precursor ion down to 50 Da determined by the desire to see immonium ions that indicate the presence of individual amino acids Each fragment ion spectrum is referred to as a segment and is collected with a discrete focusing region controlled by the Mirror Ratio setting Segments are combined to generate a composite spectrum Number of The number of PSD segments you must collect depends on segments the quality of data needed The PSD software allows you to collect any number of segments A larger number of fragment spectra generally yield better resolution in the composite spectrum For more information see Section 8 4 2 Determining the Number of Segments to Acquire for a Complete Composite Spectrum Composite When you view the PSD data in the Data Explorer software spectrum the software automatically assembles or stitches together the best portions of fragment ion spectra referred to as PSD segments to generate the full composite PSD spectrum Voyager Biospectrometry Workstation User s Guide 8 25 Chapter 8 PSD Analysis Region of segments included in composite The composite spectrum is generated from portions of the segment traces The upper mass limit of the composite region in each segment is determined by the P
176. DAT file where the spectrum is stored when you save a spectrum This tab displays a running list of DAT files You can clear the window by right clicking on the window and selecting Clear e Automatic Control lf you are operating in Automatic Control mode the Automatic Control tab displays current laser setting search pattern position and spectrum evaluation results For information on Automatic Control mode see Section 6 6 Acquiring in Automatic Mode from the Instrument Control Panel Voyager Biospectrometry Workstation User s Guide 4 5 Chapter 4 Voyager Instrument Control Panel Basics Displaying The Output window is automatically displayed when you e Acquire data Store data To display the Output window manually select Output Window from the View menu To close the Output window deselect Output Window from the View menu or right click in the Output window and select Hide Maximum number The Output window can contain a maximum of 1 000 lines If of entries the total number of lines exceeds 1 000 the oldest 500 lines are automatically deleted The most recent 500 entries are retained 4 1 2 Manual and Automatic Control Modes Overview The Instrument Control Panel allows two modes of operation e Manual Control mode Enables manual control of the laser sample positioning and data storage e Automatic Control mode Enables automated control of the laser sample positioning data storage after you set s
177. EXTERNAL STD CAL 46 SAMP5 SAMP BIC EXTERNAL STD CAL 54 56 SAMP6 8 STD BIC EXTERNAL STD CAL Voyager Biospectrometry Workstation User s Guide 7 35 Chapter 7 Acquiring Spectra from the Sequence Control Panel To run samples spotted on a 400 position or 96 x 2 position plate as illustrated in Figure 7 8 set up the run list as shown in the following table 400 well 96 2 well Base File Instrument Calibration Type Calibration Position Position Name Settings File File A1 Ai_a STD1 STD BIC INTERNAL UPDATE STD CAL A2 Ai_b SAMP1 SAMP BIC EXTERNAL STD CAL A3 A2_a STD2 STD BIC INTERNAL UPDATE STD CAL A4 A2_b SAMP2 SAMP BIC EXTERNAL STD CAL A5 A3_a STD3 STD BIC INTERNAL UPDATE STD CAL A6 A3_b SAMP3 SAMP BIC EXTERNAL STD CAL A7 A4_a STD4 STD BIC INTERNAL UPDATE STD CAL A8 A4 _b SAMP4 SAMP BIC EXTERNAL STD CAL Sample and standard in the same sample position 7 36 The number and placement of standards needed depend on your application See Mass calibration standards on page 7 33 Hint You can use the same calibration file more than once in a sequence run You may see improved mass accuracy by spotting sample and standard in as close together as possible within a sample position Figure 7 9 A standard spot within 1 mm of a sample spot can yield mass accuracy close to that expected for internal calibration Applied Biosystems Automatic
178. Extraction Velocity focusing of ions is controlled by variable voltage grid in the ion source and the delay time applied to acceleration See Velocity focusing on page 1 15 Resolution and mass accuracy are improved Initial velocity distribution of ions Minimal energy loss from collision of ions lon acceleration is delayed the ion neutral plume becomes less dense and ion collision is minimized Substantial energy loss from collision of ions lons are accelerated immediately after sample is ionized and collide in the dense ion neutral plume Fragmentation is greatly reduced by acceleration delay Background noise is reduced and the effect of laser intensity on performance is minimized Background noise from fragmentation in the source prompt fragmentation related to ion collision Less analyte matrix dependent performance because initial velocity distribution and energy loss is corrected or minimized Analyte matrix dependent performance because initial velocity distribution and energy loss conditions are specifically related to the sample and matrix used 1 14 Applied Biosystems Velocity focusing Linear mode Voyager DE Delayed Extraction Technology Delayed Extraction technology facilitates tuning modes when the time of flight of an ion is independent of the initial velocity After ions are released from the sample surface their position in the ion source is c
179. FTWARE DELAY IN REPAIR OR REPLACEMENT OR FOR LOSS OF REVENUE OR PROFITS LOSS OF GOOD WILL LOSS OF BUSINESS OR OTHER FINANCIAL LOSS OR PERSONAL INJURY OR PROPERTY DAMAGE NO AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS HAS ANY AUTHORITY TO BIND APPLIED BIOSYSTEMS TO ANY AFFIRMATION REPRESENTATION OR WARRANTY CONCERNING THE PRODUCT THAT IS NOT CONTAINED IN THIS LIMITED WARRANTY STATEMENT ANY SUCH AFFIRMATION REPRESENTATION OR WARRANTY MADE BY ANY AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS WILL NOT BE BINDING ON APPLIED BIOSYSTEMS THIS WARRANTY IS LIMITED TO THE BUYER OF THE PRODUCT FROM APPLIED BIOSYSTEMS AND IS NOT TRANSFERABLE Limited Warranty for refurbished units Damages Claims Returns The limited warranty period for refurbished units that are less than one 1 year old is one 1 year for parts labor and travel The limited warranty period for refurbished units that are more than one 1 year old is ninety 90 days for parts labor and travel All of the foregoing terms obligations and exclusions regarding the purchase of new products shall apply to refurbished units B 2 Damages Claims Returns Damages Claims Returns Please examine any shipments promptly after receipt to check for damage Contact Applied Biosystems Service Department if you have questions about checking for damage If you discover damage stop unpacking Contact the shipping carrier and request inspectio
180. From the Instrument menu select Reinitialize The software resets all system components and sets up communication between the computer and workstation 2 74 Applied Biosystems Startup and Shutdown Powering down Leave the Voyager mass spectrometer powered up unless system You need to perform maintenance on internal parts components You need to move the system To power down Software and 1 Close the Instrument Control Panel by selecting Exit computer from the File menu shutdown 2 On the Windows desktop start task bar click Start then click Shut down In the Shut Down Windows dialog box click Shut down the computer and click Yes Hardware is reinitialized NOTE If you select Close all programs and log on as a different user hardware is not reinitialized 3 Power down all other system components oscilloscope or digitizer monitor and so on Wait until a message is displayed indicating that you can shut down your computer Power down the computer and computer monitor Powering down if you need to perform maintenance on internal parts or move the mass the system power down the spectrometer spectrometer 1 Power down the system components 2 Turn off the main power switch The power switch is located e On the right side panel of the mass spectrometer cabinet on Voyager DE and Voyager DE PRO systems e On the back panel of the mass spectrometer cabinet on the Voyager DE STR system Voyag
181. Gas requirements You can use room air helium argon or xenon as the collision gas If you are using a compressed gas source regulate the gas source between 2 and 5 psi 1 Meth Enzymol McCloskey J A ed 1990 193 886 Voyager Biospectrometry Workstation User s Guide 8 33 Chapter 8 PSD Analysis Purging collision 8 34 gas lines Applied Biosystems Before turning on the collision gas purge the lines to prevent disruption of the vacuum CAUTION If you do not purge the lines the CID gas introduction may increase the pressure in the vacuum and cause an Interlock error Perform the following procedure if the CID gas has been off for more than 15 minutes 1 Observe the vacuum gauge and allow the pressure on BA1 Source Chamber pressure to reach the typical pressure listed below Model Pressure Voyager DE PRO 8x10 7 Torr Voyager DE STR 2x107 Torr Turn the top 3 way valve on the CID box to the Purge Cell position for 1 to 2 seconds Turn the top 3 way valve to the Gas Inlet position for 1 second then turn immediately to the Purge Cell position Observe BA1 on the vacuum gauge panel When BA1 reads approximately 1 x 106 Torr turn the top 3 way valve to the Gas Inlet position for 2 seconds then turn immediately to the Purge Cell position Observe BA1 on the vacuum gauge panel When BA1 reads approximately 1 x 10 6 Torr turn the top 3 way valve to th
182. H 2 Obtaining Good Spectra in Continuous Extraction Mode This section describes e Spectra resolution signal to noise ratio and laser threshold e Determining laser threshold e Checking Resolution e Fine Tuning the Laser Setting H 2 1 Spectra Resolution Signal to Noise Ratio and Laser Threshold H 8 What is a good spectrum Resolution and signal to noise ratio Applied Biosystems A good spectrum is one that is acceptable for your purposes In general it e Contains sharp symmetrical well defined peaks e Has acceptable resolution e Has acceptable signal to noise ratio For some applications for example when you are looking for detailed structural information you may require very well separated peaks and the maximum resolution possible For other applications for example when looking for an estimate of molecular weight your requirements may be less strict There is a balance between good resolution and a good signal to noise ratio To achieve maximum resolution you may a see lower signal to noise ratio Conversely to maximize the signal to noise ratio you may see less than maximum resolution Figure H 1 through Figure H 4 are examples of poor and good spectra NOTE Dimer in a spectrum may indicate that the molecular ion is saturated or that sample is too concentrated Decrease laser intensity or sample concentration to minimize the dimer Obtaining Good Spectra in Continuous Extraction
183. ICAL HAZARD Wear appropriate personal protection and always observe safe laboratory practices when operating your system Une remarque fournit une information importante l op rateur er se pr sente ainsi REMARQUE Si on vous demande d ins rer la disquette de d marrage dans le lecteur ins rez la puis appuyez sur n importe quelle touche Une recommandation fournit une information destin e viter des d t riorations du syst me ou la perte de donn es RECOMMANDATION La lampe peut tre endommag e N y touchez pas Un avertissement fournit une information indispensable la s curit de l operateur et se pr sente ainsi AVERTISSEMENT Conformez vous toujours aux r glements du laboratoire quand vous utilisez votre syst me Safety and Compliance Information Safety symbols The following symbols may be displayed on the system These symbols may also appear next to associated warnings in this document Electrical Symbols The following chart is an illustrated glossary of electrical symbols that may be displayed on your instrument Whenever such symbols appear on instruments please observe appropriate safety procedures This symbol indicates the on position of the main power switch This symbol indicates the off position of the main power switch This symbol indicates the on off position of a push push main power switch This symbol indicates that a terminal may
184. L UPDATE STD CAL 92 SAMP1 SAMP BIC INTERNAL STD CAL 93 STD2 STD BIC INTERNAL UPDATE STD CAL 94 SAMP2 SAMP BIC INTERNAL STD CAL 95 STD3 STD BIC INTERNAL UPDATE STD CAL 96 SAMP3 SAMP BIC INTERNAL STD CAL During the run the external calibration CAL file is applied then the internal calibration is performed Applied Biosystems Customizing the Sequence Control Panel 7 8 Customizing the Sequence Control Panel This section includes e Customizing the sequence display e Setting Sequence Control Preferences 7 8 1 Customizing the Sequence Display Using Workbook Workbook mode displays the run list contains sample mode information and conditions for acquisition and processing in tabbed framed format To use Workbook mode 1 Open or create a sequence run list 2 Open or create another sequence run list 3 Select Workbook mode from the View menu Figure 7 11 displays the Sequence Control Panel in Workbook mode two tabs at bottom of run list Voyager Biospectrometry Workstation User s Guide 7 43 Chapter 7 Acquiring Spectra from the Sequence Control Panel Workbook tabs Figure 7 11 Showing or hiding columns Positioning windows 7 44 Applied Biosystems Sequence Control Panel in Workbook Mode 4 Switch between the run lists by clicking the appropriate tab You can select Grid Columns from the View menu then select the columns that are displayed Checked col
185. Laser Sample Positioning control page see page 4 27 Sample To test mix the sample with a contaminated standard of known sensitivity If the standard no longer exhibits the expected sensitivity a contaminant in the sample is affecting sensitivity Clean up sample See Section 3 1 5 Sample Cleanup Not averaging Average more spectra enough spectra Manual Control mode Collect more spectra shots spectra before stopping acquisition e Automatic Control mode lIncrease number of spectra to acquire continued Voyager Biospectrometry Workstation User s Guide 9 13 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor signal to noise ratio Matrix peaks are Turn on Low Mass Gate to or sensitivity saturating the suppress matrix peaks continued detector Guide Wire Voltage Adjust See Section 5 3 4 too high lower Understanding Guide Wire masses or too low Voltage higher masses Accelerating Voltage Adjust See Section 5 4 4 2 too low Setting Accelerating Voltage Too much salt or Clean up sample See buffer in sample Section 3 1 5 Sample Cleanup Decrease salt or buffer Sample matrix ratio Try ratios higher and lower not optimal than the recommended 1 10 ratio May want to prepare a dilution series factors of 10 Accelerating Voltage Call Applied Biosystems
186. M 50 peak height Figure 6 12 Resolution Calculator 6 30 Applied Biosystems Evaluating Data in the Instrument Control Panel Type in up to four Mass Charge values for which to calculate resolution For each Mass Charge enter the window for calculation AMU NOTE To label isotopes set the AMU value low enough to prevent the calculation windows for each isotope peak from overlapping If the calculation windows overlap only the highest peak is labeled If you set too low the peak of interest may not be labeled However if you set AMU too low the peak of interest may not appear in the window and resolution will not be calculated Click OK The peaks are labeled with RXXXxX next to the peak mass where XXXX is the resolution Figure 6 13 To turn off resolution labels see Labeling spectrum peaks on page 6 29 NOTE If you do not apply peak detection parameters after acquisition is complete resolution labels are not displayed Voyager Biospectrometry Workstation User s Guide 6 31 Chapter 6 Acquiring Spectra from the Instrument Control Panel VPTE peptidr_linenr hir SampePlee Dipoy Tol Wrdou lej oe ela ere leek S lee se mlm e fe Aleleele ssl IE Spoc 42 TIC 31475649 BP 907 50 59151 1301 6546 1 pins Resolution result Figure 6 13 Resolution Calculator Results The table below lists a general rating s
187. Mirror ratio PSD ions for varies for PSD precursor analysis mass 1347 74 Mass Range specified for acquisition may be wider NOTE For CID applications use the Substance_P_Precursor BIC with the appropriate precursor mass 1347 74 Da 5 6 Applied Biosystems Loading Modifying and Saving Instrument Settings 5 1 3 Opening and Viewing Instrument Settings Overview There are two ways to open an instrument settings file e Directly open a BIC file Select a DAT file that contains the instrument settings of interest and the software loads the BIC Opening From the Instrument Control Panel 1 Select Open Instrument Settings from the File menu The Open dialog box is displayed Figure 5 1 Open 2x Look in Factory c H 8 ngiotensin_linear bic E Mix2_reflector bic Fa PSD_reflector bic x angiotensin_reflector bic E Myoglobin_linear bic a Sensitivity_linear bic a BSA_linear bic a Negitive_linear bic si Sensitivity_reflector b sl lgG_linear bic a Negitive_reflector bic E test bic E Insulin_linear bic a pitest_002 bic sal Thiredoxin_reflector E sl Insulin_reflector bic a PSD_precursor bic fl TIS_reflector bic H File name __ Op Files of type instrument Configuration Files bic z Cancel Figure 5 1 Open Dialog Box Standard instrument settings files provided are located in the C VOYAGER FACTORY directory Openin
188. NA Fragments from Conventional and Microfabricated PCR Devices using Delayed Extraction MALDI TOF Mass Spectrometry Anal Chem 1998 70 2067 2073 Ross P L Hall Smirnov and L Haff High Level Multiplex Genotyping by MALDI TOF Mass Spectrometry Nature Biotechnology 1998 16 1347 1351 Ross P L K Lee and P Belgrader Discrimination of Single Nucleotide Polymorphisms in Human DNA Using Peptide Nucleic Acid Probes Detected by MALDI TOF Mass Spectrometry Anal Chem 1997 69 4197 4202 Smirnov P M T Roskey P Juhasz E J Takach S A Martin and Lawrence A Haff Sequencing Oligonucleotides by Exonuclease Digestion and Delayed Extraction MALDI TOF Mass Spectrometry Anal Biochem 1996 238 19 25 Taranenko N I C N Chung Y F Zhu S L Allman V V Golovlev N R Isola S A Martin L A Haff and C H Chen Matrix assisted laser desorption ionization for Sequencing Single stranded and Double stranded DNA Rapid Comm Mass Spectrom 1997 11 386 392 Taranenko N I K J Matteson C N Chung Y F Zhu L Y Chang S L Allman L A Haff S A Martin and C H Chen Laser Desorption Mass Spectrometry for Point Mutation Detection Genetic Analysis Biomolecular Engineering 1997 Taranenko N I K J Matteson C N Chung Y F Zhu L Y Chang S L Allman L Haff S A Martin and C H Chen Laser Desorption Mass Spectrometry for Point Mutation Detection Genet Ana
189. ON The plastic tabs that hold the voltage selector in place are fragile Do not exert force when removing this piece 9 4 Applied Biosystems Maintenance Fuses Volt ey Ky A Fuse holder ps Figure 9 1 Changing Fuses WARNING FIRE HAZARD Using a fuse of the wrong type or rating can cause a fire Replace fuses with those of the same type and rating Voyager Biospectrometry Workstation User s Guide 9 5 Chapter 9 Maintenance and Troubleshooting 6 Insert two fuses of the proper rating Electrical Rating Volts Amps EF Fuse 5 x 20 mm 100 V 10A T10A 250V 120 V 10A T10A 250V 220 V 6 3A T6 3A 250V 240 V 5A T5A 250V 7 Insert the voltage selector fuse holder into the receptacle 8 Plug in the mass spectrometer and power up 9 1 3 Backing Up and Archiving Data Back up data weekly or as needed Archive data as needed To operate properly the Voyager software requires e Disk space for data and instrument setting files e Disk space for printing If the disk is full writing data files printing or saving instrument settings can fail and result in loss of data The computer also operates more slowly as free disk space decreases To maintain adequate disk space back up and archive data on a routine basis e Back up Temporarily store data in case of a hard disk problem e Archive Permanently
190. Preparing matrix on page 3 5 and combine 700 ul of 0 1 TFA solution in water and 300 ul acetonitrile to 10 mg of solid matrix If sample is contaminated with buffer salt or detergent instead of the proportions listed above combine 500 ul 0 1 TFA and 500 ul acetonitrile Air dry sample plate after loading sample and matrix continued Voyager Biospectrometry Workstation User s Guide 3 7 Chapter 3 Preparing Samples Table 3 1 Sinapinic Acid Matrix Information Continued NOTE The organic concentration above assumes you are premixing sample solution matrix solution in a 1 10 dilution If you are mixing sample solution matrix solution on the plate in a 1 1 dilution increase the proportion of acetonitrile to 50 percent to prevent the matrix from precipitating Adjust the other components accordingly NOTE A higher concentration of TFA up to 3 may improve sample solubility Crystals Uniform rhomboid shape see Figure 3 1 on page 3 45 Stability Prepare daily 3 8 Applied Biosystems CHCA CHCA for dried droplet application Preparing Samples Use a cyano 4 hydroxycinnamic acid CHCA for Dried drop application Peptides proteins lt 10 000 Da e Thin layer application Peptides lt 3 000 Da Use for peptides proteins lt 10 000 Da WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instru
191. Quick Start see also PSD mode accumulating spectra 8 55 acquiring segments in any order 8 55 experiment stopping 8 56 Voyager Biospectrometry Workstation User s Guide Index 29 I N D E X Index PSD acquisition manual mode continued fragment ion identity determining 8 19 instrument settings disabled during 8 53 overview 8 52 precursor in Reflector mode 8 38 process that occurs during 8 54 reacquiring a spectrum 8 56 saving spectra 8 55 segment selecting 8 54 segments appended to DAT 8 52 settings 8 43 starting 8 38 8 53 stopping 8 54 PSD acquisition Quick Start acquiring 8 9 angiotensin 8 3 angiotensin expected masses and ion types 8 13 determining if calibration is needed 8 12 experiment stopping 8 11 8 18 fragment ion identity determining 8 19 overview 8 2 precursor in Reflector mode 8 7 precursor external calibration 8 7 precursor ion mass 8 9 8 16 PSD calibration generating 8 13 saving spectra 8 11 8 18 segment selecting 8 10 8 17 settings 8 7 8 16 spectrum examining for fragments 8 10 starting 8 10 8 17 stopping 8 11 8 18 unknown 8 5 PSD Acquisition settings automatic mode 8 59 manual mode 8 44 Index 30 Applied Biosystems PSD calibration CAL file selecting 8 46 8 60 default 8 28 equation 8 28 in Instrument Settings 8 45 8 60 in PSD Acquisition Settings 8 46 8 60 standard preparing 8 65 two types 8 2 PSD composite spectrum see also PSD mode see also PSD se
192. RO Mass Spectrometer Single Stage Reflector Voyager Biospectrometry Workstation User s Guide 1 21 Chapter 1 Introducing the Voyager Biospectrometry Workstations Parts of the mass The Voyager DE and Voyager DE PRO mass spectrometers spectrometer include Laser attenuator and prism A nitrogen laser that operates at 337 nm and ionizes sample It produces 3 nanosecond duration pulses Laser rate can be set to Default 3 Hz or Optimized see page 5 26 Maximum possible rate is 20 Hz actual rate depends on the digitizer installed and the digitizer Bin Size setting 1 NOTE Optimized laser rate is available only on systems with the following serial numbers Voyager DE 1171 and later Voyager DE PRO 6131 and later The laser attenuator varies the intensity of the laser beam reaching the sample The prism deflects the laser beam into the ion source e lon Source A high voltage region used to accelerate ions Includes e Sample plate and sample stage An area supplied with voltage 0 to 25 000 V for acceleration of ions into the flight tube e Variable voltage grid A grid supplied with additional voltage to fine tune ion acceleration e Ground grid Ground surface for formation of potential gradient e Grounded aperture Entrance to flight tube For more information on the ion source and voltages see Section 5 4 4 2 Setting Accelerating Voltage and Section 5 4 3 5 Optimizing Grid Volta
193. Run Status Displays overall run status Possible states are e Off Not running e Running Acquiring a sample e Pausing Paused Pause button clicked Stopping Stopped Siop button clicked Finished Sample acquired and processed e Error Error occurred during acquisition that terminated the sequence Elapsed Time Displays the time that has elapsed during sequence acquisition Current Entry Index Displays the row number that is being acquired Remaining Entries Displays the remaining number of rows selected for acquisition 7 30 Applied Biosystems Running a Sequence Checking status You can monitor the following parameters in the Instrument in the Instrument Control Panel during a sequence run Control Panel Parameter Where to check Laser intensity Status bar of Instrument Control Panel Search Pattern Position Automatic Control tab in Output window Data file name Data Storage tab in Output window Current Peak Intensity Spectrum window Signal to Noise of Automatic Control tab in Output window previous peak Voyager Biospectrometry Workstation User s Guide 7 31 Chapter 7 Acquiring Spectra from the Sequence Control Panel 7 7 Automatic Calibration During a Sequence Run This section includes e Calibration options in a sequence External calibration standard requirements e Performing close external calibration e Internal standard calibratio
194. SD Mirror Ratio Rp with which the segment was acquired and the mass of the spectrum s H precursor ion mp Figure 8 6 included in composite Seg 1 included in composite Seg 2 A Loo R x Mp _ included in composite A i Seg 3 If first segment acquired R2 x Mp with PSD Mirror Ratio 1 0 Loo upper limit is Seg 4 a A slightly higher than Rg x Mp precursor ion mass 8 26 Applied Biosystems Figure 8 6 Portions of Segment Traces Included in the Composite Spectrum For more information see the Data Explorer Software User s Guide Chapter 8 Viewing Voyager PSD Data Overview of PSD Analysis 8 2 4 PSD Data Files PSD data DAT files Figure 8 7 include e Precursor ion mass _ All segments acquired during a PSD experiment Composite spectrum Segments are stored in the data file in the order in which they are collected Precursor ion mass PSD Segment 1 PSD Segment 2 Raw data PSD Segment 3 PSD Composite Results PSD DAT File Figure 8 7 PSD DAT File Structure Voyager Biospectrometry Workstation User s Guide 8 27 Chapter 8 PSD Analysis 8 2 5 Mass Calculation for Fragment lons PSD calibration PSD calibration specifies the mass of a fragment ion as a equation function of e Mass and flight time of the precursor ion e Mirror Ratio setting a B and y calibration constants The equation that the Vo
195. Sample loading chamber Mirror chamber Three vacuum pumps create the vacuum environment e Fore pump Creates a vacuum in the sample loading chamber creates a lower than atmospheric pressure condition before the turbo pumps start and provides backing pressure to the turbo pumps e Turbo pump 1 Creates a high vacuum condition in the main source chamber e Turbo pump 2 Creates a high vacuum condition in the mirror chamber Vacuum is maintained in the main source chamber and sample loading chamber by valves that isolate the chambers Vacuum is maintained in the mirror chamber by a differential pumping baffle Voyager Biospectrometry Workstation User s Guide 1 37 Chapter 1 Introducing the Voyager Biospectrometry Workstations Mirror chamber high vacuum Foreline valve 1 Main Turbo source pump 1 E chamber mhigh vacuum Figure 1 19 Voyager DE STR Biospectrometry Workstation Vacuum Chambers Top View 1 38 Applied Biosystems Parts of the Voyager DE STR System Vacuum gauges The Voyager DE STR Biospectrometry Workstation include three vacuum gauges e BA1 Bayard Alpert Gauge Monitors pressure in the main source chamber e BA2 Bayard Alpert Gauge Monitors pressure in the mirror chamber e TC2 Monitors pressure in the sample loading chamber Readings from the vacuum gauges are displayed in the System Status Control page in the In
196. Spectral accumulation see Accumulation spectra Spectrum Acceptance Criteria accessing 5 36 description 6 43 resolution 6 44 resolution filtering 6 44 setting 6 42 signal intensity 6 43 signal to noise filtering 6 43 6 44 Voyager Biospectrometry Workstation User s Guide Index 39 Index Spectrum Accumulation options description 5 39 selecting 5 35 Spectrum see Spectra Spectrum window see also Instrument Control Panel see also Traces accumulated spectrum data definition 4 14 Current Spectrum trace during acquisition 6 16 cursor displaying 4 21 description 4 5 display range adjusting 4 10 does not update with every laser shot 6 16 9 21 Grid displaying 4 21 live data definition 4 14 Live Spectrum trace during acquisition 6 16 Low Mass Gate spike 5 91 mass range in Spectrum window does not update with every laser shot 6 16 9 21 mass scale not accurate 9 15 peak detection parameters setting 6 29 peak labels enabling and disabling 6 29 resolution calculating 6 30 right axis displaying Absolute counts 5 70 scaling signal to noise ratio calculating 6 33 trace displaying as vertical bars 4 22 traces displayed 4 5 traces do not print 4 19 4 22 traces previewing and printing 4 18 using 4 10 zooming 4 13 Index 40 Applied Biosystems SPIRAL SP 6 49 Spotting sample see sample loading Standard compounds see also Calibration standards acquiring 6 10 conversion of mass to time F 4 molecular wei
197. Spectrom 1993 7 576 580 Krone et al Anal Biochem 1997 244 124 132 Standing K G and W Ens Methods and Mechanisms for Producing lons from Large Molecules Plenum Press New York 1991 Youngquist R S GR Fuentes M P Lacey and T Keough Matrix assisted Laser Desorption lonization for Rapid Determination of the Sequences of Biologically Active Peptides Isolated from Support bound Combinatorial Peptide Libraries Rapid Commun Mass Spectrom 1994 8 77 81 MS MS Interpretation Falick A M W M Hines K F Medzihradszky M A Baldwin and B W Gibson Low Mass lons Produced from Peptides by High Energy Collision Induced Dissociation in Tandem Mass Spectrometry J Am Soc Mass Spectrom 1993 4 882 893 Hines W M A M Falick A L Burlingame and B W Gibson Pattern Based Algorithm for Peptide Sequencing from Tandem High Energy Collision Induced Dissociation Mass Spectra J Am Soc Mass Spectrom 1992 3 326 336 Kaufman R B Spengler and F L tzenkirchen Mass Spectrometric Sequencing of Linear Peptides by Product ion Analysis in a Reflectron Time of flight Mass Spectrometer Using Matrix assisted Laser Desorption lonization Rapid Commun Mass Spectrom 1993 7 902 910 McCloskey J A ed Meth Enzymol 1990 193 886 Papayannopoulos I The Interpretation of Collision Induced Dissociation Tandem Mass Spectra of Peptides Mass Spectrometry Reviews 1995 14 4
198. Spectrometry Instrumentation and Applications in Biological Research ACS Professional Reference Book 1997 Amer Chem Society Feigl P B Schueler and F Hillenkamp Int J Mass Spectrom lon Phys 1983 47 15 Mamyrin B A V J Karatajev D V Smikk and V A Zagulin Soviet Phys JETP 1973 37 45 48 Middleditch B Practical Mass Spectrometry Plenum Press New York 1979 Watson J Throck ntroduction to Mass Spectrometry Raven Press New York 1985 Wiley W C and I H McLaren Rev Sci Instrum 1953 26 1150 1157 Zhou J W Ens K Standing and A Verentchikov Rapid Commun Mass Spectrom 1992 6 671 678 Introduction to MALDI Hillenkamp F and M Karas Methods in Enzymol 1990 193 280 295 Karas M D Bachmann U Bahr and F Hillenkamp Int J Mass Spectrom lon Processes 1987 78 53 68 Tanaka K H Waki Y Ido and S Akita Rapid Commun Mass Spectrom 1988 3 151 153 Matrixes Beavis R C B T Chait Anal Chem 1990 60 1836 1840 Beavis R C B T Chait Rapid Commun Mass Spectrom 1989 3 432 435 Beavis R C B T Chait Org Mass Spectrom 1992 27 156 158 Chen C H et al Rapid Commun Mass Spectrom 1994 8 673 677 Juhasz P C E Costello K Biemann J Am Soc Mass Spectrom 1993 4 399 409 Juhasz P C E Costello Rapid Commun Mass Spectrom 1993 7 343 351 Karas M U Bahr K Strupat F Hillenkamp A Tsourbopoulos and
199. User s Guide 2 13 Chapter 2 Installing the Voyager Biospectrometry Workstations Connection on right side panel of mass spectrometer see Figure 2 2 Connection on rear panel of computer see Figure 2 4 Cable CH2 Voyager DE PRO only CH 2 Ch 2 SMA to Ch 2 BNC between blue box and computer Ch 2 BNC to Ch 2 BNC between blue box and mass spectrometer Ground wire to computer chassis ground screw 2 14 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations 2 3 4 Connecting the LSA1000 LeCroy Digitizer If you have an oscilloscope or an internal digitizer on your system disregard this section This section describes the connections for the LSA1000 LeCroy digitizer that has been previously installed by an Applied Biosystems Technical Representative NOTE If your LeCroy digitizer has not been installed by an Applied Biosystems Technical Representative contact Applied Biosystems before installing or connecting it to your system Figure 2 5 shows the connections on the LSA1000 LeCroy digitizer CH2 Voyager DE PRO only Network 10 100 Base T PB100790 Figure 2 5 LeCroy Digitizer Connections Voyager Biospectrometry Workstation User s Guide 2 15 Chapter 2 Installing the Voyager Biospectrometry Workstations Refer to the following table when you connec
200. Voyager Biospectrometry Workstation With Delayed Extraction Technology User Guide Version 5 1 Series Software D Applied KS Biosystems NOTICE Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose charges for supporting non standard computer configurations that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support Information in this document is subject to change without notice and does not represent a commitment by Applied Biosystems Applied Biosystems assumes no responsibility for any errors that may appear in this document This manual is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this manual or the operation and use of the instrument 2000 Applied Biosystems Printed in the United States of America All rights reserved This book or parts thereof may not be reproduced in any form without the written permission of the publisher Applied Biosystems Delayed Extraction SymBiot and the fractal icon are
201. You must calibrate the mass scale for each Grid Voltage you use See Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating for more information Grid Voltage works in conjunction with Accelerating Voltage described in Section 5 4 4 2 Setting Accelerating Voltage to define an adjustable potential gradient or electric field in the ionization region of the ion source This adjustable potential gradient can be fine tuned to obtain optimum performance e In Delayed Extraction mode you must fine tune Grid Voltage in conjunction with Delay Time to optimize resolution See Section 5 4 3 Optimizing Resolution In PSD mode you can use higher Grid Voltage settings than the optimum Reflector mode settings Higher Grid Voltage improves fragment ion resolution and the resolution at the position of the Precursor lon Selector see Figure 8 20 on page 8 74 The standard BIC files provided on your system for PSD mode include Grid Voltage settings that are approximately 5 percent higher than the Grid Voltage settings in the BIC files provided for Reflector mode For more information see Chapter 8 PSD Analysis For more information on Grid Voltage see e Section 5 4 3 5 Optimizing Grid Voltage e Grid Voltage on page 5 17 Voyager Biospectrometry Workstation User s Guide 5 51 Chapter 5 Optimizing Instrument Settings Potential gradient The potential gradient in the ionization region Figure 5
202. a Cyano 4hydrorycinnamic acid Le F Default ExtemalFile Ey EAE El Laser Increment used during auto cntrol only 4 Eu x Mirror Ratio lt 1 0 Vertical Scale Input Bandwidth Laser Increment Max Stitch Mass Default PSD calibration for fragments ni o jm 0 750 0 020 200 200 o 972525 PSD Calibration mee 0 020 200 200 0 729 394 e mi 0 422 0 020 200 200 0 547 045 LEE me mj 0316 0 010 200 200 0 410 284 s Ex PSD Calibration File mj 0 237 0 010 50 25 0 307 713 ESRI High Voltage OFF SONSES Ac siion OFF Control Mo 7 External CAL file for precursor Figure 8 1 PSD Instrument Control Panel with Acquisition Settings Control Page 8 8 2 Inthe Calibration section of the Instrument Settings control page select e The matrix you are using External File then select the ANGIO CAL file you created in Generating an external calibration for the precursor ion on page 8 7 Applied Biosystems Acquiring PSD segments 1 PSD Quick Start In the PSD Acquisition Settings control page type the angiotensin Precursor mass NOTE Precursor mass is used for PSD calibration Type in an accurate value with appropriate precision for example type 1296 68 not 1297 Ensure that PSD calibration is set to Default Select Save Instrument Settings As from the File menu then save the instrument settings file with a new name NOTE The instrument settings files provid
203. a Explorer software For information see the Data Explorer Software User s Guide Section 5 9 Truncating a Spectrum Voyager Biospectrometry Workstation User s Guide 5 91 Chapter 5 Optimizing Instrument Settings 5 5 Converting Version 4 Methods and Search Pattern Files To use method MNU files and search pattern SP files created in version 4 x of the Voyager software convert them for use with the current version 5 x software using the Voyager 4 x File Converter This utility e Converts MNU files to instrument settings BIC files e Inserts a Units 1 line in search pattern SP files to indicate that units are Steps NOTE The convert function does not change values in the SP file from step values to micron values It inserts a line in the SP file that identifies the values as step values When the system runs a search pattern file it checks for the presence of the Units 1 line e If the line is present the software converts the step values to micron values and displays position information in microns e Ifthe line is not present the software assumes the values are micron values and uses the values as is NOTE If you use a search pattern file that contains step values but has not been converted that is it does not include the Units 1 line the software assumes the values in the file are microns and a smaller area than expected in the sample position will be analyzed 1 step 3 175
204. able unless you select Use Automated Laser Intensity Adjustment and Use Automated Sample Positioning For more information on the Prescan process see Prescan mode on page 6 58 7 If you enable Use Automated Laser Intensity Adjustment set adjustment criteria as described in Setting spectrum acceptance and laser adjustment criteria on page 6 42 NOTE If you do not specify laser adjustment criteria the laser is not adjusted The mid range laser setting between the minimum and maximum specified in step 5 is used Voyager Biospectrometry Workstation User s Guide 6 39 Chapter 6 Acquiring Spectra from the Instrument Control Panel Spectrum 8 Specify Spectrum Accumulation parameters accumulation and e Acquire X Spectra Specifies the number of saving spectra to acquire The software compares the value you enter with the possible number of positions in the selected search pattern and displays a message if you enter a value greater than the number of positions e Under Conditions Determines if spectra are saved individually or accumulated and if spectra are evaluated against Acceptance Criteria NOTE If you select Save All Spectra or Save All Spectra that Pass Acceptance Criteria Save Conditions which save individual multiple spectra in one data file the recommended maximum number of spectra to acquire is 500 Acquiring more than 500 individual spectra in one file generates a very lar
205. able voltage grid located above the sample plate Grid Voltage is a percentage of Accelerating Voltage For a list of Delay Time and Grid Voltage pairs see Appendix E Grid Voltage and Delay Time Settings Optimum resolution for a given mass range is obtained by finding the ideal Delay Time and Grid Voltage combination for the mass range NOTE Although Guide Wire Voltage does not affect velocity focusing of ions it has a strong impact on resolution in Reflector mode and sensitivity in all modes See Section 5 4 3 3 Optimizing Guide Wire Voltage Relationship Delay Time and Grid Voltage are interactive parameters For between DE each Delay Time there is an optimum Grid Voltage and for parameters each Grid Voltage there is an optimum Delay Time Linear mode For each optimum Delay Time and Grid Voltage combination there is a sharp optimum in Linear mode performance Over a wider mass range performance is less optimized Reflector mode For each optimum Delay Time and Grid Voltage combination the mass range across which resolution is optimized is wider in Reflector mode Voyager Biospectrometry Workstation User s Guide 5 73 Chapter 5 Optimizing Instrument Settings Effects of mass Note the following and matrix Ata fixed Delay Time higher masses require a lower Grid Voltage See Appendix E Grid Voltage and Delay Time Settings for a graphic representation of the relationship between mass Grid V
206. aces 4 14 The Spectrum window can contain two types of traces see Figure 4 5 on page 4 15 Live Current A live real time trace of data The display updates as you view or acquire data When acquisition is complete the trace name changes from Live to Current NOTE On systems with Signatec LeCroy or Acqiris digitizers the Live Trace dynamically updates as data is acquired On systems with Tektronix oscilloscopes a complete Current trace is displayed only after acquisition is complete Accumulated Spectrum A trace that is made up of one or more spectra that you have manually or automatically accepted Adding traces The Spectrum window can display a total of four traces at one time To add a new trace to a window Applied Biosystems 1 Activate click on the Spectrum window 2 Click in the toolbar NOTE You can add traces by selecting Add Remove Traces from the Display menu If the trace position has not previously been used for the current acquisition the software adds a Not Used trace to the window If the trace position has previously been used for the current acquisition the software adds a trace that contains the data originally displayed For example if you add a second trace then remove the trace then add the second trace again the original trace is displayed again instead of a Not Used trace Using the Spectrum Window Hint Resize the window to view all added traces
207. acy e Section 3 4 2 Using the Mass Accuracy Optimization Option e Section 3 4 4 Loading Sample Plates Running OptiPlate to Optimize Mass Accuracy 2 8 2 Requirements Standard and To run the OptiPlate software you need matrix G cyano 4 hydroxycinnamic acid CHCA matrix e Standard mixture containing the components in Table 2 2 Table 2 2 Standard Requirements Final concentration after mixed Component h with matrix Angiotensin 2 0 pmol ul ACTH 1 17 clip 2 0 pmol l ACTH 18 39 clip 1 5 pmol ul ACTH 7 88 clip 3 0 pmol l Insulin bovine 3 5 pmol ul WARNING CHEMICAL HAZARD Alpha cyano 4 hydroxycinnamic acid CHCA matrix may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Angiotensin ACTH and Insulin may cause an allergic skin and respiratory reaction Exposure may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Voyager Biospectrometry Workstation User s Guide 2 55 Chapter 2 2 56 High voltage warmup Applied Biosystems Installing the Voyager Biospectrometry Workstations NOTE You can use Calibration Mixture 2 from the Sequazyme Peptide Mass Standards Kit See A
208. adjusting laser 6 5 Acquiring data Sequence Control Panel see also Acquiring data Instrument Control Panel see also Sequence Control Panel list of data files acquired 7 14 multiple data files with different instrument settings 1 44 4 32 pausing 7 28 process that occurs during 7 28 sample plate aligning 7 24 Sequence parameters 7 14 starting 7 25 status 7 20 7 29 stopping 7 28 Index Acquisition Adding traces 4 14 batch 6 3 7 2 Adduct peaks 3 24 checking resolution in Continuous Adobe Acrobat Reader Extraction mode H 17 Current Spectrum trace displayed 6 16 determining laser threshold in Continuous Extraction installing from Voyager CD 2 31 opening PDF Voyager files 2 31 Advanced parameters PSD PSD Mirror to Accelerating Voltage mode H 12 Ratio 5 32 Live Spectrum trace displayed 6 16 Alpha cyano 4 hydroxycinnamic acid mass range in Spectrum window chemical structure and molecular does not update with every weight C 7 laser shot 6 16 9 21 concentration 3 3 3 9 3 11 multiple spectra in one data crystals 3 3 3 9 3 11 3 45 file 5 35 5 39 initial velocity setting 5 22 multiple spectra recommended laser intensity relative 5 67 maximum in one data mass spectrum C 2 file 5 35 organic concentration 3 8 process that occurs in Automatic organic concentration dried droplet Control mode 6 58 application 3 10 process that occurs in Manual preparing dried droplet Control mode 6 16 application 3 3 3 5 3 9 PSD aut
209. age grid in the ion source which affects calibration An accurate well depth increases the accuracy of the default calibration external calibration and mass optimization with OptiPlate Corresponds to WellDepth parameter in the PLT file see Table 3 13 PLT File Parameters on page 3 69 continued Voyager Biospectrometry Workstation User s Guide 3 81 Chapter 3 Preparing Samples Table 3 14 Create PLT File Parameters Continued Parameter Description Position Shape Ellipse or Rectangle Corresponds to WellShape parameter in the PLT file see Table 3 13 PLT File Parameters on page 3 69 Position Numbering If you select Numeric positions are numbered sequentially from 1 to 32 766 If you select Alphanumeric rows are assigned letters and positions are numbered sequentially within the row for example A1 through An for the first row B1 through Bn for the second row and so on If the number of rows exceeds 26 double letters are used for example AA1 through AAn Corresponds to PositionName parameter in the PLT file see Table 3 13 PLT File Parameters on page 3 69 First position upper left X and Y and Last position lower right X and Y Absolute coordinates of starting and ending position centers relative to the lower left corner of the sample plate The software extrapolates to determine coordinates of all other positions To determine
210. ake sure the gas supply is regulated to 2 to 5 psi Voyager Biospectrometry Workstation User s Guide 8 35 Chapter 8 PSD Analysis Turning off collision gas Adjusting collision gas Adjusting Guide Wire Voltage To turn off the collision gas 1 Turn the top 3 way valve on the CID box to the Purge Cell position 2 Wait approximately 20 seconds for the gas to evacuate 3 Turn the top 3 way valve on the CID box to the Off position To optimize fragmentation adjust the flow of the collision gas Turn the metering valve on the CID box until you observe the desired fragmentation If gas pressure is too high signal is suppressed If gas pressure is too low you will not obtain the desired fragmentation When using the CID option you may observe enhanced performance by increasing the Guide Wire Voltage above the setting used for PSD analysis without CID NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later 8 36 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Manual Control Mode 8 4 Acquiring PSD Data with Standard BIC Files in Manual Control Mode Steps to acquire The steps to acquire PSD data in Manual Control mode are PSD data Step Determine the precursor ion mass 8 38 Determine the number of segments to acquire for a complete 8 40 composite spectrum Set the PSD Acquisition parameters for Manua
211. alculate the Mirror Ratio settings needed for each segment To do so it uses a Mirror Ratio setting of 1 000 and a Decrement Ratio parameter that you enter The correlation between the Mirror Ratio and the Decrement Ratio is the inverse percentage That is if you want to obtain 20 percent segments set the Decrement Ratio to 0 8000 If you want to obtain 80 percent segments set the Decrement Ratio to 0 2000 The default Mirror Ratio is 1 0000 The default Decrement Ratio is 0 7500 Therefore the default Mirror Ratios yield the following 25 percent segments 1 000 0 237 0 750 0 178 0 563 0 133 0 422 0 100 0 316 0 075 Voyager Biospectrometry Workstation User s Guide 8 41 Chapter 8 PSD Analysis Size of segments Default number of segments Acquiring only select segments 8 42 Applied Biosystems Increase the Decrement Ratio to collect smaller segments Decrease the Decrement Ratio to collect larger segments You can acquire segments of different sizes by varying the Mirror Ratio setting For example you can set the first Mirror Ratio to collect a 20 percent segment then set the next Mirror Ratio to collect a 10 percent segment If you change the default Decrement Ratio fill down subsequent existing rows and the Mirror Ratio values are automatically recalculated Fill down is described in Using the Fill Down function on page 8 50 The standard instrument settings BIC file provided with the
212. alibration 7 10 side panel Voyager DE and internal calibration 6 26 Voyager DE PRO 2 9 maximizing 6 26 obtaining maximum 6 6 6 26 troubleshooting 3 38 9 11 9 12 troubleshooting 9 23 Mass spectrometry overview 1 7 9 17 H 23 H 24 Mass standards kit B 6 Mass accuracy optimization Mass to time conversion F 4 see also OptiPlate software Mass to charge ratio 1 7 benefits 3 52 Matrix enabling 3 57 3 60 3 HPA 3 3 3 15 C 10 how it is applied 3 52 see also Matrix Initial Velocity if no optimization found 3 53 alpha cyano 4 hydroxycinnamic if positions not optimized 3 53 acid 3 3 3 9 3 11 C 7 number and location of and HPLC grade water 3 5 3 13 standards 3 39 3 15 using a different PLT file 3 53 applications C 1 Voyager Biospectrometry Workstation User s Guide Index 23 I N D E X Index Matrix continued chemical structures C 1 crystallization examples 3 45 crystallization troubleshooting 9 9 DHB 3 3 3 16 3 17 3 19 C 7 C 8 DHBs 3 3 3 18 C 9 dithranol 3 3 3 19 C 11 HABA C 10 IAA 3 3 3 19 C 12 nicotinic acid C 12 ordering information B 6 picolinic acid C 12 premixing with sample dried droplet application 3 33 preparing 3 4 proteins 3 3 reference file modifying 5 23 reference information C 1 relative laser intensities 5 67 role of 1 8 salt contamination 3 5 3 13 3 15 selecting 3 3 C 1 selecting type in calibration 5 20 sinapinic acid 3 3 3 7 C 6 solutions C 6 stability 3 4 storage
213. align the boundaries of the sample positions with the laser position mark on your monitor e Transparency mylar overhead or acetate e Marking pen NOTE View the video monitor from a consistent angle when determining laser positions Voyager Biospectrometry Workstation User s Guide 2 49 Chapter 2 Installing the Voyager Biospectrometry Workstations Corner positions Different sample position numbers are associated with in PLT files different PLT files To determine which numbers correspond to the four corners of each PLT file see Table 2 1 Table 2 1 Four Corner Positions on Sample Plates Four Corner Position PLT file N mbers 64 well disposable plate PLT 82 12 19 89 100 well plate PLT 91 11 19 99 384 well plate PLT P1 C1 C21 P21 400 well plate PLT T1 A1 A20 T20 96 well x 2 plate PLT H1_b A1_a A12_a H12_b Using the control To align a sample plate you must use the control stick For stick details on using the control stick see Appendix Using the Oscilloscope and Control Stick Before aligning Before aligning the sample plate 1 Spotthe sample plate with matrix as described in What you need on page 2 49 2 Load the sample plate into the system and select the Plate ID that corresponds to the plate e From the Sample Plate menu select Select Sample Plate to display the Select Sample Plate Type dialog box e Select a Plate ID
214. an 150 MB for data file storage a message is displayed during the run when free disk space is no longer available Acquiring To acquire sequence data sequence data 1 Perform the steps in Section 7 3 Before Creating a Sequence 2 Create a sequence as described in Section 7 4 Creating a Sequence or open an existing sequence Voyager Biospectrometry Workstation User s Guide 7 25 Chapter 7 Acquiring Spectra from the Sequence Control Panel NOTE You can have more than one sequence open at the same time However only one sequence can be active For example you cannot edit one sequence while another sequence is running 3 Select the rows to run by clicking the check box in the Run column Only rows with a check in the Run column are acquired when the sequence runs Hint You can select Run Column State from the Edit menu then select Set All or Clear All to quickly check or uncheck all rows 4 To start the sequence click in the toolbar or select Start Sequence from the Control menu 7 6 2 What the System Checks When You Start a Sequence What the system When you start a sequence the software examines the checks contents of all selected rows checked in the Run column to make sure specified files exist and entries are valid If the system if the system finds invalid parameters it finds invalid Unchecks all rows containing invalid parameters parameters ee e Changes the color of the in
215. and C5 6131 and later PRO Ground stud On Off _ Video alee ower receptacle Power P j receptacle PB100501 Figure 2 2 Right Side Panel of Voyager DE and Voyager DE PRO Mass Spectrometer Voyager Biospectrometry Workstation User s Guide 2 9 Chapter 2 Installing the Voyager Biospectrometry Workstations Computer Depending on your digitizer option the computer has one of the following installed when you receive it e Signatec 500 MHz digitizer board e Dedicated ethernet for LeCroy LSA 1000 digitizer e Acqiris 500 MHz and 2 GHz digitizer boards e GPIB board for oscilloscope option Figure 2 3 shows the rear panel of the computer Figure 2 4 shows the boards that can be installed in your computer depending on the digitizer option selected for your system NOTE The computer layout may change without notice Boards may be located in slots that differ from those shown in Figure 2 3 and Figure 2 4 The braided ground cable connection may be located in a different position 7 O D Printer 1 0 Power LPT1 or Parallel Joy stick VAC gauge control serial2 COM2 Step motor control Network 2 Audio input output not used Serial COM1 not used Network 1 PB100776 Digitizer options see Figure 2 4 Network 1 Connection for LAN
216. and saving spectra e Search patterns e Process that occurs during acquisition in Automatic Control mode e Process that occurs when accumulating spectra from multiple search pattern positions PSD analysis in For information on performing PSD analysis in automatic Automatic mode mode see Section 8 5 Acquiring PSD Data with Standard BIC Files in Automatic Control Mode Voyager Biospectrometry Workstation User s Guide 6 35 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 6 1 Before Acquiring in Automatic Control Mode Background Before acquiring spectra in Automatic Control mode become information familiar with the information in e Section 6 1 Before You Begin e Section 6 2 Acquiring in Manual Mode from the Instrument Control Panel Aligning the Sample plate alignment is necessary for automated sample sample plate positioning in Automatic Control mode if the laser is not striking the center position of the sample position Sample plate alignment may not be necessary on your system particularly if you use the SPIRAL SP search pattern file provided with the system See Section 2 7 Aligning the Sample Plate to determine if sample plate alignment is necessary Using Mass In a typical analysis run that requires optimum mass accuracy Accuracy you include samples interspersed with many calibration Optimization standards and externally calibrate the samples using the closest calibration standard
217. arch Pattern Editor opens Figure 6 18 displaying e Pathname of the PLT file selected in the Instrument Control Panel in the title bar e Sample position area size and shape defined by the PLT file e Coordinates of the cursor position 6 50 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel C VOYAGER 100 well plate BE f Ea Title bar Menu button u displays PLT name Sample position area size and shape defined by PLT Cursor coordinates Figure 6 18 Search Pattern Editor 3 If you are creating a new SP file skip to step 4 If you are editing an SP file click Menu select Open then select an SP file NOTE Instead of clicking Menu you can right click to display the list of available commands 4 Create or edit the search pattern in any of the following Ways e Use the Search Pattern Generator to automatically generate a search pattern See Using the Search Pattern Generator on page 6 52 e Draw a new search pattern using the mouse See Drawing a search pattern on page 6 56 e Edit or add spots by setting x y coordinates See Setting x y coordinates on page 6 57 5 To add and store descriptive comments in the new SP file click Menu then select Properties Type text that describes or identifies this particular search pattern Voyager Biospectrometry Workstation User s Guide 6 51 Chapter 6 Acquiring Spectra fro
218. ards Kit P2 3143 00 e Voyager lgG1 Mass Standard GEN602151 e Sequazyme BSA Test Standard 2 2158 00 Sequazyme Pinpoint SNP Assay Kit Applied 4315924 Biosystems Adrenocorticotropic hormone ACTH 7 38 Sigma A 1527 Adrenocorticotropic hormone ACTH 18 39 Sigma A 0673 Angiotensin human Sigma A9650 Bradykinin Sigma B3259 Insulin Sigma 1 5500 Myoglobin horse heart Sigma M 1882 a cyano 4 hydroxycinnamic acid CHCA Sigma C 2020 Sinapinic acid 3 5 dimethoxy 4 Aldrich D13 460 0 hydroxycinnamic acid B 6 Applied Biosystems Spare Parts Sample plates The following sample plates are available from Applied Biosystems Description Part Number Welled Sample Plates Gold 100 well V700401 Gold 100 well no pin for Voyager Workstations V700208 manufactured in 1995 or earlier Flat Sample Plates Laser Etched Stainless steel 100 position indicated by numbers only V700664 Stainless steel 100 position indicated by numbers only V700665 no pin for Voyager Workstations manufactured in 1995 or earlier Stainless steel 100 position indicated by numbers and V700666 circles Stainless steel 100 position indicated by numbers and V700667 circles no pin for Voyager Workstations manufactured in 1995 or earlier Special Sample Plates Stainless steel polished blank surface V700668 Stainless steel polished blank surface no pin for Voyager V700669 Works
219. arge between two points Usually occurs when a high potential difference exists between two points Voyager Biospectrometry Workstation User s Guide BA1 and BA2 Bayard Alpert vacuum gauges Pressure gauges that operate below 1 millitorr by measuring a positive ion current produced by electrons from a hot filament Voyager DE systems include BA1 only Beam Guide Wire See Guide Wire Voltage Centroid Percentage of the peak height examined during peak identification For example with a Centroid of 10 the software examines the top 10 percent of the signal CID collision induced dissociation A technology that enhances fragmentation for PSD analysis As ions leave the source they pass through a collision cell interact with collision gas molecules and energy is transferred to the ions This transfer of energy enhances fragmentation Continuous Extraction Formation of ions in a strong electrical field with immediate acceleration Used for diagnostic purposes Daughter ion See Product ion Delayed Extraction Formation of ions in a weak electrical field with subsequent acceleration by application of a high voltage pulse after a predetermined time delay Glossary 1 lt II VUOTO G L O S S A R Y Glossary Delay Time used in Delayed Extraction mode Time in nanoseconds after the laser ionizes the sample at which full Accelerating Voltage is applied creating t
220. arious formats currently available See B 3 Spare Parts Manual control using control stick or mouse Sequence Control software for automated analysis Dimensions 34 inches 87 cm deep 94 inches 239 cm wide 46 inches 117 cm high Table A 9 Voyager DE STR Miscellaneous Specifications Condition Specification Environmental Operating temperature 10 25 C e Relative humidity 10 80 non condensing Altitude lt 2 000 m 6 500 ft above sea level e Installation category overvoltage category ll classified as portable equipment e For indoor use only Pollution degree rating 2 and may be installed in an environment that has nonconductive pollutants only Computer Minimum configuration Pentium III 500 MHz with 9 GB hard disk and 128 MB RAM random access memory e 17 inch color monitor A 10 Applied Biosystems Digitizer Specifications A 4 Digitizer Specifications This section includes e Digitizer specifications e Laser firing rates Digitizer Table A 10 lists specifications for the digitizer options available specifications on the Voyager system Table A 10 Digitizer Specifications X Analog Available Input Bandwith Model Samples Second Bandwidth Bin Sizes ns Software Selectable Acgiris DP 105 500 Ms S 150 MHz 2 4 10 20 None megasample sec Acgiris DP211 2 Gs S 500 MHz 0 5 1 2 4
221. art Acquisition from the acquisition Acquisition menu or click LA CAUTION If you do not save data before starting a new acquisition the data in the Current trace is lost Acquisition starts and continues until the number of Shots Spectrum specified in Spectrum Acquisition on the Instrument Settings control page is collected or until you select Stop Acquisition from the Acquisition menu or click again During During acquisition acquisition The Live Current Spectrum trace in the Spectrum window updates to display the spectrum that results from each laser shot with the following exceptions e If you are acquiring a large number of data points the Spectrum window is not updated with every laser shot The exact update rate depends on the Mass Range setting and the Bin Size setting you are using e If your system includes an oscilloscope the Current Spectrum does not display a trace until acquisition is complete The spectra that result from each laser shot are displayed on the oscilloscope screen e The system averages all spectra acquired after you start acquisition NOTE If the system is set to acquire in single shot mode spectra are not averaged See page 2 44 When acauisition is complete the software displays the averaged spectrum in a Current trace in the Spectrum window 6 16 Applied Biosystems Acquiring in Manual Mode from the Instrument Control Panel Evaluating data As data is acquired
222. arts use iaeaea nent B 6 Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose charges for supporting non standard computer configurations that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support Voyager Biospectrometry Workstation User s Guide B 1 Appendix B Warranty Service Information B 1 Limited Product Warranty Limited warranty Applied Biosystems warrants that all standard components of B 2 Applied Biosystems its Voyager Biospectrometry Workstations the Product purchased new will be free of defects in materials and workmanship for a period of one 1 year Applied Biosystems will repair or replace at its discretion all defective components during this warranty period After this warranty period repairs and replacement components may be purchased from Applied Biosystems at its published rates Applied Biosystems also provides service agreements for post warranty coverage Applied Biosystems reserves the right to use new repaired or refurbished instruments or components for warranty and post warranty replacements Repair or replacement of products or comp
223. asing the Mirror Ratio This adjusts the field strength of the mirror and allows lower energy ions to travel farther into the reflector portion of the mass spectrometer and be better focused Figure 8 5 8 22 Applied Biosystems Overview of PSD Analysis Reflector LL LE LL E 2e a Mirror Ratio 0 7 BRO MH MW 1 000 not focused a i AH MW 700 correctly focused I Eoo BH MW 300 poorly focused LLLLLLLLLE F from mm Reflector LLLLLELE LE l Mirror Ratio 0 3 MH MW 1 000 not focused source p AH MW 700 not focused He BH MW 300 correctly focused Figure 8 5 Effect of Changing Mirror Ratio Immonium ions A Mirror Ratio setting of 1 0000 correctly focuses the original ion Values of 0 7 and 0 3 correctly focus the lower energy fragments Note that the best focusing conditions occur when the fragment ion mass is equal to the Mirror Ratio multiplied by the precursor ion mass Peptide PSD spectra usually include some immonium and other low mass fragment ions that are useful in determining peptide composition For a list of immonium ions for the 20 standard amino acids and for selected modified amino acids see Falick A M W M Hines K F Medzihradszky M A Baldwin and B W Gibson Low Mass lons Produced from Peptides by High Energy Collision Induced Dissociation in Tandem Mass Spectrometry J Am Soc Mass Spectrom 1993
224. ass and PSD calibration for fragment spectra Displaying the To display PSD Acquisition control page PSD Acquisition 4 Open the Angiotensin PSD BIC file provided with the control page software This is a PSD mode BIC file The PSD Acquisition Settings control page is automatically displayed if you open a BIC file that is set to PSD mode To manually display the PSD Acquisition Settings control page change the operation mode to PSD by e Clicking Mode Digitizer in the Instrument Settings control page e Clicking the Instrument Mode tab e Selecting PSD NOTE Low Mass Gate is automatically disabled if PSD is specified for Instrument Mode The Precursor lon Selector provides the selectivity needed to screen out unwanted masses Voyager Biospectrometry Workstation User s Guide 8 43 Chapter 8 PSD Analysis 2 Ifthe PSD Acquisition Settings control page Figure 8 12 is not displayed select PSD Acquisition from the View menu Mirror reno Guide were euto fi Precursor moss 1 3660 F Precursor on pelactor Loser hcremeni used during auto contol only 4 l x Decemem ratio 175 Gade wre tracks miror rato r PSD Calteaton F Detour C Eaoma PSD Celbenton Fie mj Figure 8 12 PSD Acquisition Settings Control Page Manual Mode NOTE The Precursor lon Selector in PSD mode is the same parameter as the Timed lon Selector in Reflector mode If you change the Precursor mass used by the Precursor
225. at you have h already determined an accurate mass for the precursor ion using reflector mode high resolution analysis with internal or external calibration Generate a normal single point external Normal external calibration 8 7 calibration using the spectrum acquired in for precursor ion mass accuracy step 2 You use this single point external calibration in PSD analysis to obtain maximum Precursor CAL mass accuracy for the unknown precursor ion Set PSD Acquisition Instrument Settings Angiotensin _PSD BIC with 8 7 parameters Open Angiotensin _PSD BIC Precursor CAL provided and Angio_PSD CAL In Instrument Settings Type the precursor mass and select Precursor CAL generated in step 3 for precursor In PSD Acquisition Settings Select Angio_PSD CAL generated in step 1 for fragments Acquire unknown PSD segments precursor Unknown PSD Composite spectrum 8 14 and fragment spectra with Angio_PSD with optimum fragment ion calibration mass accuracy Voyager Biospectrometry Workstation User s Guide 8 5 Chapter 8 PSD Analysis Table 8 2 Steps to Perform PSD Analysis of an Unknown Continued See Step Result page 6 Optionally confirm or investigate fragment ion Unknown PSD Composite spectrum 8 19 identity using the lon Fragment calculator or with fragment ion labels the Peptide Fragmentation macro a GC DO 2 amp 8 6 Applied Biosystems PSD Quick Start
226. ata in the Current trace is lost CAUTION If you change the laser setting after acquisition is complete but before you save the data the last laser setting is saved with the data not the laser setting used to acquire the data Saving data To save the data when you have more than one trace displayed in the Spectrum window 1 Select click on the Current trace 2 Select Save Spectrum from the Acquisition menu The data is saved using the file name specified in the Data Storage control page Information about the data file is displayed in the Data Storage tab in the Output window Figure 6 5 at the bottom of the Instrument Control Panel The spectrum was written to the file C VoyagernDataltest0001 dat Automated Acquisition Figure 6 5 Data Storage Tab in Output Window 6 18 Applied Biosystems Saving the instrument settings BIC file Acquiring in Manual Mode from the Instrument Control Panel When acauisition is complete save the instrument settings BIC file used to acquire the data if you want to store the optimized laser intensity used during this acquisition For information on saving a BIC file see Section 5 1 5 Saving and Printing Instrument Settings NOTE You can export instrument settings from a DAT file that has been saved in Data Explorer For more information see the Data Explorer User s Guide 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Accumulating
227. ated by a previous line in the Sequence The way the CAL file is used depends on the calibration type e External Constants are applied e Internal lf a CAL file is specified constants are applied e Internal update lf the specified CAL file exists constants are applied before calibration then the CAL file is updated with new constants after calibration If the specified CAL file does not exist no constants are applied before calibration and the CAL file is created containing new constants after calibration NOTE If the specified CAL file for Internal Update does not exist the software creates the CAL file during processing in the path you specify If you do not specify a path the software creates the CAL file in the C VOYAGER directory NOTE If the BIC file specifies a CAL file the CAL file in the BIC file is not updated unless it is the same CAL file specified in the Sequence Control Panel For information on how CAL files are used in the Sequence Control Panel see Section 7 2 Understanding Settings Macros and Calibration continued Voyager Biospectrometry Workstation User s Guide 7 19 Chapter 7 Acquiring Spectra from the Sequence Control Panel Table 7 1 Run List Parameters Continued Field Description Post Macro Macro to execute after calibration Select a macro by doing either of the following e Click the field and select the macro name from the list All ma
228. ater Let stand in the refrigerator overnight Filter then wash the precipitate with cold water This procedure is not optimized for yield Crystals Rounded see Figure 3 1 on page 3 45 Stability Prepare weekly 1 Other special sample preparation techniques are described in Shevchenko A M Wilm O Vorm M Mann Anal Chem 1996 68 850 858 3 12 Applied Biosystems THAP Table 3 4 THAP Matrix Information Preparing Samples Use THAP for oligonucleotides lt 3 500 Da acidic carbohydrates acidic glycopeptides acid sensitive compounds THAP provides a more even response than 3 HPA WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves THAP may cause eye skin and respiratory tract irritation Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Matrix concentration e Oligonucleotides 10 mg ml e Acidic carbohydrates 2 mg ml Additive concentration 50 mg ml diammonium citrate in deionized water Final sample concentration 1 10 pmol ul Solvents 50 percent acetonitrile and deionized water NOTE HPLC grade water may vary in salt concentration Do not use for oligonucleotide analysis c
229. atic Mode from the Instrument Control Panel The system sets the laser to the maximum setting specified in the Automatic Control dialog box and acquires a spectrum see Shots Spectrum in Prescan mode on page 6 58 at the first point specified in the search pattern If the signal intensity is Too low lower than the minimum signal intensity set in the Spectrum Acceptance Criteria dialog box with the laser at maximum The system moves to the next position specified in the search pattern and acquires again Too high higher than the maximum signal intensity set in the Spectrum Acceptance Criteria dialog box with the laser at maximum The system continues with step 2 Within range The system begins acquiring in Acquisition mode See Acquisition mode on page 6 61 NOTE If Use Automated Sample Positioning is disabled the system always uses the center of the sample position NOTE If no minimum or maximum signal intensity criteria is specified the laser is not adjusted The mid range laser setting specified in the Automatic Control dialog box is used Voyager Biospectrometry Workstation User s Guide 6 59 Chapter 6 Acquiring Spectra from the Instrument Control Panel 2 The system sets the laser to the minimum setting specified in the Automatic Control dialog box and acquires a spectrum see Shots Spectrum in Prescan mode on page 6 58 If the signal intensity is e Too low with th
230. ation User s Guide A 3 Appendix A Specifications Table A 3 Voyager DE Miscellaneous Specifications Condition Specification Environmental Operating temperature 20 25 C e Relative humidity 20 80 non condensing Altitude lt 2 000 m 6 500 ft above sea level e Installation category overvoltage category ll classified as portable equipment e For indoor use only e Pollution degree rating 2 and may be installed in an environment that has nonconductive pollutants only Computer Minimum configuration Pentium III 500 MHz with 9 GB hard disk and 128 MB RAM random access memory e 17 inch high resolution color monitor A 4 Applied Biosystems Voyager DE PRO Specifications A 2 Voyager DE PRO Specifications This section includes the following specifications for the Voyager DE PRO Workstation Performance e Mass Spectrometer e Miscellaneous Table A 4 Voyager DE PRO Performance Specifications for Installation Condition Specification Resolution FWHM Full Width at Half Maximum in Delayed Extraction mode Linear mode 21 000 for Myoglobin 4 pmol ul 22 500 for Angiotensin 1 3 pmol l Reflector mode 210 000 for bovine insulin 3 5 pmol ul 21 200 for E coli Thioredoxin 2 75 pmol ul 27 000 for Angiotensin 2 0 pmol ul 27 000 for ACTH Clips e ACTH 1 17 2 0 pmol ul ACTH 18 39 1 5 pmol ul e ACTH 7 38 3 0 pmol ul M H i
231. aximum the system decreases the laser intensity If signal intensity is not below the maximum when the laser is at the lowest setting specified in Automatic Control the system moves to the next search pattern position Signal to Noise Used for spectrum acceptance and laser adjustment Enables or disables the signal to noise filtering Type the signal to noise ratio threshold Spectrum is rejected if the base peak has a ratio below this value The base peak in the specified Mass Range is used to calculate signal The noise is calculated over the Baseline Range specified see below continued Voyager Biospectrometry Workstation User s Guide 6 43 Chapter 6 Acquiring Spectra from the Instrument Control Panel Table 6 3 Spectrum Acceptance Criteria Parameters Continued Parameter Description Signal to Noise Used for spectrum acceptance and laser adjustment Also used to determine laser adjustment When the system adjusts the laser it checks that the signal to noise ratio is above the value entered If it is not the system increases the laser intensity If signal to noise ratio is not above the specified value when the laser is at the highest continued ne hi setting specified in Automatic Control the system moves to the next search pattern position Resolution Enables or disables the resolution filtering Type the resolution threshold Spectrum is rejected if th
232. ay be wider Loading Modifying and Saving Instrument Settings Table 5 2 Reflector Mode BIC Files Mass Rangein BIC File Sample Test BIC Optimized at Da Angiotensin_Reflector BIC Low mass Calibration 500 2 000 peptide mix1 and Resolution angiotensin l ACTH_Reflector BIC Peptide mix2 Resolution 1 000 4 000 across mass optimized at range 2 500 Insulin_Reflector BIC Peptide mix2 Resolution 5 000 7 000 insulin Peptide Sensitivity_Reflector BIC Neurotensin Sensitivity 1 000 2 000 in dilute mix1 Thioredoxin_Reflector BIC Low mass Resolution 10 000 protein mix3 thioredoxin 15 000 Peptide Reflector Negative BIC Low mass Negative ion 500 2 000 peptide mix1 mode TIS_Test BIC Substance P Timed lon 1 300 1 400 mix Selector Mass Range specified for acquisition may be wider Voyager Biospectrometry Workstation User s Guide 5 5 Chapter 5 Optimizing Instrument Settings Table 5 3 PSD Mode BIC Files Mass Range in BIC BIC File Sample Test Optimized at Da PSD_Precursor BIC Angiotensin Mirror ratio 1 for 1 000 1 400 Precursor ion in Reflector mode Angiotensin_PSD BIC Angiotensin Mirror ratio PSD ions for varies for PSD precursor analysis mass 1296 69 Angiotensin_PSD_Auto BIC Angiotensin Mirror ratio PSD ions for varies for PSD precursor analysis mass 1296 69 Substance_P_PSD BIC Substance P
233. ayed on gauge controller BA1 or BA2 shut down due to sudden surge of high pressure for example when a wet sample plate is inserted Press the EMIS button on Pressure Gauge Control Panel to turn off Press again to turn on Call Applied Biosystems Technical Support if error occurs again E03 error message displayed on gauge controller E05 error message displayed on gauge controller lon gauge connection broken or disconnected Call Applied Biosystems Technical Support E09 error message displayed on gauge controller for BA1 or BA2 BA1 or BA2 gauges shut down due to high pressure May be caused by e Sudden increase in pressure when sample plate inserted Vacuum leak Press the EMIS button on Pressure Gauge Control Panel to turn off Press again to turn on Call Applied Biosystems Technical Support if error occurs again E08 error message displayed on gauge controller Vacuum gauge board failed Power down the mass spectrometer and then power up Call Applied Biosystems Technical Support if error occurs again Voyager Biospectrometry Workstation User s Guide 9 27 Chapter 9 Maintenance and Troubleshooting Table 9 7 External Laser Troubleshooting Symptom Possible Cause Action Laser does not fire when you start acquisition External laser not set after switching from internal to external laser Reset See Section 2 9 Resett
234. be exposed to contaminants Voyager Biospectrometry Workstation User s Guide 3 49 Chapter 3 Preparing Samples 3 4 Loading Sample Plates in the Mass Spectrometer This section describes e Assigning Plate IDs e Using the Mass Accuracy Optimization option Ejecting the sample holder e Loading sample plates NOTE If you load the sample plate into the Voyager Biospectrometry Workstation before the plate is dry the pressure in the sample chamber rises and a TC2 pressure too high error code may be displayed in the Instrument Control Panel Wait a few minutes for the chamber to reach pressure 3 3 4 1 Assigning Plate IDs You assign a unique Plate ID to each physical plate you use When you load a plate you select the Plate ID The following information you previously assigned is automatically loaded e PLT file Alignment information if the plate has been aligned e Mass accuracy optimization information if the plate has been optimized To assign a Plate ID select Select Sample Plate from the Sample Plate menu and enter the information described in Table 3 10 in the Select Sample Plate dialog box NOTE You can also assign a Plate ID when you load a sample plate 3 50 Applied Biosystems Loading Sample Plates in the Mass Spectrometer Table 3 10 Sample Plate Information Parameter Description Plate ID Unique identifier up to 32 alphanumeric characters that you assi
235. be connected to another instrument s signal ground reference This is not a protected ground terminal This symbol indicates that this is a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument OO A terminal marked with this symbol either receives or delivers alternating current or voltage A terminal marked with this symbol can receive or supply an alternating and a direct current or voltage I 4 This symbol appears next to the values of the fuses required by the system Voyager Biospectrometry Workstation User s Guide xV Safety and Compliance Information WARNING This symbol indicates the presence of high voltage and warns the user to proceed with caution WARNING This symbol alerts you to consult the manual for further information and to proceed with caution gt gt Non electrical The following is an illustrated glossary of non electrical safety Symbols alert symbols that may be displayed on your instrument WARNING This symbol illustrates a heater hazard Proceed with caution when working around these areas to avoid being burned by hot components This symbol indicates that a laser is present inside the instrument b b xvi Applied Biosystems Symboles des alertes de s curit Symboles lectriques Safety and Compliance Information Les
236. before calibration 2 56 6 4 7 24 impact of changing Grid Voltage 5 51 impact of Low Mass Gate 5 89 increasing accuracy 3 24 3 38 internal see Calibration internal manual see Calibration manual mass accuracy 3 24 3 38 6 7 6 10 mass accuracy optimizing 2 53 matrix reference file modifying 5 23 matrix selecting 5 20 6 13 overview 6 7 PSD see Calibration PSD Sequence Control Panel 7 5 7 6 7 18 smoothing spectra 6 10 standards see Calibration standards types of 6 7 updating see Calibration Internal update Sequence Control Panel when to calibrate 6 8 Calibration automatic external see Calibration external internal see Calibration internal Sequence Control Panel 7 32 Calibration default description 6 7 equation 6 9 PSD description 8 28 PSD selecting 8 46 8 60 selecting 5 10 6 13 Calibration external close external performing 7 32 mass accuracy optimizing 2 53 multiple spectra in one data file 7 4 overview 6 7 selecting 5 10 6 13 standards placement for optimum mass accuracy 7 33 Calibration external Sequence Control Panel close external performing 7 34 description 7 32 overview 7 5 7 6 selecting 7 18 Calibration internal and accurate mass measurements 6 26 concentration optimizing unknowns and standard 7 39 description 6 7 determining if standard suppresses signal from unknown 7 40 if concentration of standard varies 7 42 internal standard 6 26 mass accura
237. ber of spectra to acquire or the number of search pattern positions in the SP file Save the first In Save the First Spectrum To Pass Acceptance Criteria mode spectrum to pass Figure 5 10 acceptance The first spectrum that meets the selected acceptance criteria criteria is saved e Acquisition is performed on each search pattern position until a spectrum passes or until the end of the search pattern is reached if the Use Automated Sample Positioning option is disabled acquisition is performed on the positions you manually select instead of search pattern positions All search pattern positions may not be analyzed e One DAT file containing one spectrum is created If no spectra pass acceptance criteria no data file is saved 5 42 Applied Biosystems Instrument Settings Parameter Descriptions m Spectrum Accumulation Acquire fi spectra under conditions Save the first spectrum to pass acceptance criteria x 1 spectrum from each pasition Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 50 shots 50 shots 1 spectral Number of DAT files lt 1 Figure 5 10 Save the First Spectrum To Pass Acceptance Criteria Mode Automatic Control Dialog Box Example If Random search pattern or an SP file is selected the Number to Acquire is automatically set to 1 and the number of positions analyzed is equal to the position number in which
238. bly focused higher mass ion p Focused mid mass ion Reflector Flight tube Point of initial focus moved farther down flight tube by increased Grid Voltage T T A LUE 8 74 Figure 8 20 Increased Grid Voltage and lon Focusing Applied Biosystems Acquiring Exploring PSD Mode NOTE In this exercise you will observe the impact of the Grid Voltage on the precursor ion at 1 297 Da and the fragment ion at 1 181 Da At the laser intensity you optimized for the fragment ion the precursor ion will be saturated To allow you to observe precursor and fragment ions in the spectrum change the Vertical Scale setting in the BIC file Digitizer Mode dialog box to 1 000 mV full scale 1 Enable the Precursor lon Selector if it is disabled 2 Acquire a spectrum to make sure the precursor is not saturated Hint If the precursor ion peak is still saturated but the peak valleys are noticeable you can estimate the resolution at the 2 isotopic peak 3 Make a note of the resolution of the fragment ions and the precursor ion NOTE You may need to switch between Vertical Scale settings of 1 000 mV and 200 mV and reacquire spectra to see the resolution on the precursor and fragment peaks 4 From the Acquisition menu select Stop Experiment to allow you to change the Grid Voltage in the Instrument Settings control page 5 Decrease the Grid Voltage
239. c resolution across the entire mass range Voyager Biospectrometry Workstation User s Guide 5 81 Chapter 5 Optimizing Instrument Settings 5 4 3 5 Optimizing Grid Voltage Overview You can use this procedure as an alternative to the procedure in Section 5 4 3 4 Optimizing Delay Time However optimizing Delay Time is the recommended procedure Optimizing the Delay Time ensures maximum stability of the high voltage power supplies and therefore results in better mass accuracy You can use this procedure when operating in Linear mode or Reflector mode Start with a standard BIC file optimize the Grid Voltage and leave the Delay Time unchanged NOTE The Grid Voltage setting in Reflector mode is typically significantly lower than the setting in Linear mode for the same mass range For example if Grid Voltage in Linear mode is 94 Grid Voltage in Reflector mode may be 70 These values are used as an example they may not be optimal for your system For more For more information see Section 5 3 2 Understanding Grid information Voltage 5 82 Applied Biosystems Optimizing Instrument Settings Parameters Optimizing Grid To optimize Grid Voltage Voltage 1 Open a standard BIC file for the mass range you are acquiring For more information see Selecting a BIC file on page 5 8 Acquire a spectrum and observe the resolution For more information see Section 6 5 2 Calculating Mass Reso
240. cal Offset The Vertical Offset allows fine adjustment of the spectrum baseline position It is specified as a percentage of the Vertical Scale For most applications leave the Vertical Offset at 0 Figure 5 19 illustrates the effect of setting a positive or negative offset A positive offset shifts the baseline up A negative offset shifts the baseline down NOTE If you set a Negative Vertical Offset some low intensity noise signal may not be collected in the data file Some processing functions for example Baseline Correction or Noise Filter require complete collection of noise signal to generate valid results Ana AAAL a Original data noise signal below Negative Positive scale 0 Vertical Ones se Offset Figure 5 19 Effect of Vertical Offset in Digitizer Settings 5 62 Applied Biosystems Impact of Changing Instrument Settings Parameters Input Bandwidth Decreasing the Input Bandwidth decreases the response time not available with ofthe detector and can reduce higher frequency noise A Signatec lower setting can improve resolution and smooth out a digitizers baseline but may also decrease signal to noise ratio Because the Input Bandwidth is an electronic analog filter it may slightly shift peak centroids toward higher masses relative to peaks recorded with Full Bandwidth Suggested settings Use the settings listed below as a starting point for Input Bandwidth Adjust as needed to eliminate
241. cal mass spectrometer includes e lon source lonizes sample and generates gas phase ions e Analyzer Separates ions according to individual mass to charge ratios e Detector Detects and amplifies ions e Data system Converts detection of ions into a readable or graphic display Advantages of mass spectrometry technology include e Can directly measure an absolute physical constant e Provides highly sensitive detection e Can analyze mixtures and different classes of biopolymers including peptides oligonucleotides and oligosaccharides MALDI TOF mass The Voyager Biospectrometry Workstations are MALDI TOF spectrometry mass spectrometry systems e MALDI Matrix Assisted Laser Desorption lonization sample ionization mechanism e TOF Time of Flight analysis and detection mechanism Voyager Biospectrometry Workstation User s Guide 1 7 1 Chapter 1 Matrix Assisted Laser Desorption lonization MALDI 1 8 Introducing the Voyager Biospectrometry Workstations In Matrix Assisted Laser Desorption lonization MALDI sample is embedded in a low molecular weight UV absorbing matrix that enhances intact desorption and ionization of the sample The matrix is present in vast excess of sample and therefore isolates individual sample molecules Figure 1 4 illustrates the ionization of sample lonization O Laser Q Energy transfer O from matrix to sample gt ee F e ne Ma
242. cale for resolution and molecular weight ranges for compounds acquired in Linear mode Table 6 2 Resolution Rating Scale Compounds Compounds 2 000 Compounds 5 000 Compounds lt 2 000 Da to 5 000 Da to 25 000 Da gt 25 000 Da Resolution Rating Resolution Rating Resolution Rating Resolution Rating 500 Not 250 Not 500 Not 50 Acceptable acceptable acceptable acceptable 1 500 Acceptable 400 Acceptable 700 Acceptable 100 Good 2 000 Good 500 Good 900 Good gt 200 Very good 2 500 Very good gt 600 Very good 1 000 Very good 6 32 Applied Biosystems 6 5 3 Calculating Signal to Noise Ratio Overview A signal to noise ratio is typically used to describe how well a Calculating signal to RMS noise ratio Evaluating Data in the Instrument Control Panel mass of interest in a spectrum is distinguished from background chemical and electronic noise The Control Panel software measures the signal to RMS noise ratio in a user defined region of a mass spectrum To calculate a signal to RMS noise ratio 1 When acquisition is complete select the Current spectrum of interest 2 From the Tools menu select Signal to Noise Calculator 3 Inthe Signal to Noise Calculator dialog box Figure 6 14 set the Baseline Region by doing one of the following e Type in From and To values e Inthe Spectrum window right click drag over the baseline area you want to use in calculatin
243. ccuracy Optimization option not used Enable Mass Accuracy Optimization See page 3 52 Standard and sample of interest not in adjacent sample position Calibrate using standard that is in a sample position adjacent to the sample of interest Correct initial velocity not specified Specify Initial Velocity in BIC file before acquisition by selecting matrix For more information see Matrix influence on page 5 22 Voyager Biospectrometry Workstation User s Guide continued 9 11 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor mass accuracy in Delayed Extraction mode external calibration only continued Incorrect calibration constants used Recalibrate with known standards and correct masses Make sure correct external calibration file is selected in the Calibration field on the Instrument Settings control page 9 12 Applied Biosystems continued Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor signal to noise ratio Laser intensity too Decrease laser intensity to or sensitivity high threshold by using the slider controls in the Manual Laser Sample Positioning control page see page 4 27 Laser intensity too Increase laser intensity by low using the slider controls in the Manual
244. ce This section describes e Optimizing instrument settings BIC files for a sequence run e Creating macros e Creating calibration CAL files e Creating settings SET files 7 3 1 Optimizing Instrument Settings BIC Files for a Sequence Run Select BIC files based on the compound type and mass range you are analyzing See Section 5 1 2 Standard Instrument Settings BIC Files Provided You can use more than one BIC file in your sequence run if you are analyzing a wide mass range for example when analyzing a mixture of protein and peptide samples As a general guideline you may need separate BIC files for different mass ranges for example e 1 000 to 6 000 Da e 5 000 to 12 000 Da e 10 000 to 30 000 Da Voyager Biospectrometry Workstation User s Guide 7 7 Chapter 7 Acquiring Spectra from the Sequence Control Panel BIC requirements Optimize the instrument settings for Automatic Control mode All BIC files used in the Sequence Control Panel must specify e Automatic Control mode e Linear or reflector Operation mode PSD mode is not allowed The same laser type internal or external You can not switch between internal and external lasers when running a sequence For more information see Section 6 6 2 Setting Instrument Settings for Automatic Control Mode Note the following If Use Automated Laser Intensity Adjustment is disabled in the BIC file the laser intensity is not auto
245. ce Control Panel lon acceleration description 5 51 impact of Accelerating Voltage 5 87 impact of Grid Voltage 5 51 in Continuous Extraction mode 1 12 in Delayed Extraction mode 1 12 in MALDI TOF 1 8 lon focusing lens fixed voltage applied 1 35 function 1 35 starting serial number 1 33 lon Fragmentation calculator 8 19 lon kinetic energy 1 10 8 21 strategy 5 65 Instrument State in status bar 4 5 Voyager Biospectrometry Workstation User s Guide Index 19 Index lon polarity see Polarity lon source description Voyager DE and Voyager DE PRO 1 22 description Voyager DE STR 1 34 second stage voltage for 5 17 warmup 2 56 6 4 7 24 lonization sample by Na or K instead of H 9 17 Delayed Extraction 1 12 enhancing 3 23 multiple charges on ion 1 10 single charge on ion 1 10 lons multiple charge 1 10 single charge 1 10 Isotope resolution Delay Time setting 5 81 guidelines for acceptable 5 75 in Delayed Extraction mode 1 13 labels not displayed for all peaks 6 31 J Jumper settings 2 7 K K adduct ion effect on masses 9 17 from buffer 3 24 Keyboard connecting Voyager DE and Voyager DE PRO 2 22 connecting Voyager DE STR 2 25 Kinetic energy fragment ions 8 21 8 22 molecular ions 1 10 8 21 Index 20 Applied Biosystems L Labeling peaks see Peak labels Laboratory Name specifying 2 41 Landscape printer orientation setting permanently 4 20 Large proteins matrix for 3 3 Laser
246. ce display to the minimum and maximum Y values Minimum Absolute Max Y Use Limit Sets the minimum value for the maximum Y axis setting Useful to maintain relative scaling of peaks and to prevent autoscaling noise to full scale Y Display Range From To Sets the range for scaling If Display Relative or Base Peak Relative is selected range is in percent If Absolute Value is selected range is in counts 8 Click OK 4 12 Applied Biosystems Using the Spectrum Window 4 3 2 Zooming on Traces Zooming and You can expand zoom an area of a trace by click dragging a unzooming box around the area of interest You can also click buttons in the toolbar to e Zoom in oie e Zoom out to the previous zoome Full Unzoom Expanding and When you have more than one trace displayed in the same linking traces data file in a window you can _ e Select click on a trace then click in the toolbar to expand the selected trace for closer examination To display all traces click the button again e Click in the toolbar to link all traces Any zooming actions you perform on one trace affect all traces linked to it To unlink traces click the button again 4 3 3 Adding Traces to a Window This section describes e Types of traces Adding traces e Removing traces Voyager Biospectrometry Workstation User s Guide 4 13 Chapter 4 Voyager Instrument Control Panel Basics Types of tr
247. cleotides glycoconjugates and synthetic polymers e Provides very high sensitivity and requires only femtomoles of sample 1 10 Applied Biosystems Voyager DE Delayed Extraction Technology 1 4 Voyager DE Delayed Extraction D Technology In this section This section describes e Limitations of MALDI technology e Delayed Extraction e Delayed Extraction versus Continuous Extraction e Benefits of Delayed Extraction e Velocity focusing Limitations of In traditional MALDI ions exhibit a broad kinetic energy MALDI distribution which is largely due to the initial velocity imparted technology to ions during the desorption ionization process This initial velocity of desorbed analyte ions is nearly independent of mass of the analyte and the initial kinetic energy is proportional to the mass In addition when desorption occurs in a strong electrical field energy is lost by collisions with the neutral plume and further mass dependent energy dispersion results According to the theory of time lag energy focusing as originally developed by Wiley and McLaren the dependence of ion flight time on initial velocity can be corrected to the first order by delaying the extraction of ions from the source If higher order terms are insignificant then the mass resolution should be determined by the ratio of the total flight time to the uncertainty in the time measurement The observed mass resolution should increase in prop
248. conditions 3 4 THAP 3 3 3 13 C 11 thin film 6 5 Matrix example spectra 3 HPA C 4 alpha cyano 4 hydroxycinnamic acid C 2 DHB C 3 DHBs C 3 dithranol C 5 HABA C 4 IAA C 6 Sinapinic acid C 2 THAP C 5 Index 24 Applied Biosystems Matrix Initial Velocity correcting for 5 20 impact on tuning Grid Voltage and Delay Time 5 74 in Instrument Settings 5 22 selecting 6 13 Max Stitch Mass definition 8 42 8 48 optimum focus and resolution observed near this mass 8 49 Melittin mass to time conversion F 4 Membranes sample plate to use 3 63 Memory computer 1 30 1 41 Methionine Enkephalin mass to time conversion F 4 Methods converting version 4 to version 5 5 92 replaced by instrument settings 5 2 Microsoft Windows NT version 1 30 1 41 Mirror see also PSD Mirror Ratio see also Reflector Voltage adjusting 8 22 Mirror Ratio see PSD Mirror Ratio Mirror To Accelerating Voltage Ratio default values for converted MNU files 5 95 Mirror to Accelerating Voltage Ratio setting 5 32 MNU files converting version 4 to version 5 5 92 replaced by BIC files 5 2 Mode Digitizer parameters accessing 5 16 5 24 Advanced 5 31 Bin size 5 29 5 50 5 59 Digitizer Linear Reflector 5 28 Extraction Type 5 25 Input Bandwidth 5 30 5 50 5 63 Instrument Mode 5 25 Laser Rate 5 26 5 27 Laser Type 5 26 Linear Reflector PSD mode 5 25 Mirror to Accelerating Voltage Ratio 5 32 Number of Data Points Digitized 5 29 Polarity 5 25 T
249. control Le Le Le Le Le fe tap oer fey Ken eye fa Cay Ge Ob Tee Cat UE Le Lahey aetes es le Nei teh O Let SES LL A SEC UG MG an Cy Les Gate Cs Go teas se kn ay oo oo o oo oo oo oo oo SRS o Coordinates of active Relative Xx 587 500 y 1587 500 position 7 Absolute fooco v fo o00 fa Figure 4 8 Manual Laser Sample Position Control Page Plate View Voyager Biospectrometry Workstation User s Guide 4 27 Chapter 4 Voyager Instrument Control Panel Basics 4 28 Manually adjusting laser intensity Applied Biosystems You can adjust the laser intensity using any of the following e Slider control Use to set laser intensity to an exact setting To use the slider control click drag the slider to adjust laser intensity k Fine laser controls lt gt Use to adjust the intensity in small increments e Coarse laser controls ep Use to adjust the intensity in large increments You can set the small and large increments in the Laser Configuration dialog box For more information see Laser Configuration on page 2 42 NOTE After adjusting the laser intensity the slider and fine and coarse adjusting buttons are disabled while the system adjusts the laser to the intensity specified Hint You can also set laser intensity by pressing Ctrl PgUp and Ctrl PgDn on the keyboard Pressing these keys adjusts inten
250. cquiring PSD Data with Standard BIC Files in Manual Control Mode Guidelines for Guide Wire Voltage Vertical Scale and Input Bandwidth Typing or selecting new values As a general guideline when setting Guide Wire Voltage Vertical Scale and Input Bandwidth use decreasing values to compensate for decreasing ion energy seen at lower Mirror Ratios You need to experiment to determine the optimum values for these settings at lower Mirror Ratios NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later To type or select new values in the segment list 1 Type in new values for Mirror Ratio or Guide Wire in any row in the table You can specify Mirror Ratios in any order in the table NOTE When you click the Mirror Ratio field the entry is displayed with more than 3 digit precision which is the precision used to calculate the mass range for the segment during the analysis You can specify the same Mirror Ratio for multiple segments if needed When the composite spectrum is generated the software uses the most recently acquired segment if duplicate Mirror Ratios are present Observe the Max Stitch Mass when you type a Mirror Ratio Optimum focus and resolution is achieved for fragment ions close to this mass To select new values for Vertical Scale or Input Bandwidth click the cell A drop down list of values appropriate for your system is displayed
251. cros and scripts you created in the Data Explorer software are listed e Type the button number to which you assigned the macro For more information see Creating Macros on page 7 8 Hint Display the Data Explorer software then place the cursor over a macro button to display the macro name assigned to the button Macro buttons are numbered sequentially from left to right If the Macro toolbar is not displayed select Toolbar from the View menu select Macros then click Close Sample Text that describes the sample Description optional Comments Additional descriptive text optional Acquisition Displays the status of the current acquisition in the run list For Status information see Checking the Acquisition Status field on page 7 29 Run If checked the row is acquired when the sequence runs If it is not checked the row is skipped 4 When all necessary rows are filled in save the sequence by selecting Save Sequence from the File menu The run list is saved as part of the SEQ file 7 20 Applied Biosystems Creating a Sequence Modifying and You can modify and customize the run list using the following customizing the commands on the Edit menu run list Cut Copy Paste Use to cut copy and paste information _ Insert Row Insert Multiple Rows Delete Row Use to insert and delete rows Fill Down Use to automatically fill in run list grid entries Click drag to selec
252. ct labels and set the number of decimal points to display For information see the Data Explorer Software User s Guide To disable select Peak Label from the Display menu and disable labels as needed Voyager Biospectrometry Workstation User s Guide 6 29 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 5 2 Calculating Mass Resolution You can calculate mass resolution for up to four peaks in the spectrum currently being acquired The resolution values are displayed in the trace next to the mass value for the peak Calculating To calculate mass resolution resolution for live 4 When acquisition is complete select the Current data spectrum 2 From the Tools menu select Resolution Calculator 3 Inthe Resolution Calculator dialog box Figure 6 12 set the percentage of peak height at which to calculate resolution The default is 50 which calculates the resolution at the full width half maximum of the peak height FWHM NOTE Measuring resolution at the full width half maximum is the industry standard Use 50 Peak Height for most applications 4 Set Minimum Peak Intensity Signals below this intensity absolute counts are not included in the calculation Resolution Calculator Live Data xi m Calculation Parameters Peak Height pa Minimum Peak Intensity 50 Select Peaks x Mass Charge AMU Peak 1 fi 300 1 211 Peak 2 fo i fi Peak 3 fo 1 Peak4 0 Pa FWH
253. cting a Matrix Purpose of matrix In MALDI TOF the matrix plays a key role in the ionization process The well developed crystals of matrix material assist in ionizing the molecules you are analyzing Common matrixes For most applications you can use one of the following matrixes Matrix Application Sinapinic acid 3 5 Dimethoxy 4 hydroxy cinnamic acid Peptides and proteins gt 10 000 Da o cyano 4 hydroxycinnamic acid CHCA Dried drop application Peptides proteins lt 10 000 Da e Thin layer application Peptides lt 3 000 Da 2 4 6 Trihydroxy acetophenone THAP Small oligonucleotides lt 3 500 Da acidic carbohydrates acidic glycopeptides acid sensitive compounds 3 hydroxypicolinic acid 3 HPA in ammonium citrate Large oligonucleotides gt 3 500 Da 2 5 dihydroxybenzoic acid 2 5 DHB Peptides neutral or basic carbohydrates glycolipids negative ions polar and non polar synthetic polymers small molecules 2 5 dihydroxybenzoicacid and 5 methoxysalicylic acid DHBs Peptides and proteins gt 10 000 Da glycosylated proteins Dithranol and Ag TFA Aromatic polymers trans 3 indoleacrylic acid IAA Non polar polymers Procedures for preparing matrix are included in Section 3 1 2 Preparing Matrix Voyager Biospectrometry Workstation User s Guide 3 3 3 Chapter 3 Preparing Samples Mixtures Specialized app
254. ctions Wear appropriate protective eyewear clothing and gloves Alpha cyano 4 hydroxycinnamic acid CHCA may cause eye skin and respiratory tract irritation Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Trifluoroacetic acid causes severe burns to the eyes skin and respiratory tract Table 3 2 CHCA Matrix Information for Dried Droplet Application Matrix concentration 10 mg ml Final sample concentration 0 1 5 pmol ul Solvents Acetonitrile 0 1 TFA in deionized water Preparation Follow the procedure in Preparing matrix on page 3 5 and combine one part 0 1 TFA solution with one part acetonitrile Dissolve the matrix ata concentration of 10 mg ml for example add 500 ul 0 1 TFA solution and 500 ul acetonitrile to 10 mg of solid matrix Air dry sample plate after loading sample and matrix or use gentle air flow to speed drying continued Voyager Biospectrometry Workstation User s Guide 3 9 Chapter 3 Preparing Samples Table 3 2 CHCA Matrix Information for Dried Droplet Application Continued NOTE If the dry matrix is a mustard yellow color instead of bright yellow it may contain impurities To recrystallize purify dissolve the a cyano matrix in warm ethanol Filter and add about two volumes of deionized water Let stand in
255. ctor mode 8 38 entering in PSD setting 8 45 8 60 precision required 8 45 8 60 Precursor spectrum see PSD precursor spectrum Preface xxvii Prescan mode description 6 58 enabling 5 34 6 39 Pressure ranges for vacuum gauges 8 34 time required 2 77 Voyager DE and Voyager DE PRO 2 76 Voyager DE and Voyager DE PRO 9 26 Voyager DE DTR 2 76 Voyager DE STR 9 26 Pressure vacuum 2 76 Pressures displaying 2 77 4 5 Preventative maintenance 9 2 Previewing traces before printing 4 18 Print preview Instrument Control Panel 4 18 Print Setup 4 20 Printer connecting Voyager DE and Voyager DE PRO 2 22 connecting Voyager DE STR 2 25 dedicating to landscape orientation 4 20 Index Printing changing colors to black before 4 18 instrument settings 5 12 landscape orientation 4 20 traces 4 18 traces do not print 4 19 4 22 Product spectra see PSD mode see PSD segments Prompt fragments acceleration and flight time 8 71 description 6 24 8 71 example 8 70 mass 8 71 Proteins Input Bandwidth setting 5 30 matrix for 3 3 C 6 C 10 Starting Mass recommendations 5 90 PSD acquisition automatic mode see also PSD mode experiment automatically closed 8 64 fragment ion identity determining 8 19 instrument settings disabled during 8 63 process that occurs during 8 64 segment selecting 8 63 segments appended to DAT 8 57 settings 8 58 starting 8 63 stopping 8 64 PSD acquisition manual mode see also PSD acquisition
256. ctrum is discarded e After all positions have been analyzed one DAT file containing the current best spectrum displayed in the accumulated trace is created If no spectra pass acceptance criteria no DAT file is created gt Spectrum Accumulation Acquire E spectra under conditions Save the best spectrum 7 1 spectrum from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 50 shots 50 shots 1 spectra Number of DAT files lt 1 Figure 5 11 Save the Best Spectrum Mode Automatic Control Dialog Box 5 44 Applied Biosystems Example Accumulate all spectra Instrument Settings Parameter Descriptions If Random search pattern or an SP file is selected and Number to Acquire is 5 the number of positions analyzed is five One DAT file containing the spectrum with the highest Signal to Noise result is created If no spectra meet criteria no DAT file is created NOTE If you set the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified number of spectra to acquire or the number of search pattern positions in the SP file In Accumulate All Spectra mode Figure 5 12 One spectrum is acquired from a search pattern position if the Use Automated Sample Positioning option is disabled acquisition
257. curs when Accumulating Spectra from Multiple Search Pattern Positions In Accumulate All Spectra That Pass Acceptance Criteria mode Figure 5 13 e Acquisition is performed on the same search pattern position until Acceptance Criteria fail if the Use Automated Sample Positioning option is disabled acquisition is performed on the positions you manually select instead of search pattern positions e Acquisition continues until the Number of Spectra to Acquire have been acquired All search pattern positions may not be analyzed e Each spectrum that meets the specified acceptance criteria is accumulated into one DAT file that contains one spectrum If no spectra pass acceptance criteria no DAT file is created NOTE In this mode acquisition moves to the next search pattern position only if acceptance criteria fail Instrument Settings Parameter Descriptions gt Spectrum Accumulation Acquire E spectra under conditions Accumulate all spectra that pass acceptance criteria x 1 of more spectra from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 250 shots 50 shots 5 spectra Number of DAT files lt 1 Figure 5 13 Accumulate All Spectra That Pass Acceptance Criteria Mode Automatic Control Dialog Box Example If Random search pattern is selected and Number to Acquire is 5 the number of positions analyzed dep
258. cy optimizing 2 53 multiple spectra in one data file 7 4 overview 6 7 search pattern file example 7 41 search pattern file for separate spots creating 7 38 7 40 Index Calibration internal Sequence Control Panel description 7 32 overview 7 5 7 6 selecting 7 18 with external cal backup 7 42 Calibration Internal update Sequence Control Panel description 7 11 how to program every X minutes 3 39 Calibration internal update Sequence Control Panel overview 7 5 7 6 selecting 7 18 selecting for close external calibration 7 32 Calibration PSD default 8 28 default selecting 8 46 8 60 equation 8 28 in Instrument Settings 8 45 8 60 in PSD Acquisition Settings 8 46 8 60 two types 8 2 Calibration Sequence Control Panel close external enhanced by Mass Accuracy Optimization 7 34 Calibration standards see also Internal standard see also Standard compounds fewer needed with Mass Accuracy Optimization 3 39 internal if concentration varies 7 42 location on plate 3 38 H 24 molecular weights F 2 number and location required 3 38 ordering information B 6 reducing number needed 3 52 requirements 6 10 Voyager Biospectrometry Workstation User s Guide Index 7 Index Calibration standards continued requirements for a sequence run 7 33 Voyager mass standards kit B 6 with optimized plate 3 39 with unoptimized plate 3 40 Camera see Video camera Carbohydrates matrix for 3 3 C 7 Carbonic anhydrase m
259. d Biosystems Acquiring PSD Data with Standard BIC Files in Manual Control Mode The information in the first row of remaining columns is copied to all rows below e The Max Stitch Mass is calculated and displayed for each row Any rows for segments that have already been saved are skipped Adding or To add rows select any existing row then click A row is deleting rows added to the end of the list and the settings from the selected row or the first row if no row is selected are copied to the added row Change settings as needed To delete rows select the rows to delete and click You cannot delete a row if the segment has been saved Saving the BIC To save the instrument settings BIC file select Save file Instrument Settings As from the File menu then save the BIC file with a new name Voyager Biospectrometry Workstation User s Guide 8 51 Chapter 8 PSD Analysis 8 4 5 Acquiring and Saving PSD Segments in Manual Mode This section includes e Overview e Acquiring PSD segments e Selecting and acquiring a segment e During acquisition e Changing settings e Accumulating or saving the segment e Selecting and acquiring remaining segments e Reacquring a segment e Stopping the experiment Overview When you start acquiring in PSD mode the software automatically opens a PSD experiment When a PSD experiment is open e You cannot change instrument settings This ensures that all PSD segments
260. d Biosystems Move to a new area of the sample well that contains the same sample Acquire a spectrum to verify that the setting is valid for the laser power selected and not caused by e Sample surface excitement caused by the previous higher laser power setting e Sample consumption If the setting is valid you see a spectrum similar to the one just acquired You may need to adjust the laser slightly 5 to 10 counts Examine several spots in the sample well to make sure you have not tuned the laser intensity on a cold spot You can further fine tune the laser setting by setting a smaller laser step size in Hardware Configuration For example instead of a step size of 10 change the step size to 2 Make sure peak shape and resolution are acceptable See Section H 2 3 Checking Resolution If you do not see a signal at the current setting increase the laser power NOTE If peak intensity of the final scan is near maximum intensity it is very possible that some scans averaged into the final scan are saturated Reacquire using a lower laser power Obtaining Good Spectra in Continuous Extraction Mode H 2 3 Checking Resolution After you determine laser threshold calculate the resolution Determine if the resolution is acceptable for your application See Section 6 5 2 Calculating Mass Resolution Table H 6 lists a general rating scale for resolution and molecular weight ranges for compounds acquired in Linear
261. d for your system For more information see Standard instrument settings files on page 5 3 2 30 Applied Biosystems Installing Software e User Guides Includes PDF portable document format versions of the Voyager documentation that you can view online using Adobe Acrobat Reader 3 Click Next 4 A series of dialog boxes in which you specify the hardware options and serial number for your system are displayed Leave settings at the defaults or change the settings if needed Click Next The Select Program Folder dialog box appears 5 Leave the Program Folder set to Voyager Click Next The installation proceeds 6 A message is displayed asking if you want to install Data Access Packs Click Yes 7 A message is displayed asking if you want to install Adobe Acrobat Reader Click Yes When the software is installed a message is displayed The following icons are created and displayed on the Windows desktop e Voyager Control Panel e Data Explorer e Voyager Sequence Control Panel Accessing the To access the PDF versions of the Voyager documentation online User s 4 From the Windows Start menu select Programs Guides 2 Select Voyager then select User Guides and select the document to open Voyager Biospectrometry Workstation User s Guide 2 31 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 5 2 Starting the Software 2 32 Starting Instrument
262. d samples 4 Dispense 0 5 ul of matrix prepared for thin layer application on the sample plate to form a thin layer It will dry in seconds For information see CHCA for thin layer application on page 3 11 2 Load 0 5 to 1 ul of the sample in each sample position onto the thin matrix layer applied in step 1 3 Dry the plate with a heat gun 4 After drying load 2 to 3 ul of double distilled water on top of each dried sample to wash the sample 5 Wait 5 seconds and blow off the water droplet with compressed air 6 Repeat step 4 and step 5 two more times NOTE During acquisition use the surfing method moving the sample plate after every 5 to 10 shots to anew position on the sample 1 Shevchenko A M Wilm O Vorm M Mann Anal Chem 1996 68 850 858 3 44 Applied Biosystems Loading Samples on Sample Plates 3 2 5 Examining Crystals on Sample Plates Overview if you are using polished blank or laser etched sample plates see Types of sample plates on page 3 36 you can look at the crystallization of sample and matrix under a microscope A magnification of 30X is sufficient to see crystals You can also view crystallization on the video monitor on the instrument The morphology of crystals is a critical element for successful analysis Ideally you want small crystals that are evenly distributed in the sample well a cyano or Figure 3 1 illustrates good patterns of crystal
263. data file to acquire During acquisition the software appends to the base name a 4 digit suffix starting at 0001 For example if you specify a base name of SAMPLE the final data file name is SAMPLE_0001 DAT By default the position name number is also appended and precedes the 4 digit suffix For example if you specify a base name of SAMPLE the final data file name is SAMPLE_A1_0001 DAT for the data file acquired from position A1 To suppress the position name number in the file name deselect Append Sample Position to File Name in Preferences described on page 7 45 Instrument Settings File required entry BIC file to use for the current row Click the down arrow and select an instrument settings BIC file that you have optimized for Automatic Control mode For more information see Section 6 6 2 Setting Instrument Settings for Automatic Control Mode NOTE If you are editing a BIC file save it before selecting it in the Sequence Control Panel If you make changes to the BIC file after you select it in the Sequence Control Panel the Sequence Control Panel does not recognize the changes NOTE All BIC files specified in a sequence must specify either the internal laser or the external laser You cannot switch between internal and external lasers when running a sequence If you specify an internal laser BIC file then specify an external laser BIC the second BIC is flagged as invalid when you try to run the se
264. definition 5 2 exporting from DAT file 6 19 Linear mode 5 4 loading 5 7 location 5 3 Manual Control mode setting 5 9 name displayed 5 8 opening 5 7 opening from DAT file 5 7 preventing changes 5 13 protecting 5 13 provided with system 2 30 5 3 PSD mode 5 6 8 43 8 58 read only 5 3 5 13 Reflector mode 5 5 Index 18 Applied Biosystems Instrument Settings files BIC continued requirements for Sequence run 7 8 7 27 saving 5 11 saving after acquisition to store laser intensity 6 19 selecting standard 5 8 setting to read only 5 13 Instrument Settings parameters see also Automatic Control mode parameters see also Instrument Settings files BIC see also Instrument Settings parameters optimizing see also Mode Digitizer parameters see also Spectrum Acceptance Criteria Accelerating Voltage 5 17 5 50 Automatic Control mode 5 33 6 37 Calibration 5 20 Continuous Extraction parameters H 2 Control mode 5 16 Delay Time 5 18 5 49 5 54 E 1 Delayed Extraction parameters 5 73 Digitizer settings 5 57 displaying 5 9 6 12 Grid Voltage Continuous Extraction H 6 Grid Voltage Delayed Extraction 5 17 5 50 5 51 E 1 Guide Wire Voltage Continuous Extraction H 6 Guide Wire Voltage Delayed Extraction 5 18 5 50 5 56 impact of changing Linear and Reflector mode 5 49 impact of changing PSD mode 8 76 Instrument Settings parameters continued Instrument mode 5 16 Low Mass Gat
265. des e Overview e Two ways to start the Instrument Control Panel e During acquisition e Keeping both control panels open e Organizing the desktop and windows Overview The Sequence Control Panel works in conjunction with the Instrument Control Panel to allow you to acquire multiple samples using different instrument settings BIC files Two ways to start You can start the Instrument Control Panel in either of two the Instrument ways Control Panel Double click the Instrument Control Panel icon e Double click the Sequence Control Panel icon Voyager Biospectrometry Workstation User s Guide 4 33 Chapter 4 Voyager Instrument Control Panel Basics 4 34 During acquisition Applied Biosystems If you start the Instrument Control Panel using the Sequence Control Panel icon it functions the same way as if you started it using the Instrument Control Panel icon with the following exceptions e Warning and error messages are not displayed during operation e The Instrument Control Panel will close when you close the Sequence Control Panel The preferred method is to start the Instrument Control Panel using the Instrument Control Panel icon create instrument settings files then open the Sequence Control Panel using the Sequence Control Panel icon or to start the Sequence Control Panel by clicking on the toolbar button in the Instrument Control Panel If you are acquiring data from one Control Panel the acqu
266. determine the laser threshold or the appropriate laser setting for your application You may need to move the signal up using the Vertical Position or the Vertical Scale knobs so that you can see the tops of the peaks If peak intensity of the final scan is near maximum intensity Input range 50 200 or 1000 mV it is very possible that some scans averaged in to the final scan are saturated Reacquire using a lower laser power Voyager Biospectrometry Workstation User s Guide 1 3 Appendix Using the Oscilloscope and Control Stick 1 2 Scaling Initial scaling Initial scaling of the oscilloscope is determined by the settings in the Mode Digitizer dialog box See Section 5 2 2 Mode Digitizer Dialog Box for more information You can adjust the initial scaling after acquisition starts by using the knobs on the front panel of the oscilloscope Figure l 2 Vertical Position Horizontal Position Select Cursor Control VERTICAL SO SS66e 00000 BOO OOM PB100276 Reset Button Vertical Scale Horizontal Scale Figure l 2 Front Panel of Oscilloscope l 4 Applied Biosystems Use these knobs to adjust the signal on the oscilloscope Vertical position Moves the signal up and down Vertical scale Adjusts the amplitude of the signal Horizontal scale Adjusts the width of the signal Horizontal position Moves the s
267. digitizer signal 5 30 Oligonucleotides matrix for 3 3 C 11 Online user guides accessing 2 31 Operating temperature and humidity A 4 A 7 A 10 Operation mode see Instrument mode Optimized laser rate 1 22 1 34 affected by number of data points A 12 not available on all systems 5 26 selecting 5 26 Optimized sample plates see Optiplate software Mass accuracy optimization Optimizing Instrument Settings parameters see Instrument Settings parameters optimizing OptiPlate software acceptance criteria required 2 60 benefits 2 54 BIC files provided 2 60 color codes 2 69 data files deleting when run complete 2 70 display settings required for best color results display 2 63 during a run 2 67 high voltage warmup required 2 56 how mass accuracy optimization is applied 3 52 if no optimization found 3 53 if positions not optimized 3 53 laser intensity determining setting needed for minimum signal intensity 2 61 matrix required 2 55 number of positions to spot 2 59 overview 2 53 Index 26 Applied Biosystems OptiPlate software continued pausing and resuming a run 2 68 PLT file requirements 2 57 reference position location 2 59 results color codes 2 69 results evaluating 2 70 results saving 2 70 running 2 63 sample plate preparing 2 59 standards required 2 55 system preparing 2 60 using unoptimized PLT 3 53 OptiPlate_Linear BIC 2 60 OptiPlate_Reflector BIC 2 60 Oscilloscope activating cursors I 5 adjusting display
268. djust the laser in large steps for example 100 counts As you begin to fine tune the laser use smaller steps Fragment ion yield initially increases with higher laser intensities and then decreases at very high settings To make sure that the signal decrease is not due to sample exhaustion move around in the sample position 7 Decrease the laser intensity and observe the signal Signal intensity decreases with lower laser intensities When laser intensity is too low signal intensity is weak 8 Continue decreasing the laser intensity until signal is no longer visible 9 Continue experimenting with laser settings until you are familiar with the appearance of a spectrum acquired when the laser intensity is set too high or too low Determining Fine tune the laser setting until you find a laser intensity that optimum laser provides a signal strength for the angiotensin fragment ions intensity for observed between 1 180 and 1 1 90 Da that is around 20 000 fragments to 40 000 counts in the Spectrum window Record this laser setting NOTE The precursor ion peak will be saturated at a laser intensity that yields adequate intensity for fragment ions 8 68 Applied Biosystems Exploring PSD Mode 8 6 2 Observing the Effects of Precursor lon Selector Turning on the Precursor lon Selector eliminates prompt fragments in a spectrum In this section In this section you will e Turn off the Precursor lon Selector ca
269. ds refer to Section 3 1 3 Matrix Information to application determine the appropriate concentration for this phase of 3 22 Applied Biosystems sample preparation Preparing Samples Note the following When diluting sample keep in mind that you will be further diluting when you mix sample with matrix If you are unsure of the starting concentration of sample make a serial dilution to prepare various dilutions of the same sample Different samples are soluble in different liquids Try water first then add acetonitrile and then add 0 1 TFA to increase solubility if required WARNING CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves A higher concentration of TFA may enhance sample ionization and improve sensitivity in samples prepared in buffers WARNING CHEMICAL HAZARD Trifluoroacetic acid causes severe burns to the eyes skin and respiratory tract Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Voyager Biospectrometry Workstation User s Guide 3 23 Chapter 3 Preparing Samples Preparing samples for thin layer application Internal standa
270. e Appendix C Data Explorer Toolbox Visual Basic Macros Voyager Biospectrometry Workstation User s Guide 8 19 Chapter 8 PSD Analysis Before you begin 8 2 Overview of PSD Analysis This section includes e Post source decay analysis e Differences from regular analysis e Segments and composite spectra e PSD data files e Mass calculation for fragment ions e Optimizing the Precursor lon Selector NOTE Analysis of post source decay is available on Voyager DE PRO and Voyager DE STR workstations only For successful analysis of post source decay you must be familiar with the information in e Chapter 5 Optimizing Instrument Settings e Chapter 6 Acquiring Spectra from the Instrument Control Panel 8 2 1 Post Source Decay Analysis What is post source decay analysis 8 20 Applied Biosystems You can obtain valuable structural information about a sample by analyzing the fragment ions generated from the original ions in the flight tube On a MALDI TOF system this is known as post source decay PSD analysis PSD analysis on the Voyager system is an acquisition method tailored to the analysis of fragment ions PSD mode allows you to better analyze ions that fragment in the flight tube by optimizing the following settings Mirror Ratio setting Brings different mass ranges of fragment ions into focus e Precursor lon Selector Selectively analyzes the ion of interest without interfe
271. e components Parts of the Voyager DE STR System Minimum computer configuration of Pentium III 500 MHz CPU 9 GB hard disk and 128 MB RAM random access memory 3 5 inch HD high density 1 44 MB floppy disk drive CD ROM drive integrated sound card 17 inch color monitor WIN95 Spacesaver Quiet Key 104 key keyboard Microsoft compatible mouse Control stick Optional laser printer Software The Voyager DE STR Biospectrometry Workstation includes the following software components Microsoft Windows NT version 4 0 or later Voyager software includes Voyager Instrument Control Panel Voyager Sequence Control Panel and Data Explorer software Microsoft Office 2000 Problems using Do not enable screen savers on the Voyager computer screen savers Screen savers use computer memory and may decrease system performance or cause other system problems Voyager Biospectrometry Workstation User s Guide 1 41 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 7 Software Overview The Voyager Biospectrometry Workstation software includes control software Voyager Instrument Control Panel Voyager Sequence Control Panel and post processing software Data Explorer software e Voyager Instrument Control Panel Controls the mass spectrometer for calibration and acquisition of single samples e Voyager Sequence Control Panel Works in conjunction with the Instrument Control Panel to allow you
272. e laser intensity needed to ionize sample Molecular weight of sample Higher masses require a higher laser intensity to generate an acceptable signal Sample characteristics Different classes of compounds in the same matrix may require different laser intensities For example carbohydrates in DHB require a higher laser intensity than peptides Sample contaminants Buffers salts and detergents in sample can substantially increase the laser intensity needed Sample exposure Fresh sample surfaces ionize better than surfaces that have been exposed to irradiation The significance of sample exposure varies widely between matrices and changes across the sample surface Obtaining Good Spectra in Continuous Extraction Mode Determining Determining laser threshold becomes easier with threshold experience Sample is consumed when exposed to the laser so minimize the number of spectra you acquire to determine threshold H 2 2 2 Adjusting the Laser Intensity This section describes e Adjusting the laser Fine tuning threshold Adjusting the NOTE If settings are not listed for the matrix set the laser laser to the lowest high setting listed for another matrix and start at step 2 1 Set the laser to the high laser setting for your system 2 Start an acquisition and acquire about 10 spectra At this high laser setting you may see a saturated spectrum where peaks are full scale and may be truncated Figure H
273. e sample position area e Width and Height Set the width and height of the generation area If you are creating a new SP file units are in microns If you are editing an SP file units are based on the units in the existing SP file Default values are the width and height of the sample position as defined in the PLT file If you set Center X Center Y Width and Height values so that any part of the generation area falls outside the sample position area defined in the PLT file an error message is displayed when you generate the pattern and spots that fall outside the sample position area are not created see Figure 6 20 NOTE Center X Y and generation area are not shown on the screen continued Voyager Biospectrometry Workstation User s Guide 6 53 Chapter 6 Acquiring Spectra from the Instrument Control Panel Table 6 4 Search Pattern Generator Parameters Continued Parameter Description Generation Area Parameters Generation area Generation area overlaps falls outside continued sample position area sample position area No spots created outside sample position area Center XY 0 0 Center XY 100 100 Width Height 2540 2540 Width Height 2540 2540 Figure 6 20 Impact of Changing Center X Y Number of Spots Type the number of spots up to 10 000 you want the search pattern to generate automatically You can add or insert more spots in a search pattern manually as de
274. e small molecules for a shorter time than larger molecules In general do not dialyze for more than two hours 9 When the size of the sample spot stabilizes remove the sample and place it in a microcentrifuge tube NOTE The size of the sample drop can increase by a factor of 10 when salt concentration is high 3 28 Applied Biosystems Preparing Samples 3 1 5 3 Cation Exchange When to use Use this technique for non polar proteins or DNA when you know the sample contains only a salt contaminant This technique is faster than dialysis but does not remove other contaminants What you need Use 200 mesh cation exchange beads Cation exchange beads in the ammonium form work best for MALDI applications Preparation of To prepare cation exchange beads in the ammonium form beads iN 4 Place beads in twice the bead volume of ammonium form 1M ammonium acetate 2 Leave beads in ammonium acetate overnight IMPORTANT Ammonium acetate Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 3 Remove beads and wash with water acetone and then hexane through a Buchner funnel under vacuum WARNING CHEMICAL HAZARD Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and h
275. e 5 20 Manual Control mode 5 9 5 15 mass range 5 19 Matrix 5 20 matrix and Initial Velocity 6 13 optimizing for Sequence run 7 7 parameters that affect Delayed Extraction 5 73 printing 5 12 PSD mode 8 43 8 58 resolution 6 25 Shots Spectrum 5 19 5 50 signal to noise ratio 6 25 Start and End Mass 5 19 summary Linear and Reflector mode 5 49 summary PSD mode 8 76 viewing all 5 8 Instrument Settings parameters optimizing see also Instrument Settings parameters Accelerating Voltage 5 84 5 87 before you begin 5 64 Continuous Extraction H 2 H 5 Delay Time 5 77 Delayed Extraction parameters 5 73 for Sequence run 7 7 Grid Voltage 5 82 5 83 Guide Wire Voltage 5 84 5 88 laser setting 5 67 Low Mass Gate 5 89 overview 5 65 PSD mode 8 43 8 58 resolution 5 71 Shots Spectrum 5 89 signal to noise ratio 5 85 Insulin BIC file 5 4 5 5 mass to time conversion F 5 molecular weight F 3 Insulin B oxidized mass to time conversion F 5 molecular weight F 3 Integration peak 6 29 Index Intensity laser see Laser intensity Intensity signal see also Sensitivity 5 67 see also Signal intensity definition 1 9 finding hot spots 6 5 peaks near maximum H 16 I 3 Interlock error description 1 40 with CID 8 34 Internal jumper settings 2 7 Internal standard calibration 6 7 6 26 concentration 3 24 if concentration varies 7 42 mass range 3 24 Internal Update calibration see Calibration internal update Sequen
276. e 5 75 resolution compared to continuous mode 1 13 resolution troubleshooting 9 10 setting mode 5 25 troubleshooting 9 10 9 11 9 12 Deleting text annotation 4 17 des Arg Bradykinin molecular weight F 2 Detector high current 1 23 1 35 linear 1 23 1 35 reflector 1 36 DHB chemical structure and molecular weight C 7 C 8 concentration 3 16 3 17 3 19 crystals 3 16 3 17 3 19 3 46 initial velocity setting 5 22 laser intensity relative 5 67 mass spectrum C 3 preparing for neutral carbohydrates 3 16 preparing for polymers 3 19 preparing for small molecules 3 17 sample concentration 3 16 3 17 3 19 stability 3 16 when to use 3 3 DHBs chemical structure and molecular weight C 9 concentration 3 18 crystals 3 18 3 46 Index 12 Applied Biosystems DHBs continued mass spectrum C 3 preparing 3 18 sample concentration 3 18 stability 3 18 when to use 3 3 Dialysis sample cleanup 3 27 Digitizer see also Oscilloscope configuring 2 44 description 1 19 1 32 options connecting to computer 2 11 Signatec connecting 2 13 Single shot mode 2 45 specifications A 11 type determining 2 44 Digitizer Acqiris connecting to Voyager DE and Voyager DE PRO 2 17 connecting to Voyager DE STR 2 26 optimized laser rate supported 5 26 A 12 Digitizer LeCroy LSA 1000 connecting 2 15 optimized laser rate supported 5 26 A 12 Digitizer settings adjusting 5 57 Bin size 5 29 description 5 57 impact on resolution and signal to noise 5
277. e All or the Save All Passing option in Automatic Control the CAL file is applied to all spectra in the DAT file 7 4 Applied Biosystems How file types and calibration you specify affect the data Understanding Settings Macros and Calibration Figure 7 2 and Figure 7 3 illustrate how the type of calibration and the files you specify affect the data that is acquired ina sequence run If you specify this Calibration Type These SET CAL and macros And a data file are applied if specified is created with in the order shown No Calibration DAT file with Peak detection settings from SET or defaults if no SET e Acquisition calibration specified in BIC External Calibration CAL calibration constants DAT file with Peak detection settings from SET or defaults if no SET e Calibration constants settings from CAL Internal Calibration SET peak detection DAT file with Peak detection settings from SET Calibration constants SET generated after reference calibration constants masses i from CAL applied Autocal then calibration with reference masses in SET we NOTE If the DAT file contains multiple spectra generated using the Save All or the Save All Passing option in Automatic Control the CAL fi
278. e Gas Inlet position for 3 seconds then turn immediately to the Purge Cell position At this point the lines should be purged If pressure is greater than 2 x 10 6 Torr repeat step 7 Turn the top 3 way valve to the Gas Inlet position then turn on the collision gas as described on page 8 35 Enhancing Fragmentation with CID Turning on To turn on the collision gas after purging collision gas 4 ifthe metering valve on the side of the CID box is set at zero turn the metering valve approximately 1 4 turn If the metering valve is not set at zero do not turn the valve 2 Wait 1 to 2 minutes for BA1 on the vacuum gauge panel to stabilize at 3 x 10 6 Torr or at the optimum pressure for CID operation that you have determined and recorded for your system 3 Ifthe pressure is lower than the optimum turn the metering valve one notch clockwise to a higher setting Wait for BA1 to stabilize at the optimum pressure If the pressure is higher than the optimum turn the metering valve one notch counterclockwise to a lower setting Turn the top 3 way valve to the Purge Cell position then to the Gas Inlet position Wait for BA1 to stabilize at the optimum pressure 4 Continue adjusting the metering valve until BA1 is stable at the optimum pressure NOTE If BA is below the optimum pressure when using a compressed gas supply and you cannot increase the pressure by turning the metering valve clockwise to a higher setting m
279. e PSD_Precursor BIC file provided with the software This is a reflector mode instrument settings BIC file All other instrument settings are identical to the settings in the PSD mode Angiotensin_PSD BIC file you use to acquire segments 2 Acquire using a laser intensity that does not saturate the reflector spectrum 3 Save the precursor ion DAT file by clicking EJ in the toolbar To obtain maximum mass accuracy for the precursor ion generate an external calibration file using the spectrum acquired in the previous section You will use this external calibration when you perform the PSD acquisition If you do not obtain maximum mass accuracy for the precursor ion during the PSD analysis you will not obtain maximum mass accuracy for the fragment ions NOTE This is not a PSD calibration that affects fragment ion masses It is an external calibration that is applied before PSD analysis to ensure accurate mass ofthe precursor Voyager Biospectrometry Workstation User s Guide 8 15 Chapter 8 PSD Analysis 1 Click in the Instrument Control Panel toolbar to open the precursor ion data file in the Data Explorer software 2 Create a single point calibration using the precursor ion mass and save the calibration file as PRECURSOR_UNKNOWN CAL by exporting the calibration constants from the data file You may need to add the mass for the unknown to your calibration reference file before creating the single point calib
280. e X Y coordinates of the sample positions and quickly and easily create a PLT file Follow the procedure in Creating a PLT file using Create PLT File on page 3 79 If you spot sample plates in more complex patterns you must create the PLT file manually Follow the procedure in Creating or editing a PLT file using Notepad editor on page 3 83 Sample Plate Types 3 5 5 Adjusting the Laser Position for a Custom PLT File After you have defined your own sample plate format 1 Spot sample on the four corner positions defined in the PLT file For optimum mass accuracy do not spot sample on the outer edges of the plate 2 Load the sample plate and PLT file as described in Section 3 4 4 Loading Sample Plates 3 Check the laser beam position on these sample positions as described in Figure 3 14 on page 3 78 If the beam does not strike the center of the sample position align the sample plate as described in Section 2 7 Aligning the Sample Plate Voyager Biospectrometry Workstation User s Guide 3 85 Chapter 3 Preparing Samples 3 86 Applied Biosystems 4 Voyager Chapter Instrument Control 4 Panel Basics This chapter contains the following sections 4 1 Instrument Control Panel 4 2 4 2 Using the Control Pages s 4 8 4 3 Using the Spectrum Window 4 10 4 4 Customizing the Instrument Control Panel 4 21 4 5 Controlling the Workstation 4 24
281. e all relevant precautions AVERTISSEMENT RISQUE CHIMIQUE Avant de manipuler des produits chimiques veuillez consulter la fiche de s curit du mat riel fournie par le fabricant et observer les mesures de pr caution qui s imposent Voyager Biospectrometry Workstation User s Guide xix Safety and Compliance Information To order MSDSs Then Over the Internet Go to our Web site at www appliedbiosystems com techsupport Click MSDSs Enter keywords or partial words or a part number or the MSDSs Documents on Demand index number Click Search Click the Adobe Acrobat symbol to view print or download the document or check the box of the desired document and delivery method fax or e mail By telephone in the United States Dial 1 800 327 3002 then press 1 XX Applied Biosystems General Warnings Safety and Compliance Information WARNING FIRE HAZARD Using a fuse of the wrong type or rating can cause a fire Replace fuses with those of the same type and rating AVERTISSEMENT DANGER D INCENDIE L usage d un fusible de type ou de valeur nominale diff rents risque de provoquer un incendie Il convient donc de remplacer les fusibles usag s par des fusibles du m me type et de la m me valeur nominale WARNING LASER HAZARD The laser emits ultraviolet radiation Lasers can burn the retina and leave permane
282. e an appropriate CAL file in the Data Explorer software then select the CAL file on the Instrument Settings page of the BIC file Voyager Biospectrometry Workstation User s Guide 5 97 Chapter 5 Optimizing Instrument Settings 5 98 Applied Biosystems Chapter 6 6 Acquiring Spectra from the Instrument Control Panel This chapter contains the following sections 6 1 Before You Begin 6 2 6 2 Acquiring in Manual Mode from the Instrument Control Panel 6 11 6 3 Obtaining Good Spectra in Delayed Extraction Mode 6 21 6 4 Making Accurate Mass Measurements 6 26 6 5 Evaluating Data in the Instrument Control Panel 6 28 6 6 Acquiring in Automatic Mode from the Instrument Control Panel 6 35 Voyager Biospectrometry Workstation User s Guide 6 1 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 1 Before You Begin This section includes e Overview of acquisition options e Guidelines for acquiring e Calibrating the mass scale 6 1 1 Overview of Acquisition Options NOTE You cannot operate the mass spectrometer with the front or side panels off Safety interlocks prevent operation when panels are not in place The Voyager system provides the following options for acquiring data Table 6 1 Voyager Acquisition Options Acquisition ar For more Description Option information see Manual
283. e appropriate settings instead of entering new settings Converting version 4 methods and search pattern files Loading Modifying and Saving Instrument Settings NOTE Data storage parameters are not stored in BIC files See Setting Data Storage parameters on page 6 14 for more information Before using methods MNU or search pattern files SP created using version 4 0 software convert to instrument settings BIC and version 5 compatible search pattern SP files For information see Section 5 5 Converting Version 4 Methods and Search Pattern Files 5 1 2 Standard Instrument Settings BIC Files Provided Standard instrument settings files Location of BIC files This section includes e Standard instrument settings files e Location of BIC files e BIC files and control mode e List of BIC files Standard read only instrument settings files are provided on your system These instrument settings files have been optimized for your instrument CAUTION These instrument settings files have been used to test and optimize your system Do not delete or change these instrument settings files Standard instrument settings BIC files are initially located in the C VOYAGER DATA FACTORY directory NOTE You can also export BIC files from an existing data DAT file in the Data Explorer software The standard BIC files provided are initially located in the C VOYAGER FACTORY direct
284. e base peak has a resolution below this value Resolution is measured at 50 percent of the most intense peak in the specified mass range Criteria Evaluation Mass Range Specifies the mass range to evaluate Signal to Noise Baseline Range Mass range in which to calculate noise if Signal to Noise is enabled above Saving the instrument settings BIC file 6 44 Applied Biosystems 3 Click OK to close the Spectrum Acceptance Criteria dialog box 4 Click OK to close the Automatic Control dialog box From the File menu in the Instrument Control Panel select Save Instrument Settings All instrument settings including Automatic Control mode setting Spectrum Acceptance Criteria and Laser Intensity Adjustment Criteria are saved Acquiring in Automatic Mode from the Instrument Control Panel 6 6 3 Automatically Acquiring Evaluating and Saving Spectra To automatically acquire a spectrum 1 Inthe Instrument Control Panel open or create an instrument settings file with the appropriate parameters See Section 6 6 2 Setting Instrument Settings for Automatic Control Mode for information 2 Set Data Storage parameters as described in Setting Data Storage parameters on page 6 14 NOTE You must specify a file name in the Data Storage contro page before acquiring in Automatic Control mode 3 Inthe Manual Laser Sample Positioning control page select the Active Position to anal
285. e clean particulate free spectrometer sample plate sample plate Arcing caused by negative Decrease Accelerating ion mode Voltage See Section 5 4 4 2 Setting Accelerating Voltage Arcing caused by excess Decrease amount of matrix in sample matrix in sample preparation may be preparation required for ionization of an certain samples If excess matrix is required to ionize sample decrease Accelerating Voltage See Section 5 4 4 2 Setting Accelerating Voltage Arcing caused by faulty Do not restart computer or grid in ion source software Call Applied Biosystems Technical Support Arcing caused by faulty Do not restart computer or electronic components software PCBs or cables Call Applied Biosystems Technical Support continued 9 24 Applied Biosystems Troubleshooting Table 9 5 Mass Spectrometer Troubleshooting Continued Symptom Possible Cause Action Sample holder empty when you click Eject CAUTION Do not load another plate if the sample holder is empty when you eject Problem with sample ejecting mechanism Call Applied Biosystems Technical Support Sample plate jammed in system Call Applied Biosystems Technical Support Vacuum gauge The Vacuum Gauge Panel Figure 9 2 is located on Voyager DE and Voyager DE PRO On the right front of the mass spectrometer cabinet panel Voyager DE STR On the front lower left of the mass spectro
286. e is described in Figure 6 1 t nsec to AVmz higher order terms where t Flight time of the ion Difference in time between the start time of the analysis and the time of ion extraction in Delayed Extraction or the time of ionization in Continuous Extraction Also called Effective Delay A Effective Length mm A a x 105 vV Accelerating Voltage V where m 1 dalton mass in SI units 1 66054 x 10 27 kg e charge of electron in SI units 1 602177 x 10 Coulomb Effective Length Length of flight tube corrected for ion acceleration through the stages of the source and flight tube and for the impact of the guide wire m z mass to charge ratio Figure 6 1 Calibration Equation Default The default theoretical calibration is a multi parameter calibration equation that uses calculated values for t and A Calculated values are based on the configuration of the system such as flight tube length and accelerating voltage Voyager Biospectrometry Workstation User s Guide 6 9 Chapter 6 Acquiring Spectra from the Instrument Control Panel Generated if you are performing an internal standard calibration the calibration software determines the constants as listed below Internal Standard Constant Value Used Calibration One point A Calculates from standard mass to Uses value from default calibration Two point tp and A Calculates from standard mass
287. e laser at minimum The system continues with step 3 Too high with the laser at minimum The system begins acquiring in Acquisition mode e Within range The system begins acquiring in Acquisition mode 3 The system sets the laser halfway between the minimum and maximum and acquires a spectrum see Shots Spectrum in Prescan mode on page 6 58 For the first spectrum minimum and maximum laser settings are taken from the Automatic Control dialog box For subsequent spectra minimum and maximum laser settings are determined as described below e Ifthe signal is too low the current setting is used as the new minimum setting e Ifthe signal is too high the current setting is used as the new maximum setting 4 The system repeats step 3 until either e The signal falls between the specified maximum and minimum values e The difference between the maximum and minimum laser intensity in step 3 is smaller than 2 times the laser step size specified in the Automatic Control dialog box 6 60 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Acquisition mode Acquisition mode starts after Prescan mode determines the laser setting or immediately if Prescan mode is disabled NOTE Each time the system begins acquiring in Acquisition mode it acquires the number of Shots Spectrum specified in the Instrument Settings control page In Acquisition mode the system does the foll
288. e minimizes the distance the plate must travel to reanalyze the reference position 3 Allow the sample plate to dry Voyager Biospectrometry Workstation User s Guide 2 59 Chapter 2 Installing the Voyager Biospectrometry Workstations Preparing the To prepare the system system 4 Assign a Plate ID if you have not already assigned one for the plate For information see Assigning Plate IDs on page 3 50 2 Load the plate into the mass spectrometer as described in Section 3 4 Loading Sample Plates in the Mass Spectrometer Verifying laser Two instrument settings BIC files are provided for use with intensity OptiPlate e OptiPlate_Linear BIC e OptiPlate_Reflector BIC These files are set with values that have been optimized for your system and contain the following acceptance criteria to ensure best results when you run OptiPlate Parameter Setting Minimum 20 000 Signal Intensity Maximum 60 000 Signal Intensity Stop Stop after 8 consecutive failing Conditions acquisitions Criteria e Linear mode 1 000 to 6 000 Da Evaluation e Reflector mode 1 000 to Mass Range 3 000 Da 2 60 Applied Biosystems Running OptiPlate to Optimize Mass Accuracy Before running OptiPlate you need to e Determine the laser intensity that yields a signal intensity between 20 000 and 60 000 counts see below e Set the Minimum and Maximum Laser Intensity settings in Automatic Control to
289. e plate and insert it into the main source chamber The Load Eject Sample Plate dialog box Figure 3 8 is displayed If you are removing a plate but not loading a new plate click Load No Plate and skip the remaining steps Voyager Biospectrometry Workstation User s Guide 3 59 Chapter 3 Preparing Samples Load Eject Sample Plate x Plate ID ha Plate Type Last Aligned Tre sample plate has not been aligned Optimization No Plate ID selected Created a UMass Ecua Upinzania Warming Be sure hands are free of sample loading mechanism PIN Load Cancel Load No Plate Figure 3 8 Load Eject Sample Plate Dialog Box 5 Select a Plate ID The PLT file plate alignment information and plate optimization information associated with the Plate ID are automatically loaded For more information see Section 2 7 Aligning the Sample Plate and Section 2 8 Running OptiPlate to Optimize Mass Accuracy Alternatively you can specify a new Plate ID then select a PLT file For more information see Assigning Plate IDs on page 3 50 6 Select Use Mass Accuracy Optimizations to apply calibration corrections to the data acquired from the plate Before selecting this option read Section 3 4 2 Using the Mass Accuracy Optimization Option to understand how mass accuracy optimization is applied See Optimization strategy on page 3 53 to understand the options you have when using Mass Accuracy Optimi
290. e pulses Laser attenuator Device that controls laser intensity Controlled by laser step setting Laser intensity See Intensity Laser step unit of movement of laser attenuator The size of the laser step is set in Laser Configuration The number of laser steps is controlled by using the slider controls on the Manual Laser Control page Voyager Biospectrometry Workstation User s Guide Glossary Low Mass Gate Mechanism for suppressing low mass ions The voltage in the detector is not turned on until ions below the specified starting mass have passed Ladder sequencing Peptide or DNA sequencing in which amino acid sequence is read from one of the ends of the peptide chain M H _Molecular ion M 2H 2 _Doubly charged ion Appears in spectrum at approximately half the molecular weight of the ion M 3H 3 Triply charged ion Appears in spectrum at approximately one third the molecular weight of the ion MALDI Matrix assisted laser desorption ionization Described in Section 1 3 MALDI TOF MS Technology Overview Mass accuracy The error between the observed mass and the calculated mass from atomic composition Can be expressed as mass units or the ratio of mass assignment error divided by ion mass in percent terms Mass resolution Describes the separation between adjacent mass peaks Expressed as the mass m of the ion signal divided by the width of the peak at half height
291. e signal intensity However varying the laser intensity during acquisition may affect mass accuracy Voyager Biospectrometry Workstation User s Guide 6 5 Chapter 6 Acquiring Spectra from the Instrument Control Panel e Observe the Spectrum window or Oscilloscope screen to determine if data is acceptable See Section 4 3 Using the Spectrum Window or Appendix I Using the Oscilloscope and Control Stick for more information Obtaining The quality of the data you acquire is directly affected by acceptable data ionization properties of the sample e Sample preparation discussed in Section 3 1 Preparing Samples e Laser intensity e Laser position on the sample e Acquisition instrument settings It may take some practice before you observe spectra that are acceptable for your analyses For examples of acceptable data and guidelines for obtaining acceptable data refer to Section 6 3 Obtaining Good Spectra in Delayed Extraction Mode Obtaining For maximum mass accuracy note the following maximum mass Use the Mass Accuracy Optimization option when accuracy acquiring For information see Section 3 4 2 Using the Mass Accuracy Optimization Option e Acquire at least six replicates of each sample for a statistically significant sampling of data e Collect at least 50 shots of data determined by the Shots Spectrum parameter in the Instrument Settings control page e Set Centroid at 50 for most application
292. e type on area available for analysis Sample Plate Types When creating search pattern SP files to analyze sample positions defined in custom PLT files note that the area available for analysis around the absolute X Y coordinate of the position specified in the PLT file is determined by three factors e Plate being used e Tolerances and non systematic errors e Search pattern radius Search pattern files are described in detail in Section 6 6 4 Search Patterns NOTE You can correct for systematic errors introduced by a sample preparation device by aligning the sample plate in the Voyager Workstation See Section 2 7 Aligning the Sample Plate If you are using different sample preparation devices you can compensate for different systematic errors by creating different PLT files and sample plate alignments for each system When creating search pattern files consider the plate types used e Ifyou are using laser etched position plates the area available for analysis is limited to the well or laser etched position size 2 540 um on a 100 well plate e If you are using plates with polished blank surfaces the area available for analysis is determined by the proximity of other positions specified Approximately half the distance between the X Y coordinates of two adjacent positions is available for analysis of each position For example if the center X Y coordinates of the two positions are 5 080 um apart plus
293. e water with the appropriate side up refer to manufacturer s information Do not use your fingers because you can contaminate the membrane with oil and salt Place about 10 parts of sample for example 10 ul in the center of the membrane Voyager Biospectrometry Workstation User s Guide 3 27 Chapter 3 Preparing Samples 5 Place 1 part of pure acetonitrile for example 1 ul on top of the sample spot to increase surface area Do not exceed a 10 percent concentration of organic It may dissolve the membrane WARNING CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 6 Cover the container to prevent drying and allow it to sit for 30 minutes 7 Note the size of the sample organic spot then let it sit for 30 more minutes The size of the sample spot increases as dialysis is occurring This can be used to check for completion 8 Observe the size of the sample organic spot If the size of the spot is larger than the first time you checked it allow the sample to sit for 15 more minutes Observe the size of the sample organic spot again NOTE Sample also passes through the membrane during dialysis particularly low mass samples Dialyz
294. eadache and so on Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Voyager Biospectrometry Workstation User s Guide 3 29 Chapter 3 Preparing Samples WARNING CHEMICAL HAZARD Hexane is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on and damage the peripheral nervous system numbness of the extremities Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves After washing beads can be stored indefinitely and used as needed Procedure To perform cation exchange 1 Place about 0 1 mg a small spatula tip full of cation exchange beads on a piece of laboratory film 2 Add 5to 10 ul of sample for 200 mesh cation exchange beads If sample quantity is limited you can make up the difference with deionized water 3 Add 5 to 10 ul of matrix on top of the sample as matrix may also contain salt 4 Mix by withdrawing and expelling about 20 times with a pipet NOTE The volume of sample decreases as the beads absorb water 5 Allow the beads to settle for 30 seconds 6 Remove the supernatant with a clean pipet tip and spot on the sample plate 3 30 Applied Biosystems Prepa
295. eating and Editing SP Files Using the Search Pattern Editor on page 6 50 7 7 4 Internal Standard Calibration Considerations Optimizing unknown and standard concentration This section includes Optimizing unknown and standard concentration Determining if standard suppresses signal from unknown Creating a search pattern file for internal calibration on separate spots Performing internal standard calibration with an external calibration backup Before combining sample and internal standard determine the concentration of each that yields optimum signal intensity without saturation To optimize relative peak intensities determine the peak intensity of the sample and adjust the concentration of the standard to bring the standard peak intensity into the same range Voyager Biospectrometry Workstation User s Guide 7 39 Chapter 7 Acquiring Spectra from the Sequence Control Panel Determining if standard suppresses signal from unknown Creating a search 7 40 pattern file for internal calibration on separate spots Applied Biosystems In some cases standard signal can suppress unknown signal Mix optimized concentrations of standard and unknown and analyze to determine if you see adequate signal for all compounds If standard signal suppresses unknown signal you have two options for analysis Perform internal calibration using separate spots Spot standard and sample as close to eac
296. echnique described in Section 3 2 4 Loading Samples Thin Layer Application Mix sample and matrix in microcentrifuge tubes before applying sample to the sample plate when you are e Working with concentrated samples with a high salt concentration and need to make dilutions e Preparing many samples e Analyzing non polar samples and matrix prepared in high organic concentration that evaporates rapidly For peptides and proteins mix 1 ul of sample concentration of 0 1 to 100 pmol l and 9 ul of matrix in a microcentrifuge tube for a final concentration of 0 1 to 10 pmol ul Mix well on a vortex mixer or shake by hand For other compounds refer to Matrix Information on page 3 6 to determine the volume of sample and matrix to mix to yield the necessary final sample concentration NOTE If sample concentration is too high the sample signal may be suppressed If sample concentration is too low sample signal may not be present Voyager Biospectrometry Workstation User s Guide 3 33 Chapter 3 Preparing Samples Mixing sample and matrix on the 3 34 sample plate Applied Biosystems Mix sample and matrix directly on the sample plate when you are e Working with dilute samples and can make a 1 1 preparation of sample matrix e Analyzing only a few samples e Concerned about sample adhering to plastic tubes When mixing directly on the plate you may need to use a higher concentration of organic
297. ect Exit to return to the Window NT desktop 9 22 Applied Biosystems Troubleshooting 9 2 3 Hardware Troubleshooting This section includes Mass spectrometer troubleshooting Vacuum gauge panel troubleshooting External laser troubleshooting Table 9 5 Mass Spectrometer Troubleshooting Symptom Possible Cause Action Internal stepper motor making noise when the sample plate is moving Normal operation of the sample plate stepper motor No action Normal occurrence High whining sound when you power up the mass spectrometer Normal startup operation of the turbo pump No action Normal occurrence Internal stepper motor making grinding noise when the sample plate is not moving or is moving erratically Home or Load position not found may be displayed on startup Problem with sample positioning system Do not restart computer or software Call Applied Biosystems Technical Support In Automatic Control mode center of sample position is not aligned with laser spot as observed on the video monitor Sample plate not aligned Align See Section 2 7 Aligning the Sample Plate Voyager Biospectrometry Workstation User s Guide continued 9 23 Chapter 9 Maintenance and Troubleshooting Table 9 5 Mass Spectrometer Troubleshooting Continued Symptom Possible Cause Action Cracking sound in mass Arcing caused by dirty Us
298. ection includes the following specifications for the Voyager DE STR Workstation with Delayed Extraction technology Performance e Mass Spectrometer e Miscellaneous NOTE For specifications on Voyager DE STR systems with serial number 4153 and earlier refer to the original Voyager Workstation User s Guide provided with your system Table A 7 Voyager DE STR Performance Specifications for Installation Condition Specification Resolution FWHM Full Width at Half Maximum in Delayed Extraction mode Linear mode 23 000 for Angiotensin I 1 3 pmol ul e 23 500 for ACTH 18 39 1 5 pmol l Reflector mode 220 000 for bovine insulin 3 5 pmol ul 21 200 for E coli Thioredoxin 2 75 pmol l e 212 000 for Angiotensin 2 0 pmol ul e 212 000 for ACTH Clips e ACTH 1 17 2 0 pmol l e ACTH 18 39 1 5 pmol ul ACTH 7 38 3 0 pmol ul M H ion analyzed Signal to Noise Ratio e 2200 1 for IgG 0 6 pmol l e 2100 1 for BSA 4 pmol ul Sensitivity Routine detection of 5 fmol of neurotensin with a signal to noise ratio gt 80 1 A 8 Applied Biosystems Voyager DE STR Specifications Table A 7 Voyager DE STR Performance Specifications for Installation Condition Specification Mass Accuracy in Delayed Extraction mode difference between the mean of six measurements External Calibration 0 05 and the theoretical mass of the sample Linear mode Internal Calibra
299. ecular research and facilitate data interpretation Features Features of the Voyager DE STR Biospectrometry Workstation include e High speed digitizer options and laser rates for optimum sample throughput e Positive and negative ion detection e Modular horizontal design e High performance reflector e Post source decay PSD analysis capability Linear or reflector mode operation e lon path length e Linear mode 2 0 meter e Reflector mode 3 0 meter e Timed ion selection e m z range in excess of 300 kDa e Sensitivity to less than 5 femtomoles with dried droplet application e Variable two stage ion source e Automated single plate sample loading system sample plates of various formats available e lon focusing lens technology for increased sensitivity serial number 4154 and later e Video camera and monitor for sample viewing e Low mass gate matrix suppression e Intuitive Microsoft Windows NT based software Collision induced dissociation CID option e Second laser option 1 6 Applied Biosystems MALDI TOF MS Technology Overview 1 3 MALDI TOF MS Technology Overview Mass Mass spectrometry measures the mass of molecules by spectrometry measuring the mass to charge ratio m z Mass is a molecular attribute that can help identify or confirm the identity of a molecule Molecular weight measurements by mass spectrometry are based upon the production separation and detection of molecular ions A typi
300. ed 2 To save the changes under the current instrument settings file name select Save Instrument Settings from the File menu The name of the current instrument settings BIC file is displayed in the title bar of the Instrument Control Panel NOTE If you try to save a read only instrument settings file under the current instrument settings file name a message is displayed indicating that the file cannot be saved The current settings can be used for analysis but cannot be permanently saved to the instrument settings file You can save the changes by saving the file with a new name by selecting Save Instrument Settings As from the File menu Changes to the instrument settings file are lost if you do not save them before you open a different instrument settings file or exit the Voyager Instrument Control Panel 3 To save the settings under a different instrument settings file name select Save Instrument Settings As Enter anew name and click Save Saving BIC files The laser intensity required to yield optimum resolution and for different signal to noise ratio for a sample can vary substantially for matrixes different matrixes Other instrument settings do not vary for the same sample in different matrixes Because the laser setting is saved in the BIC file you can create and save different BIC files for different matrixes For more information on laser intensity settings see Section 5 4 2 Determining the Las
301. ed and add the correction information to the existing optimization file Replace existing correction factors for positions you select Positions selected for optimization are indicated with a white dot in the center of the position Starting acquisition 8 Click Start Acquisition Acquisition starts in the Instrument Control Panel Acquisition and processing status are displayed at the bottom of the OptiPlate screen for each position as it is acquired The Data Acquisition mode is automatically set to Append Data when you start acquisition 2 66 Applied Biosystems Running OptiPlate to Optimize Mass Accuracy During an During an OptiPlate run the software OptiPlate run e Acquires a spectrum at the Reference position e Internally calibrates the reference spectrum using monoisotopic masses in Reflector mode or average masses in Linear mode e Acquires a spectrum at each specified position and applies the reference calibration to the data e Calculates and displays the Average Error ppm for the position compares the observed masses to the observed reference masses then determines the average error across all expected peak masses for the components listed in Table 2 2 Standard Requirements on page 2 55 e Calculates and displays the Extraction Correction mm for the position by performing a least squares fit of the data e Displays a color code for each position see Color coding on page 2 69 and
302. ed with your system PSD_Precursor BIC and Angiotensin _PSD BIC are read only You cannot save changes to these files unless you assign a new name In the Manual Laser Intensity Sample Positioning control page select the same sample position from which you acquired the precursor spectrum From the View menu select Data Storage Set parameters as needed See Setting Data Storage parameters on page 6 14 for information Hint Include a _PSD suffix when you name PSD data files to help you distinguish them from non PSD data files For example if you type in Experiment1_PSD as the file name the complete data file name will be Experiment1_PSD DAT or Experiment1_PSD_0001 DAT if Autosequence File Names is enabled Voyager Biospectrometry Workstation User s Guide 8 9 Chapter 8 PSD Analysis Selecting and 3 Inthe PSD Acquisition Settings control page select the acquiring a first row click the number box in the Segment column segment that corresponds to the segment you want to acquire 4 To start acquiring select Start Acquisition from the Acquisition menu or click Al 5 Adjust laser intensity to optimize signal intensity You typically need a higher laser intensity to optimize signal intensity for segments with lower Mirror Ratio settings The laser intensity needed for the first segment the segment with the highest Mirror Ratio setting is typically 150 to 200 counts higher than the laser intens
303. eeeeeceeeeeeeeeeeeeeeeeeneees 5 71 5 4 3 1 OVervieW iii 5 72 5 4 3 2 Acceptable Resolution in Delayed Extraction Mode 5 75 5 4 3 3 Optimizing Guide Wire Voltage 5 76 5 4 3 4 Optimizing Delay Time seses 5 77 5 4 3 5 Optimizing Grid Voltage eeeeceeeeeeeeeeeeee ease etenees 5 82 5 4 4 Optimizing Signal to Noise Ratio ccceceeeeeeeeeeeeeeeeeeeeeeees 5 85 5 4 4 1 OVOIVIGW ne ra tan tete test sas tues 5 86 5 4 4 2 Setting Accelerating Voltage 5 87 5 4 4 3 Setting Guide Wire Voltage seenen 5 88 5 4 4 4 Setting Shots Spectrum sesser 5 89 5 4 4 5 Setting Low Mass Gate assecc 5 89 Converting Version 4 Methods and Search Pattern Files 5 92 Chapter 6 Acquiring Spectra from the Instrument Control viii 6 1 6 2 6 3 6 4 Panel Befor You BEGI creses rarte nn dde andre ta porn nanas an 6 2 6 1 1 Overview of Acquisition Options c ceeeeeeeeeeeeeeeeeeeeeaaaee 6 2 6 1 2 Guidelines for Acquiring cccceeeeeeeee eee eeeeeeeeeeeeeeeeeeeaeeeaeeaea 6 4 6 1 3 Calibrating the Mass Scale cccceceseeeeeeeeeeeeeeeeeeeeeeeeeeaeaaaes 6 7 Acquiring in Manual Mode from the Instrument Control Panel 6 11 6 2 1 Manually Acquiring Evaluating and Saving Spectra 6 11 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions 6 19 Obtaining Good Spectra in Delayed Ex
304. efresh the spectrum view until averaging is finished Unlike the real time update in the spectrum view the oscilloscope screen displays the detector signal on a time scale and the signal is inverted on the screen Following acquisition the signal is inverted and calibrated onto the mass scale in the spectrum view Figure I 1 shows the front panel of the oscilloscope Only the knobs you use to adjust the signal while acquiring spectra are labeled Other knobs are not used for general operation Vertical Position Horizontal Position Select Cursor Control 585850 00000 o o oa es oe oe PB100276 Reset Button Vertical Scale Horizontal Scale Figure I 1 Front Panel of Oscilloscope Appendix Oscilloscope overview N l 2 Applied Biosystems Guidelines for Acquiring 1 1 Guidelines for Acquiring Consider the following as you acquire a spectrum and use the oscilloscope When you start acquiring you should see a signal that contains matrix peaks and sample peaks Make sure the full range of the signal is displayed Brackets must overlap the end range markers see Figure l 3 If brackets do not overlap turn the Horizontal Scale and Horizontal Position knobs until the left bracket overlays the left marker Start with a high laser power to saturate the initial signal This is a good starting point from which you can
305. eft corner of the sample plate NOTE If the coordinates are not displayed you can click drag the bottom border of the control page to make them visible Voyager Biospectrometry Workstation User s Guide 4 29 Chapter 4 Voyager Instrument Control Panel Basics Switching between Plate You can use these coordinates when you create a search pattern file For information on creating an SP file see Creating and Editing SP Files Using the Search Pattern Editor on page 6 50 You can change the view of the sample plate between the whole sample plate and a single sample position Refer to the and following table for plate view choices Sample view In this view If you The view Plate Single click on a position Moves to the selected position Figure 4 8 on Displays position number in Page ET the Active Position field Turns the Active Position white Double click on the active Moves to the selected position position Displays position number in NOTE If you double click the Active Position field DeLee poses ANG Switches to Sample View software zooms on the location on which you Turns the Active Position clicked and selects the white closest defined position as the Active Sample Position Double click on a non active Moves to the selected position position Makes it the active position Displays position number in the Active Position field Sample Single click or double click Moves
306. elector 8 69 8 6 3 Observing the Effects of Grid Voltage 8 73 8 6 4 SUMMANY weve eel sev etn ti ia dii iiie ii Heidi teeters 8 76 Viewing PSD Data 3 222 nn ate ieee gia desde date 8 77 Chapter 9 Maintenance and Troubleshooting 9 1 9 2 Maintenance nEaN Ta E KENNEN A E KENANA PAE AA RE nel 9 2 9 1 1 Maintenance Schedule cc cceeeeeeeeeeeeeaeeeaeeaaeeeeeeeeeereeeeeees 9 2 9 1 2 Hardware Maintenance ssseeeessesesssrrirsrssrrriissrrrrressrrrressrrrnns 9 3 9 1 3 Backing Up and Archiving Data aeseeeeeseeerrreersrrrressrrrnn 9 6 Troubleshooting a aea rea dis KAEVAS ae ca dee oe ieee 9 7 9 2 1 Spectrum Troubleshooting esssssssssessessesssrserirrrrrrrrereerssrernnns 9 7 9 2 2 Software Troubleshooting ccceceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeaaes 9 19 9 2 3 Hardware Troubleshooting 9 23 Voyager Biospectrometry Workstation User s Guide xi Table of Contents Appendix A Specifications 2 0 ccc ee eeeteeeeteees A 1 Appendix B Warranty Service Information B 1 Appendix C Matrixes 00 00 00 cs esse neeteseeteeneeeaseeseenen C 1 Appendix D Log Sheets D 1 Appendix E Grid Voltage and Delay Time Settings E 1 Appendix F Reference Standard Information F 1 Appendix G Maintenance Log G 1 Appendix H Continuous Extraction Mode 0
307. ends on whether or not Acceptance Criteria fail acquires from same search pattern position until Acceptance Criteria fail and on how many times the system must acquire until it obtains five spectra that pass acceptance criteria One DAT file containing one accumulated spectrum is created NOTE If you set the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified number of spectra to acquire or the number of search pattern positions in the SP file Voyager Biospectrometry Workstation User s Guide 5 47 Chapter 5 Optimizing Instrument Settings If an SP file with 7 positions is selected and Number to Acquire is 5 the number of positions analyzed is determined by whether or not acceptance criteria fail e If all fail seven positions are analyzed total number of positions in SP no DAT file is created e If the first position fails but all remaining pass the first two positions are analyzed one DAT file containing one accumulated spectrum is created e If the first and second positions fail but all remaining pass the first three positions are analyzed one DAT file containing one accumulated spectrum is created and so on e If acceptance criteria never fail the first search pattern position is analyzed and one DAT file containing one accumulated spectrum is created NOTE If you set
308. ength Voyager DE PRO 1 4 path length Voyager DE STR 1 6 sensitivity 5 25 setting 5 25 when to use H 20 Linking traces 4 13 Live Spectrum trace definition 4 14 during acquisition 6 16 not displayed on oscilloscope systems 6 16 Load No Plate 3 57 3 59 Load Eject dimmed 9 19 Loading sample plate in mass spectrometer 3 50 sample plates Instrument Control Panel 4 25 samples on plates 3 35 Log file Sequence Control Panel 7 14 Log sheet maintenance G 1 sample loading D 1 Logging on to Windows NT Username and password 2 74 without initializing hardware 2 75 Low Mass Gate description 5 20 5 89 function 5 89 impact on calibration 5 89 improving signal to noise ratio 5 89 PSD mode disabled in 8 43 spike in Spectrum window 5 91 starting mass recommendations 5 90 when to use 5 20 5 89 5 90 Index 22 Applied Biosystems Low Mass Gate spike description 5 91 eliminating 5 91 Low mass ions acceptable resolution 6 32 H 17 cannot see in PSD mode 9 21 improving peak definition 5 88 LSA1000 LeCroy digitizer see Digitizer M m z 1 7 Macros example for multispectrum data file 7 11 Macros Sequence Control Panel creating 7 8 description 7 3 selecting 7 17 7 20 Maintenance archiving 9 6 computer 9 6 hard disk 9 6 log sheet G 1 mass spectrometer hardware 9 3 preventative 9 2 schedule 9 2 MALDI TOF advantages 1 10 definition 1 7 description 1 8 limitation of traditional 1 11 Manual Control mode see also Acquiring data
309. ent instrument settings BIC files The Sequence Control Panel Figure 1 22 is displayed when you start the Voyager Sequence Control software or click a toolbar button in the Instrument Control Panel The Sequence Control software and how the Instrument and Sequence Control panels interact are described in Chapter 4 Voyager Instrument Control Panel Basics Foret sea sea De ES Yow Gated Minden Doit tee Disc FBS Ss lle amp Figure 1 22 Sequence Control Panel Data DAT file Data DAT file format incorporates all information into a format single file about how a data file is acquired and processed This format improves data processing and data storage efficiency Voyager DAT files can contain one or more spectra You can also store parameters in separate files by extracting information from a DAT file as needed for use with other files For more information the Data Explorer Software User s Guide Section 1 2 File Formats and Types 1 44 Applied Biosystems Software Overview 1 7 2 Post Processing Software Data Explorer The Data Explorer software is a powerful software module that allows you to graphically and interactively manipulate spectral and chromatogram multispectrum data Using the Data Explorer software you can e Automatically and manually calibrate spectrum data e Set peak detection parameters and custom labels for regions of the trace e Calculate centroid mass ele
310. er Biospectrometry Workstation User s Guide 2 75 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 11 Checking System Status and Pressures System pressures Display the System Status page for complete status information In the Instrument Control Panel select System Status from the View menu to display the System Status page Figure 2 29 System Status Instrument JON High Voltage OFF Source Chamber BA1 Pressure 252006 Mirror Chamber BA2 Pressure 252006 OFF OFF Acquisition Data Storage Figure 2 29 System Status Control Page System and acquisition status are represented by colored indicators and read only text fields described in Table 2 3 Colors represent Green OK Yellow Fault e Gray Off Before acquiring data allow the pressure in the Source Chamber BA1 and Mirror Chamber BA2 to reach Model Source Chamber BA1 Pressure Torr Mirror Chamber BA2 Pressure Torr Voyager DE and Voyager DE PRO Less than 10 6 Less than 10 8 Voyager DE STR Less than 5x10 7 Less than 5 x10 2 76 Applied Biosystems Checking System Status and Pressures NOTE At initial startup or after venting the system it will require one to two days after startup to reach these vacuum level Status Status parameters are described in Table 2 3 parameters Table 2 3 System Status Parameters Parameter Descr
311. er C L U LI E ll PB100427 Figure 1 17 Voyager DE STR Biospectrometry Workstation Voyager Biospectrometry Workstation User s Guide 1 31 Chapter 1 Introducing the Voyager Biospectrometry Workstations Parts of the Voyager DE STR system include e Mass spectrometer A time of flight mass spectrometer described in Section 1 6 2 Mass Spectrometer The high vacuum system of the mass spectrometer is described in Section 1 6 3 Vacuum System e Computer Data System A computer that operates the Voyager control software and the Voyager processing software You control the mass spectrometer using the computer e Digitizer not shown An analog to digital converter that allows the signal from the mass spectrometer to be transferred to the computer The standard digitizer provided is a 4 GHz digitizer e Video monitor A monitor that displays a real time video image of the sample spot for examination during acquisition e Control stick A peripheral device that controls position of the sample plate in the mass spectrometer Allows you to start and stop acquisition and transfer data to the processing software NOTE You can also contro sample position start and stop acquisition and transfer data using the Voyager Instrument Control Panel software 1 32 Applied Biosystems Parts of the Voyager DE STR System 1 6 2 Mass Spectrometer The parts of the Voyager DE STR Bio
312. er Setting Voyager Biospectrometry Workstation User s Guide 5 11 Chapter 5 Optimizing Instrument Settings Saving BIC files 5 12 for different modes Printing Applied Biosystems When saving BIC files for use in different operating modes make sure to create an identifier so that you will know which instrument settings BIC files are optimized for which experiments For example e Linear mode Use LIN BIC e Reflector mode Use _REF BIC PSD mode Use PSD BIC To print instrument settings 1 Open the instrument settings file in the Instrument Control Panel 2 Select Print Instrument Settings from the File menu The instrument settings file prints Loading Modifying and Saving Instrument Settings 5 1 6 Setting Instrument Settings Files to Read Only Status Standard instrument settings files are read only files Read only files cannot be changed and saved However they can be changed temporarily and used without saving them or saved with a new name You can set any instrument settings file to read only status To set an instrument settings file to read only status 1 Display the Windows NT Explorer 2 Select the instrument settings BIC file you want to set to read only status 3 Select Properties from the File menu The Properties dialog box is displayed Select Read only from the Attributes section Click OK The file is listed with r next to the file
313. ers have the advantage that they have no upper mass limit and are well suited to the analysis of large molecules Torr A unit of pressure equal to one millimeter of mercury Turbo pump A vacuum pump that operates in the pressure range where the mean free path is large compared to the chamber dimensions Molecules that diffuse into the throat are knocked to the bottom by a fan operating at supersonic speeds The collected gas is further compressed to atmospheric pressure by a mechanical fore pump Turbo pumps operate well up to about 10 Torr Voyager Biospectrometry Workstation User s Guide Glossary Velocity focusing in Delayed Extraction the tuning of the Grid Voltage and Delay Time to account for the different initial velocities of ions as they are released from the sample plate Velocity focusing provides improved resolution See Velocity focusing on page 1 15 V volt X Y and z ions Generic description of potential ions that are formed by fragmentation of a parent peptide protein x y and z ions are fragments that retain the charge at the carboxy end c terminus of the molecule See also a b c ions zn xn yn p cofin Rn Rn 1 Glossary 5 lt II UVODNO Glossary G L O S S A R Y Glossary 6 Applied Biosystems Bibliography General Mass Spectrometry Beavis R C and B T Chait Chem Phys Lett 1991 181 479 Cotter R J Time of Flight Mass
314. es samples Are prepared in phosphate buffers Ammonium salts or derivatives of organic amines ammonium bicarbonate TRIS HCl do not usually cause a problem in low concentrations less than 50 mM e Contain salt for example from cation or anion exchange purification e Are contaminated with detergent Symptoms that indicate the need for sample cleanup include e Poor crystallization on the sample plate e Large tail on the high mass side of peaks which may be unresolved salt or buffer adducts e Poor sensitivity with a sample concentration that should yield a strong signal To detect this condition mix the sample with a standard that you know yields a strong signal If the standard no longer exhibits the expected signal a contaminant in the sample is affecting sensitivity Voyager Biospectrometry Workstation User s Guide 3 25 Chapter 3 Preparing Samples 3 1 5 1 Washing 3 26 When to use What you need Procedure Applied Biosystems Use this technique when you know the solubility of the contaminant You can wash a dried sample directly on the sample plate Select a solvent in which the contaminant is more soluble than the matrix and the sample For example use e Cold water to prevent sample dissolving with 0 1 TFA for a polar contaminant such as a salt or buffer e 5 isopropanol in water for a non ionic detergent such as octyl glucoside WARNING CHEMICAL HAZARD Isop
315. es 1 42 1 7 2 Post Processing Software Data Explorer cee 1 45 Voyager Biospectrometry Workstation User s Guide iii Table of Contents Chapter 2 Installing the Voyager Biospectrometry iv 2 1 2 2 2 3 2 4 2 5 2 6 2 7 Workstations Installing the Syste ni is ns aa denies sae aaa E SEA EEKE aaa evs 2 2 Selecting the Site as a aa a A ahs aa aaae 2 2 2 2 1 Voyager DE and Voyager DE PRO Workstations 2 2 2 2 2 Voyager DE STR Workstation 0 cceceeeeeeeeeee eee aeeeneeneeeeeeeees 2 7 Connecting Voyager DE and Voyager DE PRO Workstations 2 8 2 3 1 Side Panel Diagrams for Mass Spectrometer and Computer 2 9 2 3 2 Connecting the Mass Spectrometer to the Computer 2 12 2 3 3 Connecting the Signatec 500 MHz Digitizer Board 2 13 2 3 4 Connecting the LSA1000 LeCroy Digitizer 0 0 0 0 ee 2 15 2 3 5 Connecting the Acajiris Digitizers ccecceecesssseeeeeeeaaeeeeeees 2 17 2 3 6 Connecting the Tektronix Oscilloscope 2 19 2 3 7 Connecting the Video Monitor sseeeeeeeeeeeeeeeaeeaeeaees 2 21 2 3 8 Connecting Devices to the Computer ceeeeeeeeeeeeeeeeees 2 22 Connecting the Voyager DE STR Workstation 2 23 Installing Software r sise men Menu mien ru res 2 29 2 5 1 Installing the Voyager Software ceeeeeeceeee eee eeteeeteeeeeeeeeeees
316. ese values are not exact Use them as a guide Time seconds Standards Z Re Li 25 000 V 10 000 V 28 000 V 15 000 V 10 000 V 13m 1 3 m 2 0 m 2 0 m 2 0 m Leucine 1 556 61 1 40E 05 2 21E 05 2 03E 05 2 77E 05 3 40E 05 Enkephalin Methionine 1 574 65 1 42E 05 2 24E 05 2 06E 05 2 82E 05 3 45E 05 Enkephalin Bradykinin 1 1061 2 1 93E 05 3 05E 05 2 80E 05 3 83E 05 4 69E 05 2 531 1 1 36E 05 2 16E 05 1 98E 05 2 71E 05 3 32E 05 Angiotensin Il 1 1047 18 1 92E 05 3 03E 05 2 78E 05 3 80E 05 4 66E 05 human 2 524 09 1 35E 05 2 14E 05 1 97E 05 2 69E 05 3 30E 05 Angiotensin 1 1297 48 2 13E 05 3 37E 05 3 10E 05 4 23E 05 5 19E 05 human 2 649 24 1 51E 05 2 38E 05 2 19E 05 3 00E 05 3 67E 05 Melittin 1 2847 5 3 16E 05 4 99E 05 4 59E 05 6 27E 05 7 68E 05 2 1424 25 2 23E 05 3 53E 05 3 25E 05 4 44E 05 5 43E 05 F 4 Applied Biosystems Conversion of Mass to Time for Typical Standards Time seconds Aver MW Standards un 25 000 V 10 000 V 28 000 V 15 000 V 10 000 V 1 3m 1 3m 2 0 m 2 0m 2 0 m Bovine Insulin B 3496 9 3 50E 05 5 53E 05 5 09E 05 6 95E 05 8 51E 05 Oxidized 1748 95 2 48E 05 3 91E 05 3 60E 05 4 92E 05 6 02E 05 Bovine Insulin 5734 5 4 48E 05 7 09E 05 6 52E 05 8 90E 05 1 09E 04 2867 75 3 17E 05 5 01E 05 4 61E 05 6 30E 05 7 71E 05 Insulin like 7650 76 5 18E 05 8 19E 05 7 53E 05 1 03E 04 1 26E 04 Growth Factor lgF 382
317. etry Workstations Detection Extraction delay and the magnitude of the extraction pulse can set that ions of a given mass to charge ratio with different initial velocity reach the detector exactly at the same time Detector t t slow fast Figure 1 10 Velocity Focusing of lons in Linear Mode Detection Phase Voltage U PB100773 In Reflector mode ions are velocity focused at the exit of the ion source instead of at the detector By tuning the variable voltage grid and the acceleration delay time you can alter the point at which ions are focused and enhance resolution This initial focus is refocused by the reflector which you can tune for second order velocity focusing Parts of the Voyager DE and Voyager DE PRO Systems 1 5 Parts of the Voyager DE and Voyager DE PRO Systems This section describes System components Mass spectrometer e Vacuum system Computer components 1 5 1 System Components Voyager DE The Voyager DE Biospectrometry Workstation is shown in Figure 1 11 Mass ra spectrometer Video monitor Computer Control stick Figure 1 11 Voyager DE Biospectrometry Workstation Voyager Biospectrometry Workstation User s Guide 1 17 Chapter 1 Introducing the Voyager Biospectrometry
318. etween sample loader the Instrument menu and software Acquisition timeout Communication problem 1 Make sure all cables message displayed between digitizer and between digitizer and software mass spectrometer is securely connected see Figure 2 4 on page 2 11 If your system includes a LeCroy digitizer check network connections between the digitizer and the computer 2 Select Reinitialize from the Instrument menu 3 If the problem continues call Applied Biosystems continued Voyager Biospectrometry Workstation User s Guide 9 19 Chapter 9 Maintenance and Troubleshooting Table 9 2 Instrument Control Panel Troubleshooting Continued Symptom Possible Cause Action Slider control does not change laser setting Voyager Instrument Control Panel is not the active window Click on the Instrument Control Panel to activate the window before using slider controls Active position number does not reflect actual position under laser Sample plate is not aligned Align the sample plate See Section 2 7 Aligning the Sample Plate Number of times the laser fires is greater than Shots Spectrum Delay in transferring data from the digitizer to the computer No action Normal occurrence Negative ion selection not displayed in Instrument Settings control page and you have the optional hardware Instrument not configured for negative ion hardware
319. eusable no wells PLT file 5 Enter or edit WellUnits WellShape WellWidth WellHeight and WellDepth if necessary For more information see Table 3 13 PLT File Parameters on page 3 69 6 On the second line type or edit the absolute coordinates for the first position Separate absolute x and y coordinates with a comma and include one X Y pair per line see Figure 3 11 on page 3 68 for X Y pair example Type in the position name Blank lines are allowed 7 Use the Manual Laser Sample Positioning control page or the Control Stick to move to the next position then note the absolute coordinates for that position 8 Repeat step 5 through step 7 for remaining positions 9 Save the file with a unique name and a PLT extension for example Lab01 PLT Voyager Biospectrometry Workstation User s Guide 3 83 Chapter 3 Preparing Samples Hint Some applications automatically append a TXT extension to file names To name the file with a PLT extension include the file name and extension in double quotes in the Save File dialog box for example 100Well PLT 10 Check the sample plate alignment See Section 3 5 5 Creating PLT files for SymBiot 3 84 plates Applied Biosystems Adjusting the Laser Position for a Custom PLT File If you spot sample plates in evenly spaced rows and columns using the SymBiot Sample Workstation you can use the Create PLT File function to extrapolate th
320. f Prompt PSD and PSD Prompt Prompt Prompt PSDAI PSD T 1140 T T T T T T T 1160 1180 1200 1220 1240 1260 1280 Figure 6 10 Fragment lons in Reflector Mode 6 24 Applied Biosystems Prompt fragments PSD fragments Obtaining Good Spectra in Delayed Extraction Mode Prompt fragments appear in the spectrum at masses that correspond to the theoretical masses of the fragments because they are formed in the source PSD fragments appear in the spectrum at masses slightly higher than the theoretical masses of the fragments because they are formed after the source but travel at the same speed as the precursor until they reach the reflector Because they have lower kinetic energy than the precursor they separate in mass from the precursor in the reflector 6 3 3 Parameters Affecting Resolution and Signal to Noise Ratio Parameters affecting resolution Parameters affecting signal to noise ratio These parameters have a primary impact on resolution e Delay Time e Guide Wire Voltage e Grid Voltage e Digitizer Bin size and Input Bandwidth NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later For more information on optimizing resolution Section 5 4 3 Optimizing Resolution These parameters have a primary impact on signal to noise ratio e Accelerating Voltage e Guide Wire Voltage e Shots Spectrum e Digitizer Input Ba
321. f positions Ensures correct sample placement for running in Automatic Control mode Number of sample positions corresponds to number of microtiter plate wells Sample positions are inset from the plate perimeter which minimizes variation in mass accuracy that can be caused by variation in electric field at plate edges 1 Dogruel D P Williams and R W Nelson Anal Chem 1995 67 4343 4348 2 Brockman A H and R Orlando Anal Chem 1995 67 4581 4585 3 Krone et al Anal Biochem 1997 244 124 132 3 64 Applied Biosystems Sample Plate Types 3 5 2 Editable Configuration Plate PLT Selecting plate You can select plate types PLT files in two ways types PLT files When loading the plates in the mass spectrometer e From the Sample Plate menu in the Instrument Control Panel For an example of how to select plate types see Section 3 4 4 Loading Sample Plates Customizing PLT You can select and use the plate types described below You files can also customize these plate types by editing PLT files provided with the system Each PLT file describes a plate configuration available for selection when a plate is loaded into the Voyager system For more information see e Section 3 5 3 Guidelines for Defining Custom Plate Types e Creating or editing a PLT file using Notepad editor on page 3 83 NOTE You cannot edit the original PLT files provided with the system These are read on
322. f the MNU file You accessed this parameter in Tune Mode from the version 4 x Control Panel The following default values are set for Mirror To Accelerating Voltage Ratio e DE RP 1 06 e Elite 1 06 e DE STR 1 12 DE PRO 1 2 NOTE This parameter influences the tuning characteristics and default calibration of the instrument Do not change this setting unless instructed to do so by an Applied Biosystems Technical Representative Changing this value significantly alters the optimum settings of Grid Voltage and Delay Time Before converting determine the methods and search pattern files that you want to convert It is good practice to move the files you want to convert to a new directory for example a directory called Converted for easy access All converted files are placed in the same directory as the original files Because converted search pattern files are given the same name as the original search pattern files it is important to keep track of which search pattern files you have converted Voyager Biospectrometry Workstation User s Guide 5 95 Chapter 5 Optimizing Instrument Settings 5 96 Converting Applied Biosystems i OW tee 2025 10 From the Tools menu select Voyager 4 x File Converter The Voyager 4 x File Converter dialog box is displayed Figure 5 27 Click Add and select the MNU files to convert Click Add and select the SP files to convert Click Convert If
323. f the workstation CAUTION Power down before making connections Open the right side cabinet and slide out the computer shelf Connect devices as shown in Figure 2 9 r Printer VO contro of LPT1 or Parallel Joy stick VAC gauge control serial2 COM2 Step motor control Audio input output not used USB ports not used Serial COM1 Network PB100846 Digitizer options see Figure 2 4 on page 2 11 Figure 2 9 Computer Connections for Voyager DE STR Mass Spectrometer NOTE The computer layout may change without notice Boards may be located in slots that differ from those shown in Figure 2 9 The braided ground cable connection may be located in a different position 2 24 Applied Biosystems Connecting the Voyager DE STR Workstation Connections are listed below Device Connection Keyboard 5 pin round connector VGA monitor 12 pin connector 3 rows of pins Control stick 15 pin connector Mouse COM1 or 5 pin round connector Printer LPT1 or Parallel Digitizer Depending on your digitizer option the computer will have one of the following installed when you receive it e GPIB board for the oscilloscope e Acqiris 2 GHz digitizer board Figure 2 9 on page 2 24 shows the rear panel of the computer Figure 2 4 on
324. fan organic solvent is necessary rinse with acetonitrile at the lowest percentage that will dissolve the matrix 6 Examine the plate If you see any sample or matrix residue oil or fingerprints on the plate soak the sample plate in a working solution of laboratory detergent in water for example RBS 35 detergent from Pierce or LIQUI NOX from VWR for the minimum time required to remove samples IMPORTANT RBS LIQUI NOX Please read the manufacturer s MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CAUTION Do not leave the sample plate in detergent for longer than 10 minutes Longer exposure can cause the bottom holders on the sample plate to corrode Do not sonicate sample plates or use acid to clean sample plates Both can alter the surface of the sample plate and reduce the quality of the data obtained 7 Rinse the plate thoroughly in deionized water 8 Allow the plate to dry at room temperature in an area where it will not be exposed to contaminants Allow the plate to dry completely before use to obtain the maximum hydrophobic effect Cleaning gold and NOTE Use a cleaning solvent similar to the sample stainless steel solvent plates To clean gold and stainless steel sample plate surfaces 1 Rinse the plate with a squeeze bottle of solvent 2 Rub with a lint free lab tissue to clean 3 Rinse with deionized water 3 48 Applied B
325. fference between the measured and the theoretical mass of the sample Sensitivity Routine detection of 5 fmol of neurotensin with signal to noise ratio gt 20 1 Table A 2 Voyager DE Mass Spectrometer Specifications Condition Specification Mass range 2300 000 Da Upper limit set by suitably ionized biomolecule Flight tube 1 2 m linear A 2 Applied Biosystems Voyager DE Specifications Table A 2 Voyager DE Mass Spectrometer Specifications Continued Condition Specification lon source Two stage lon source voltages Tunable Accelerating Voltage Up to 25 000 V Grid Voltage Range determined by Accelerating Voltage Laser Nitrogen 337 nm 3 ns pulse 20 Hz maximum firing rate Actual laser firing rate dependant on digitizer See Section A 4 Digitizer Specifications Digitizer 500 MHz digitization See Section A 4 Digitizer Specifications Vacuum system Automatic with turbomolecular pumping for high vacuum lon detection Positive and negative Sample analysis Automated single plate sample loading system sample plates of various formats available See B 3 Spare Parts Manual control using control stick or mouse Sequence control software for automated analysis Dimensions 27 inches 69 cm deep 25 inches 64 cm wide 65 inches 165 cm high includes cabinet and flight tube Voyager Biospectrometry Workst
326. figuration _Idle Power Off Idle Time When enabled the number of minutes after which the high voltage power supplies automatically turn off if the instrument is not used Default is 60 minutes If this value is zero the high voltage remains on until any of the following occur you select Instrument Turn off High Voltage Source Pressure exceeds Maximum Operating Pressure you click Load or Eject you align a sample plate or you exit the software 4 Click OK to exit 2 6 3 Timed lon Selector Configuration To check the Timed lon Selector called Precursor lon Selector in PSD mode configuration 1 Inthe Instrument Control Panel select Hardware Configuration from the Instrument menu 2 Click the Timed lon Selector tab to display the Timed lon Selector page Figure 2 17 Hardware Configuration x Vacuum Control Stick Laser Timed lon Selector High Voltage Instrument Digitizer Flight Length to Deflector mm 1435 Deflector Gate Width mm 30 Figure 2 17 Timed lon Selector Configuration Voyager Biospectrometry Workstation User s Guide 2 39 Chapter 2 Installing the Voyager Biospectrometry Workstations 3 Check the following values as needed Flight Length to Deflector Distance in millimeters from the grid to the deflector Deflector Gate Width Distance in millimeters that the Timed lon Selector is on CAUTION Do not alter the Timed lon Selector paramete
327. file you apply contains both processing and graphic settings If it does not the system applies default graphic settings black background with yellow traces when the macro runs If desired assign the macro to a button as described in the Data Explorer Software User s Guide Section 6 7 3 Assigning Macros to Buttons The button number to which you assign the macro is the number that you select in the Sequence Control Panel NOTE Assigning a macro to a button is not required You can select a macro by name or number in the sample list Voyager Biospectrometry Workstation User s Guide 7 9 Chapter 7 Acquiring Spectra from the Sequence Control Panel Selecting a macro NOTE Do not select in the Sequence Control Panel in a sequence macros that require user action macros in which you must enter or select values then click OK to execute If you select macros that require user action the sequence stops when the macro is activated by the Sequence Control Panel and waits for you to enter values and click OK To select a macro in the sample list 1 Create a sequence as described in Section 7 4 Creating a Sequence 2 Inthe macro field for each row select a macro by doing either of the following e Click the field and select the macro name from the list All macros and scripts you have created in the Data Explorer software are listed e Type the button number to which you assigned the macro
328. for 3 67 applications for 3 62 disposable applications 3 63 disposable maximum number of spots 3 74 disposable PLT file for 3 67 gels 3 63 gold applications 3 62 gold cleaning 3 48 membranes 3 63 overview 3 62 stainless steel applications 3 62 stainless steel cleaning 3 48 Teflon applications 3 63 Teflon cleaning 3 47 Teflon PLT file for 3 67 types supported PLT 3 61 Index Sample position including in data file name 7 16 7 46 random 5 37 selecting automatically 6 41 selecting manually 4 25 4 29 6 13 Sequence Control Panel 7 16 shape displayed in Manual Laser Sample Positioning control page 3 69 3 82 uniform edge bias or center bias 5 37 zooming 6 13 Sample positioning automated see Automated Sample Positioning Sample preparation see Sample Sample spotting see sample loading Sample throughput increasing 3 52 Sample view sample position accessing from Plate view 6 13 accessing Plate view 4 31 laser position 4 31 Save All Spectra data collection mode description 5 39 selecting 5 35 Save All Spectra That Pass Acceptance Criteria data collection mode description 5 40 selecting 5 35 Save the Best Spectrum data collection mode description 5 44 selecting 5 36 Save the First Spectrum to Pass Acceptance Criteria data collection mode description 5 42 selecting 5 36 Voyager Biospectrometry Workstation User s Guide Index 35 Index Saving data Instrument Control Panel Linear and Reflec
329. fragment ion mass based on precursor ion mass and Mirror Ratio setting described in Section 8 2 5 Mass Calculation for Fragment lons Steps to perform The steps to perform PSD analysis on angiotensin and on an PSD analysis unknown are summarized in Table 8 1 and Table 8 2 8 2 Applied Biosystems PSD Quick Start Table 8 1 Steps to Perform PSD Analysis of Angiotensin See Step Result page Generate a Reflector mode precursor Angiotensin Reflector spectrum 8 7 spectrum use PSD_Precursor BIC provided Precursor RN Generate a normal single point external Normal external calibration 8 7 calibration using the spectrum acquired in for precursor ion mass accuracy step 1 You use this single point external r calibration in PSD analysis to obtain maximum Angio CAL mass accuracy for the precursor ion Set PSD Acquisition Instrument Settings 8 7 parameters Open Angiotensin_PSD BIC Angiotensin _PSD BIC with provided and Angio CAL In Instrument Settings Type the Default PSD calibration angiotensin precursor mass and select Angio CAL generated above for precursor In PSD Acquisition Settings Select default PSD calibration for fragments Acquire PSD segments precursor and Angiotensin PSD Composite spectrum 8 9 fragment spectra 3 o NG Generate a PSD multi point external PSD calibration 8 13 calibration using the spectrum acquired
330. g signal to noise ratio For accurate results specify a flat non rising region of baseline that does not include peaks Signal to Noise Calculator Live Data E3 Baseline Region From To ae ee Select Peaks Mass Charge AMU Peak 1 0 1 Peak2 fo fi Peak 3 fo fi fo fi Peak 4 Figure 6 14 Signal to Noise Calculator Voyager Biospectrometry Workstation User s Guide 6 33 Chapter 6 Acquiring Spectra from the Instrument Control Panel 4 For each Mass Charge enter the window for calculation AMU NOTE To label peaks set the AMU value low enough to prevent the calculation windows for each peak from overlapping If the calculation windows overlap only the first peak is labeled However if you set AMU too low the peak of interest may not appear in the window and signal to noise will not be calculated 5 Click OK The peaks are labeled with SXXX next to the peak mass where XXX is the signal to noise ratio NOTE If you do not apply peak detection parameters after acquisition is complete signal to noise labels are not displayed 6 34 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel 6 6 Acquiring in Automatic Mode from the Instrument Control Panel This section includes e Before acquiring in Automatic Control mode e Setting Instrument Settings for Automatic Control mode e Automatically acquiring evaluating
331. g Sample Plates in the Mass Spectrometer Load Eject Sample Plate x Plate ID h Plate Type 5 Last Aligned Tre sample plate has not been aligned Optimization No Plate ID selected Created Jn Use Mess Accuracy a pimteation Warning Be sure hands are free of sample loading mechanism ay Load Cancel Load No Plate Figure 3 6 Load Eject Sample Plate Dialog Box If you are removing a plate but not loading a new plate click Load No Plate and skip the remaining steps Select a Plate ID The PLT file plate alignment information and plate optimization information associated with the Plate ID are automatically loaded For more information see Section 2 7 Aligning the Sample Plate and Section 2 8 Running OptiPlate to Optimize Mass Accuracy Alternatively you can specify a new Plate ID then select a PLT file For more information see Assigning Plate IDs on page 3 50 Select Use Mass Accuracy Optimizations to apply calibration corrections to the data acquired from the plate Before selecting this option read Section 3 4 2 Using the Mass Accuracy Optimization Option to understand how mass accuracy optimization is applied See Optimization strategy on page 3 53 to understand the options you have when using Mass Accuracy Optimization Click Load The sample plate is loaded and aligned as needed For more information see How the system aligns a plate on page 2 47 Voyager Bi
332. g from a 2 To open an instrument settings file directly double click BIC file the BIC file or select a file and click Open Hint If the BIC file you need is not visible click the scroll bar to view more instrument settings files Voyager Biospectrometry Workstation User s Guide 5 7 Chapter 5 Optimizing Instrument Settings Opening from a DAT file To open an instrument settings file from a DAT file select DAT from the Files of Type drop down list select the DAT file that contains the BIC of interest and click OK The instrument settings file is loaded The currently loaded instrument settings file name is displayed in the title bar of the Instrument Control Panel Viewing To view all of the instrument settings in a BIC file print the instrument settings as described in Printing on page 5 12 5 1 4 Modifying an Instrument Settings File BIC 5 8 In this section This section includes Selecting a BIC file Applied Biosystems Selecting a BIC file Modifying for Manual Control mode Optimizing Modifying for Automatic Control mode We suggest that you use standard instruments settings BIC files provided and modify as needed However you can open and modify any BIC file as needed To select a standard instrument settings BIC file hs Open a BIC file for the mass range you are analyzing For information on mass ranges in BIC files see Section 5 1 2 Standard Inst
333. ge Video camera A camera that displays a real time sample image 100 times magnification on the video monitor 1 22 Applied Biosystems Parts of the Voyager DE and Voyager DE PRO Systems Vacuum system A pumping system and sealed enclosure that creates and maintains a high vacuum environment for unobstructed ion drift Refer to Section 1 5 3 Vacuum System for more information Flight tube and beam guide wire A field free region no additional accelerating forces are present in which ions drift at a velocity inversely proportional to the square root of their masses The voltage applied to the beam guide wire overcomes the dispersion effect from the ion source and refocuses ions on the detector Linear detector A device that detects ions that travel down the flight tube The linear detector measures ion abundance over time and sends a signal to the digitizer for conversion On the Voyager DE PRO system the linear detector is used in Linear mode only It is not used in Reflector or PSD mode Linear detectors are hybrid high current detectors consisting of a single microchannel plate a fast scintillator and a photomultiplier These detectors have superior tolerance for high ion currents NOTE High current detectors are standard on Voyager DE systems with serial number 1128 and later and Voyager DE PRO systems with serial number 6007 and later Voyager Biospectrometry Workstation User s Guide 1 23
334. ge vacuum see BA1 and BA2 see IG1 and IG2 see TC2 see Vacuum gauge Gels sample plate to use 3 63 Glu Fibrinopeptide B molecular weight F 2 Glycolipids matrix for C 7 Glycopeptides matrix for 3 3 Index GPC MALDI sample plate type 3 62 Graphic options accessing 4 22 customizing the display with 4 21 setting trace colors with 4 18 turning off right axis 4 11 Grid displaying in Spectrum window 4 21 Grid Voltage see also Grid Voltage effects adjusting 5 55 5 73 affected by matrix 5 74 and Delay Time 5 55 5 73 E 1 description 5 17 5 51 for higher masses 5 74 impact of changing 5 53 5 55 in Continuous Extraction mode H 6 H 11 in Delayed Extraction mode 5 74 in Linear and Reflector modes 5 74 5 82 in PSD mode 8 73 8 76 optimizing 5 82 optimizing setting 5 83 PSD mode 5 51 range 5 17 Grid Voltage effects on fragment ions in PSD mode 8 73 fragmentation 5 50 5 53 ion acceleration 5 51 resolution 5 50 5 51 6 25 resolution and signal to noise in PSD mode 8 76 resolution in PSD mode 8 74 Grids in ion source 1 22 1 34 5 52 Guide wire see Beam guide wire Guide Wire Voltage see also Guide Wire Voltage effects adjusting for CID 8 36 description 5 18 5 56 effect of changing 5 56 Voyager Biospectrometry Workstation User s Guide Index 15 Index Guide Wire Voltage continued effect on signal to noise ratio 6 25 in Continuous Extraction mode H 6 H 11 in Delayed Extraction mode 5 84
335. ge Bias search pattern 5 37 Ejecting sample plates Instrument Control Panel 4 25 Electromagnetic compliance xxiv EMC standards xxiv EMIS button on vacuum gauge panel 9 26 Energy kinetic 1 10 1 24 1 36 8 21 minimizing spread of 1 11 1 24 1 36 5 54 spread of ions reducing 1 11 5 18 5 54 5 56 Enolase molecular weight F 3 Environmental conditions A 4 A 7 A 10 Voyager Biospectrometry Workstation User s Guide Index 13 Index Equations calibration 6 9 default theoretical calibration 6 9 drift time for multiply charged ions 1 10 drift time for singly charged ions 1 10 PSD calibration 8 28 Error codes vacuum gauge panel 9 27 Evaluating data see also Data Explorer Software User s Guide automatically 6 42 manually 6 17 spectrum acceptance criteria 6 42 Event log see NT Event log Exiting software 2 34 Expanding traces 4 13 Experiment PSD starting 8 52 8 54 8 64 stopping 8 56 8 64 Exporting Sequence run list 7 22 External laser see Laser external optional Extraction Correction 2 53 Extraction Type description 5 25 F Factor IGF 1 mass to time conversion F 5 Fast fragments see Prompt fragments Features Voyager DE system 1 4 Voyager DE PRO system 1 4 Voyager DE STR system 1 6 File name in Instrument Control Panel 4 4 6 14 in Sequence Control Panel 7 16 including position number in 7 16 7 46 Index 14 Applied Biosystems File size and Bin size setting 5 59 impact on resolution and
336. ge data file that can be slow to open and process When you select conditions the software displays the information about the number of data files and spectra that will be acquired and saved For more information see Spectrum Accumulation on page 5 35 9 If you selected saving or accumulation conditions in step 8 that specify acceptance criteria set Spectrum Acceptance Criteria as described in Setting spectrum acceptance and laser adjustment criteria on page 6 42 Saving or accumulation conditions that specify acceptance criteria are e Save all spectra that pass acceptance criteria e Save first spectrum to pass acceptance criteria e Save best spectrum e Accumulate all spectra that pass acceptance criteria 6 40 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Sample positioning 10 Stop conditions 12 Select Use Automated Sample Positioning NOTE To manually adjust sample positioning when acquiring in Automatic Control mode deselect Use Automated Sample Positioning For more information see Section 4 5 2 Adjusting Laser Intensity and Selecting Sample Position If you enabled Use Automated Sample Positioning select Random Search Pattern or Search pattern file then select an SP file For information on search pattern files see Section 6 6 4 Search Patterns NOTE Before using search pattern files created in version 4 software convert to version
337. ghts F 2 ordering information B 6 spotting next to sample 3 38 H 24 Voyager mass standards kit B 6 Standard internal see also Calibration internal concentration 3 24 mass range 3 24 Standby mode high voltage power supplies 6 4 7 24 Standby time high voltage 2 39 Status acquisition 2 78 4 5 4 25 acquisition Sequence Control Panel 7 20 7 29 active position 4 5 Control Mode 4 5 data storage 2 78 high voltage 2 77 4 5 instrument mode 4 5 instrument state 2 77 4 5 laser intensity 4 5 mirror chamber BA2 pressure 2 78 4 5 source chamber BA1 pressure 2 77 4 5 System Status displaying control page 2 76 Status bar Instrument Control Panel description 4 5 Step size laser Automatic Control mode parameters 5 34 configuring 2 43 Stop Conditions Automatic Control mode 5 38 Stopping acquisition 4 25 6 16 PSD experiment 8 56 8 64 software 2 34 Substance P BIC file 5 5 5 6 Substance P amide molecular weight F 2 Summary of Instrument Settings parameters Linear and Reflector mode 5 49 PSD mode 8 76 Swap file required disk space 9 6 SymBiot sample plate creating PLT file for 3 84 System performance improving closing Sequence Control Panel when not in use 4 35 screen savers do not use 1 30 System Status checking 2 76 parameters 2 76 status bar 4 5 T TC2 Voyager DE description 1 27 max load pressure 2 37 pressure range 9 26 wait time 2 37 TC2 Voyager DE PRO description 1 29 max load pressure 2
338. gments improved resolution with more segments 8 25 mass range 8 25 number of segments needed 8 40 viewing see Data Explorer Software User s Guide PSD data file contents 8 27 not available for viewing until experiment closed 8 52 open until experiment closed 8 52 segments appended during acquisition 8 52 PSD Decrement Ratio and segment size correlation 8 41 correlation with PSD Mirror Ratio 8 41 default settings 8 41 setting 8 50 PSD experiment starting 8 52 8 54 8 64 stopping 8 52 8 64 PSD fragments see also Fragment ions see also PSD segments acceleration and flight time 8 71 and laser intensity 8 68 angiotensin observed F 7 calibration 8 28 description 6 24 8 21 8 71 Index PSD fragments continued PSD mode continued example 8 70 guidelines for settings 8 49 focusing 8 21 Input Bandwidth 8 48 mass 8 71 instrument settings optimized PSD Mirror Ratio for H 4 acquiring multiple spectra with ion selectivity improving 8 29 same value 8 49 laser intensity observing effects and Guide Wire Voltage 8 76 of 8 66 calculating automatically 8 41 mass range for segments 8 54 8 50 8 64 Decrement ratio 8 41 8 50 Max Stitch Mass 8 42 8 48 default settings 8 41 8 48 Mirror Ratio see PSD Mirror Ratio focusing ions 8 21 no DAT file created 8 53 8 56 function 8 20 optimum resolution observed near precision displayed when you click Max Stitch Mass 8 49 on entry 8 49 overview 8 20 PSD mode parameters 8 20 8 43 8 58
339. gn to a physical plate Allows customized alignment and mass accuracy optimization of more than one physical plate using the same PLT file For more information see PLT files and multiple alignments on page 2 47 Plate Type PLT file which contains plate configuration information All PLT files are located in the C VOYAGER directory For more information on PLT files see PLT file format on page 3 68 Last Aligned Read only field that displays the date of the last alignment performed For information see Section 2 7 Aligning the Sample Plate Optimization Created Read only field that displays the date and time that the plate associated with the Plate ID selected above was optimized for mass accuracy For information see Section 2 8 Running OptiPlate to Optimize Mass Accuracy Use Mass Accuracy Applies correction factors that optimize mass accuracy Optimizations to the data as it is acquired For information see Section 3 4 2 Using the Mass Accuracy Optimization Option Voyager Biospectrometry Workstation User s Guide 3 51 Chapter 3 Preparing Samples 3 4 2 Using the Mass Accuracy Optimization Option Benefits of using Mass Accuracy Optimization during analysis 3 52 How mass accuracy optimization is applied Applied Biosystems In a typical analysis run that requires optimum mass accuracy you include samples interspersed with many calibration standard
340. gure 2 3 Cable VO CTL VO CTL O Ctl STP MTR CTL STP MTR CTL 25 pin 25 pin mesh cable Ground stud Nut below LPT1 or the tab extension on top of the computer cover Braided screw on cable VAC GAUGE CTL Serial 2 or COM 2 9 pin Vac Gg Ctl I O POWER 3 pin round connector Not used on serial number 1171 and later DE and 6131 and later PRO I O power 5 V 1 pin round connector I O power 2 12 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations 2 3 3 Connecting the Signatec 500 MHz Digitizer Board If you have an oscilloscope or an external digitizer on your system disregard this section CAUTION Do not use the Signatec 500 MHz digitizer board without digitizer signal protection circuits blue boxes on cables Operation without digitizer signal protection circuits will result in damage to the internal digitizer Refer to the following table when you connect the Signatec 500 MHz digitizer board to the mass spectrometer Connection on right side panel of mass spectrometer see Figure 2 2 Connection on rear panel of computer Cable see Figure 2 4 TRIG Trig Trig SMA to Trig BNC CH 1 CH 1 Ch 1 SMA to Ch 1 BNC between blue box and computer Ch1 BNC to Chi BNC between blue box and mass spectrometer Ground wire to computer chassis ground screw Voyager Biospectrometry Workstation
341. h other as possible Create a search pattern file that analyzes both spots Create an instrument settings file that generates a single spectrum Accumulate All data collection mode and specify internal calibration in Sequence Control See Creating a search pattern file for internal calibration on separate spots on page 7 40 NOTE To use this strategy make sure standard and sample are compatible with the laser intensity and acceptance criteria in the instrument settings and that the standard and sample peaks do not overlap Perform close external calibration Spot standard and sample as close to each other as possible and perform close external calibration as described in Section 7 7 3 Performing Close External Calibration To create a search pattern file for internal calibration on separate spots il Spot sample and standard as close to each other as possible within 1 mm see Figure 7 9 on page 7 37 NOTE For optimum mass accuracy calibration standards must be as close to unknowns as possible An internal standard spot within 1 mm of a sample spot can yield mass accuracy close to the expected mass accuracy for internal standard calibration on your system Automatic Calibration During a Sequence Run NOTE Use plates without laser etched sample positions if you spot standard and sample as shown in Figure 7 9 on page 7 37 Load the sample plate select the position containing sample and standard
342. he Voyager Biospectrometry Workstations In the Automatic Control dialog box set the Minimum and Maximum Laser Intensity settings to 50 of the laser intensity that yields signal intensity between 20 000 and 60 000 counts The recommended 50 setting is intended to keep the laser intensity range as narrow as possible to allow OptiPlate to run as quickly as possible However if the laser intensity range is too narrow it may not yield signal intensity in the specified range which will cause OptiPlate to fail If you observe failures during the OptiPlate run you may need to increase the laser intensity range Click OK From the File menu select Save Instrument Setting As then name the file OptiPlate_Linear1 BIC or OptiPlate_Reflector1 BIC NOTE Save the BIC file before selecting it in OptiPlate If you make changes to the BIC file after you select it in OptiPlate OptiPlate does not recognize the changes Running OptiPlate to Optimize Mass Accuracy 2 8 4 Running OptiPlate This section includes e Running OptiPlate e During an OptiPlate run Running To run OptiPlate OptiPlate 4 inthe Instrument Control Panel select OptiPlate from the Tools menu to display the OptiPlate dialog box Figure 2 27 on page 2 64 By default the Plate ID for the plate loaded in the mass spectrometer is specified in the OptiPlate software If there are OptiPlate results associated with the selected Plate ID color coded well posit
343. he current entry is acquired and sequence processed and Sequence Acquisition Status is Paused To resume the sequence click again The sequence resumes with the next row selected to run Skipping a line To skip a row in the sequence select Skip Line from the Control menu NOTE The log file does not contain any information for rows you Skip Modifying the run To modify the run list after you start the sequence click to list during pause the sequence You can modify lines below the line at acquisition which the sequence has paused Process that During the sequence run occurs After each data file is acquired it is processed e Sequence status is displayed in the locations listed in Checking Sequence Status on page 7 29 After the sequence run e The run log is created in the location specified in Section 7 4 1 Setting General Sequence Parameters The run log contains all errors that are logged and a list of files that are acquired The last BIC file that was executed remains loaded in the Instrument Control Panel 7 28 Applied Biosystems Running a Sequence 7 6 4 Stopping a Sequence To stop a sequence click m The sequence stops as soon as the current processing function can safely stop You cannot resume a stopped sequence You must restart at the beginning 7 6 5 Checking Sequence Status You can check sequence status in three places e Acquisition Status field in the run list e Seq
344. he potential gradient that accelerates ions Digitizer A device that converts an analog signal to a digital word and stores the result in memory This allows the transfer of the digitized signal to a computer for additional processing Dimer A species that results from a association between two identical molecules Expressed as 2M H Appears in a mass spectrum at 2 times the protonated molecular ion mass minus one mass unit Edman degradation technique An approach to amino end group determination involving the use of a reagent phenylisothiocyanate that can be applied to the liberation of a derivative of the amino terminal residue without hydrolysis of the remainder of the peptide chain Flight tube Vvacuum chamber in which ions drift from the source to the detector Fore pump Rotary pump that maintains vacuum in the sample loading chamber creates the low vacuum condition needed by the turbo pump and provides backing pressure to the turbo pump Fragment ion dissociated from precursor ion Glossary 2 Applied Biosystems FWHM Full width at half maximum GPMAW General Protein Mass Analysis for Windows software a software program used to identify protein sequences Grid voltage Secondary voltage used to fine tune ion acceleration on a variable voltage grid above the sample plate Guide Wire voltage voltage applied to beam guide wire to focus ions on detector Particularly usefu
345. i avec la norme CSA 1010 Sp cifications de s curit du mat riel lectrique utilis pour les mesures les contr les et dans les laboratoires Partie 1 Sp cifications g n rales et il est conforme a cette norme C est un produit homologu par les ETL Testing Laboratories EMC This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur le materiel brouilleur du Canada Safety This instrument meets European requirements for safety EMC Directive 73 23 EEC This instrument has been tested to and complies with standard EN61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use EMC This instrument meets European requirements for emission and immunity EMC Directive 98 336 EEC This product has been evaluated to the EN61326 1998 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Radiated Emissions were evaluated to Group 1 Class B requirements Voyager Biospectrometry Workstation User s Guide XXV Safety and Compliance Information Laser Safety Laser The Voyager Biospectrometry Workstation uses a standard classification nitrogen laser and an optional Nd YAG laser Under normal operating conditions the instrument laser is categorized as a Class laser Under certain condit
346. ia is saved e Acquisition is performed on the same search pattern position until Acceptance Criteria fail if the Use Automated Sample Positioning option is disabled acquisition is performed on the positions you manually select instead of search pattern positions e Acquisition continues until the Number of Spectra to Acquire have been acquired or the end of the search pattern is reached All search pattern positions may not be analyzed One DAT file containing one or more spectra is created If no spectra pass acceptance criteria no data file is saved NOTE In this mode acquisition moves to the next search pattern position only if acceptance criteria fail 5 40 Applied Biosystems Instrument Settings Parameter Descriptions gt Spectrum Accumulation Acquire E spectra under conditions Save all spectra that pass acceptance criteria x 1 of more spectra from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 50 shots 50 shots 1 spectra Number of DAT files lt 1 Figure 5 9 Save All Spectra Mode That Pass Acceptance Criteria Mode Automatic Control Dialog Box Examples If Random search pattern is selected and Number to Acquire is 5 the number of positions analyzed depends on whether or not Acceptance Criteria fail acquires from same search pattern position until Acceptance Criteria fail and on how man
347. ice is a Bradbury Nielson gate positioned approximately 676 mm from the ion source This prevents ions deflected by the gate from entering the reflector and reduces background noise in the detector in PSD experiments When the Timed lon Selector is turned on voltage is applied to the Timed lon Selector to deflect ions At the time that corresponds to the ion of interest voltage is turned off and the ion of interest passes to the detector After the ion passes through the Timed lon Selector voltage is turned on again 1 5 3 Vacuum System The Voyager DE and Voyager DE PRO Biospectrometry Workstations provide a high vacuum environment for time of flight analysis The high vacuum environment Allows unobstructed ion drift e Provides conditions needed to maintain the high potential difference between the ion source and ground Voyager Biospectrometry Workstation User s Guide 1 25 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 1 5 3 1 Voyager DE Vacuum System Vacuum The Voyager DE Biospectrometry Workstation includes two chambers vacuum chambers Figure 1 15 Main source chamber e Sample loading chamber Foreline valve 1 Foreline valve 2 Main Turbo a source E chamber m ste a high vacuum chamber low vacuum Figure 1 15 Voyager DE Biospectrometry Workstation Vacuum Chambers 1 26 Applied Biosystems Vacuum pumps Vacuum gauges Pa
348. iew e Setting Accelerating Voltage e Setting Guide Wire Voltage Setting Shots Spectrum Setting Low Mass Gate For more information on measuring Signal to Noise see Section 6 5 3 Calculating Signal to Noise Ratio and the Data Explorer Software User s Guide Section 6 4 Using the Signal to Noise Ratio Calculator Manually The manual accumulation feature of the Voyager system accumulating allows you to acquire a spectrum examine the spectrum for spectra to signal quality visually or using the Resolution or Signal to Noise Ratio calculator and then accumulate the improve 5 spectrum with other previously examined spectra or discard signal to noise the spectrum This process of accumulating only acceptable ratio spectra allows you to improve the signal to noise ratio of the final spectrum you save For more information on manually accumulating spectra see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Voyager Biospectrometry Workstation User s Guide 5 85 Chapter 5 Optimizing Instrument Settings 5 4 4 1 Overview You can set the following parameters to optimize signal to noise ratio Figure 5 25 e Accelerating Voltage e Guide Wire Voltage e Shots per Spectrum e Low Mass Gate The following sections give guidelines for setting these parameters To Optimize Adjust Signal to Noise Ratio Accelerating Voltage Guide Wire Voltage Shots Spectrum Low Mass Ga
349. if your system includes a Signatec digitizer Network 2 Connection for LAN if your system includes a LeCroy Tektronix or Acqiris digitizer 1 O power not used on Serial Number 1171 and later DE and 6131 and later PRO Figure 2 3 Rear Panel of Computer 2 10 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations PB100823 LSA 1000 LeCroy dedicated ethernet network connection Signatec board Tektronix Oscilloscope IEEE 488 GPIB Acqiris board 500 MHz LED CH1 CH2 TRIG Acqiris board 2 GHz Acqiris boards require a 90V BNC spark gap and 3dB BNC attenuator between the cable and the board Figure 2 4 Digitizer Options NOTE The network connection to which the LeCroy digitizer is connected requires a specific IP address On some systems the LeCroy digitizer may be connected to a network connector other than the one shown in Figure 2 4 Do not change this connection without consulting an Applied Biosystems technical representative Voyager Biospectrometry Workstation User s Guide 2 11 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 3 2 Connecting the Mass Spectrometer to the Computer Refer to the following table when you connect the mass spectrometer to the computer Connection on side panel of mass spectrometer see Figure 2 2 Connection on rear panel of computer see Fi
350. ified Number of Spectra to Acquire are acquired e The difference between the maximum and minimum laser intensity is smaller than the laser step size specified in Automatic Control dialog box and the laser can no longer be adjusted e Any stop conditions are met The end of the search pattern file is reached NOTE If the number of positions in the search pattern file is lower than the specified number of spectra to acquire acquisition stops before the total number of spectra specified are acquired For example if you specify 5 spectra to acquire but the search pattern file specifies 4 positions acquisition stops after the fourth position is sampled even if 5 spectra have not yet been acquired Voyager Biospectrometry Workstation User s Guide 6 63 Chapter 6 Acquiring Spectra from the Instrument Control Panel 5 The system saves data files based on the Spectrum Accumulation conditions selected Acceptance criteria must be met for all collection modes except Save All and Accumulate All See Section 5 2 4 Description of Spectrum Accumulation Options for a description of the files saved in each Spectrum Accumulation condition 6 If an accumulation condition that uses Acceptance Criteria is selected and Acceptance Criteria are met the system begins a new acquisition from the current search pattern position If Acceptance Criteria are not met if an accumulation condition that does not use Acceptance Crite
351. ignal left and right Select Activates the right or left cursor Scaling Cursor Control Moves the active cursor Reset button Resets the oscilloscope screen For information on using other controls on the oscilloscope refer to the manual shipped with the oscilloscope The spectrum you see on the oscilloscope is inverted Figure l 3 NOTE The oscilloscope displays an intensity versus time spectrum Brackets indicate time range shown Marker indicates baseline position Sy Early low mass matrix peaks Intensity mV gridline changes as scale is adjusted chil im IN my Wil il NN Number of scans averaged 0 Acqs 4 i swt Wu i i NA Wi il if ik IL Ng LU l A Difference between cursors Position of active cursor solid line PB100506 Ch 1 10 mV Active Later higher mass Inactive cursor sample peak cursor solid line dotted line Figure l 3 Example of Signal on Oscilloscope Voyager Biospectrometry Workstation User s Guide l 5 l 6 Appendix I Adjusting the display Applied Biosystems Using the Oscilloscope and Control Stick The Channel indicator Ch1 in Figure l 3 displays Ch1 in Linear mode On Voyager DE PRO and Voyager DE STR systems the Channel indicator displays Ch2 in Reflector mode The top of the screen also displays Average During acqu
352. imed lon Selector 5 32 Vertical Offset 5 30 5 62 Vertical Scale 5 30 5 60 Molecular ions kinetic energy 1 10 8 21 Molecular weights conversion to time for standards F 4 estimating H 18 matrices C 7 relationship to drift time 1 8 1 10 standard compound F 2 Monitor see Video monitor Monoisotopic peak creating macro to deisotope 7 10 setting filtering in SET file 7 3 7 12 7 17 Mouse connecting Voyager DE and Voyager DE PRO 2 22 connecting Voyager DE STR 2 25 Multiple spectra calibrating in one data file 7 4 recommended maximum in one data file 5 35 saving in one data file 5 35 5 39 Voyager Biospectrometry Workstation User s Guide Index Multiply charged ions 1 10 Myoglobin BIC file 5 4 mass to time conversion F 5 molecular weight F 3 N Na adduct ion effect on masses 9 17 from buffer 3 24 Name instrument specifying 2 41 laboratory specifying 2 41 Nd YAG laser matrices C 12 Negative ion mode BIC file 5 4 5 5 selection not in BIC file 9 20 setting 5 25 Switch Delay Time 2 38 Neurotensin molecular weight F 2 Nicotinic acid C 12 Nitrocellulose in matrix 3 11 Noise reducing higher frequency 5 30 Nonpolar synthetic polymers matrix for C 11 Not Used traces 4 14 NT Event log checking 9 22 location 9 22 Number of Data Points Digitized digitizer description 5 29 Number of Data Points digitizer impact on laser firing rate 5 27 Index 25 Index O Offsetting
353. impact of changing instrument settings parameters 5 49 6 25 improving 1 14 5 56 5 59 5 88 improving by accumulating spectra 5 71 isotopes in Delayed Extraction mode 5 75 labels not displayed 6 31 Resolution mass continued obtaining maximum in Continuous Extraction mode H 18 parameters affecting in Continuous Extraction mode H 11 peak height used in Automatic Control mode 6 44 PSD mode 8 73 PSD segment optimum observed near Max Stitch Mass 8 49 rating scale for MW ranges 6 32 H 17 Reflector mode 5 25 results 6 32 troubleshooting 5 76 9 10 H 21 Resolution optimizing Accelerating Voltage 5 84 5 88 Delay Time 5 77 for a mixture 5 78 5 83 Grid Voltage 5 82 5 83 Guide Wire Voltage 5 56 5 84 Input Bandwidth 5 63 overview 5 71 parameters affecting 5 72 Results OptiPlate 2 69 resolution mass 6 32 signal to noise ratio 6 34 Return Authorization RA number B 5 Returning damaged items B 5 Right axis changing scale 4 11 5 70 turning off 4 11 turning on and off 4 21 Run list see Sequence Run Log file Sequence Control Panel contents 7 14 created 7 28 specifying 7 14 Index S Safety information laser xxvi Safety standards xxiv Salt concentration in sample 3 5 3 13 3 15 3 24 Sample buffers impact of 3 24 diluting recommended solutions 3 23 guidelines for good crystallization 3 37 high salt impact of 3 24 impact of ionization on data 6 6 internal standard 3 24 loss minimizing 3 24 mixing on sa
354. improved mass accuracy Initial laser setting Using optimized instrument settings e When acquiring a spectrum e Obtaining acceptable data e Obtaining maximum mass accuracy High voltage For maximum mass accuracy allow the high voltage power warmup for supplies to warm up for a short period of time before improved mass pele This beni a sa voltages accuracy and yields more reproducible ion flight times To turn on the high voltage power supplies click in the toolbar NOTE The high voltage power supplies are automatically turned off after 60 minutes if the mass spectrometer is not used To change the Idle Time see High Voltage Configuration on page 2 38 Initial laser After starting the software the laser intensity is set to the setting mid range default laser setting approximately 1 800 When you load a BIC file the laser intensity that was stored in the BIC file is loaded in the Instrument Control Panel See Adjusting laser intensity on page 5 68 for more information Using optimized Before analyzing samples in the Instrument Control Panel instrument open and optimize instrument settings in a BIC file You can settings optimize more than one BIC file to cover mass ranges or compounds of interest For more information see Section 5 4 Optimizing Instrument Settings Parameters 6 4 Applied Biosystems Before You Begin When acquiring a When acquiring a spectrum note the following spectr
355. in PSD mode See Section 8 2 Overview of PSD Analysis Timed lon Selector with mass Allows analysis of the mass of interest by deflecting other ions This parameter is typically only used in PSD mode but is available in Reflector mode for advanced applications See Section 8 2 Overview of PSD Analysis and Section 8 6 2 Observing the Effects of Precursor lon Selector for more information NOTE The Timed lon Selector in Reflector mode is the same parameter as the Precursor lon Selector in PSD mode If you change the mass for Timed lon Selector in Reflector mode the Precursor mass used by the Precursor lon Selector in PSD mode also changes 5 32 Applied Biosystems Instrument Settings Parameter Descriptions 5 2 3 Automatic Control Dialog Box On the Instrument Settings control page see Figure 5 3 on page 5 15 select Automatic Control mode then click the Automatic Control button to display the Automatic Control dialog box Figure 5 7 NOTE The Automatic Control button is dimmed if Automatic Control mode is not selected Automatic Control x r Laser Intensity Minimum 1o00 Maximum 2000 Step Size jio Note No criteria specified for adjustment laser midrange will always be used T Use Prescan Acquire 2 r Spectrum Accumulation spectra under conditions Accumulate all spectra that pass acceptance criteria x Number of DAT files lt 1
356. in PSD mode 8 48 8 76 not available on STR models with serial number 4154 and later 5 18 optimizing resolution 5 76 optimizing signal to noise ratio 5 88 range 5 18 Guide Wire Voltage effects on resolution 5 50 5 56 5 57 6 25 sensitivity 5 50 5 56 5 84 5 88 signal to noise ratio 5 88 Guidelines for acquiring 6 4 H HABA 3 4 chemical structure and molecular weight C 10 mass spectrum C 4 Hard disk drive backing up 9 6 maintenance 9 6 size 1 30 1 41 space required 2 29 Hardware configuring 2 35 initialized when computer is shut down 2 75 initialized when software starts 2 74 not initialized when you log on as new user 2 75 reinitializing 2 74 Help see PerSeptive Biosystems Technical Support Index 16 Applied Biosystems High current detector starting serial number 1 23 1 35 5 91 High mass ions acceptable resolution 6 32 H 17 cannot see in Reflector mode 9 17 Delay Time recommendation 5 55 fragmenting in alpha cyano 9 17 Grid Voltage recommendation 5 74 Guide Wire Voltage recommendation 5 56 Input Bandwidth setting 5 30 laser intensity required in Continuous Extraction mode H 11 moving on sample position 6 5 using Low Mass Gate 5 90 High organic sample concentration sample plate to use 3 62 High voltage Instrument Control Panel automatically turned off 4 25 configuring 2 38 idle time 2 39 On Off state displayed in status bar 4 5 polarity switch delay 2 38 standby mode time 2 39 6 4 7 24 stat
357. includes an oscilloscope wait approximately one minute for its initialization cycle to finish A message indicates that the power on self check passed then the oscilloscope screen is displayed See the Appendix I Using the Oscilloscope and Control Stick for more information Voyager Biospectrometry Workstation User s Guide 2 73 Chapter 2 Installing the Voyager Biospectrometry Workstations If your system includes a LeCroy digitizer wait approximately one minute until the digitizer completes its internal calibration before starting the Instrument Control Panel 4 Logon to the Voyager Workstation using your User Name and Password See your system administrator for your User name and Password 5 Start the Voyager Instrument Control Panel software by s 1 double clicking the Voyager Instrument Control icon on the Windows NT desktop Voyager Instrument Initializing The hardware is automatically initialized when you start the software During initialization the video image on the sample stage is displayed The sample stage moves to the home position and then to the load position If problems occur an error message is displayed when you log on to the workstation Further details on any problem can be obtained by viewing the Windows NT Event Log For more information see Checking the Windows NT Event Log on page 9 22 Reinitializing To reinitialize the hardware 1 Open the Instrument Control Panel 2
358. ing PLT files e Adjusting the laser position for a custom PLT file Voyager Biospectrometry Workstation User s Guide 3 61 Chapter 3 Preparing Samples 3 5 1 Sample Plate Types and Applications The Voyager Instrument Control software supports several types of reusable and disposable plates See Table 3 11 for the applications of different sample plates NOTE For additional information on available sample plates and ordering information see Appendix B 3 Spare Parts Table 3 11 Sample Plate Types Applications and Benefits Sample Plate Type Applications Benefits Welled Sample Plates Gold e GPC MALDI or HPLC fractions when high 100 position concentration of organic solvent provides no surface tension e Polymer analysis using highly volatile solvents e Easier automated acquisition specifically for search pattern SP file creation The wells ensure correct sample placement for running in Automatic Control mode Flat Sample Plates with laser etched circles indicating sample position Stainless steel e Increased mass accuracy allows close external 100 position and calibration procedures 400 position e Ensures correct sample placement for running in Automatic Control mode e Crystallization particularly with DHB DHBs and 3 HPA matrixes is more defined and easier to observe on the video monitor 3 62 Applied Biosystems Sample Plate Types Table 3 11 Sample Pla
359. ing Proteins from Two dimensional Gels by Molecular Mass Searching of Peptide Fragments in Protein Sequencing Database Proc Natl Acad Sci USA 1993 90 5011 5015 Clauser K R P Baker and A L Burlingame Role of Accurate Mass Measurement 10 ppm in Protein Identification Strategies Employing MS or MS MS and Database Searching Anal Chem 1999 71 2871 2882 Patterson D H G E Tarr F E Regnier and S A Martin C Terminal Ladder Sequencing via Matrix Assisted Laser Desorption Mass Spectrometry Coupled with Carboxypeptidase Y Time Dependent and Concentration Dependent Digestions Anal Chem 1995 67 3971 DNA Applications Ehring H M M Karas F Hillenkamp Org Mass Spectrom 1992 27 472 480 Kirpekar F E Nordhoff K Kristiansen P Roepstorff A Lezius S Hahner M Karas and F Hillenkamp Matrix Assisted Laser Desorption ionization Mass Spectrometry of Enzymatically Synthesized RNA up to 140 kDa Nucleic Acids Research 1994 22 3866 3870 Bibliography 4 Applied Biosystems Bibliography Juhasz P M T Roskey I P Smirnov L A Haff M L Vestal and S A Martin Applications of Delayed Extraction Matrix Assisted Laser Desorption lonization Time of Flight Mass Spectrometry to Oligonucleotide Analysis Anal Chem 1996 68 941 946 Pieles U W Z rcher M Sch r and H E Moser Matrix Assisted Laser Desorption lonization Time of flight Mass Spectrometry a Po
360. ing the Optional External Laser Laser position on the sample drifts If the laser is left on for long periods of time position may drift Press Stop then Start or Reset 9 28 Applied Biosystems A Specifications This appendix contains the following sections A1 Voyager DE Specifications A 2 A2 Voyager DE PRO Specifications A 5 A3 Voyager DE STR Specifications A 8 A 4 Digitizer Specifications A 11 NOTE The specifications for this instrument are subject to change without notice Voyager Biospectrometry Workstation User s Guide A 1 Appendix A Specifications A 1 Voyager DE Specifications This section includes the following specifications for the Voyager DE Workstation Performance e Mass Spectrometer e Miscellaneous Table A 1 Voyager DE Performance Specifications for Installation Condition Specification Resolution FWHM Full Width at Half Maximum 2800 for Insulin bovine 3 5 pmol ul in Delayed Extraction mode 21 000 for Myoglobin 4 pmol l 22 000 for Angiotensin 1 3 pmol l M H ion analyzed Signal to Noise Ratio e 2200 1 for IgG 0 6 pmol ul 2100 1 for BSA 4 pmol ul Mass Accuracy in Delayed Extraction mode External Calibration 0 05 difference between the mean of six measurements Internal Calibration 0 02 and the theoretical mass of the sample Mass accuracy is the di
361. ing the laser intensity by 10 percent until the signal appears When signal appears the current laser intensity is the high setting for the sample class matrix you are analyzing H 14 Applied Biosystems Obtaining Good Spectra in Continuous Extraction Mode Fine tuning When you determine the high and low setting for the sample threshold class matrix you are analyzing you can fine tune the threshold by setting the laser intensity midway between the high setting and low setting determined above If signal is present when you decrease the laser assume that this is the new high setting If signal is not present when you decrease the laser assume that this is the new low setting Continue adjusting until the difference between high and low settings is less than 10 laser counts NOTE If the difference between the high and low setting is equal to the laser step size decrease the laser step size in the Hardware Configuration by a factor of 2 Figure H 6 shows a spectrum generated with an acceptable laser intensity This File 3 CAVOYAGERIDATASKEITHiK0106010 MS Collected 146495 8 39 AM 15000 10000 5000 races 2 25 379 26 294 355 1297 9 146 208 72 194 500 1000 1500 2000 2500 Figure H 6 Spectrum at Lower Laser Setting Voyager Biospectrometry Workstation User s Guide H 15 Appendix H Continuous Extraction Mode H 2 2 3 Verifying Threshold Setting H 16 Applie
362. inuous Extraction Mode H 1 Optimizing a Continuous Extraction Standard Instrument BIC Setting Before you begin H 2 Instrument settings parameters Applied Biosystems NOTE Due to the superior results obtained during Delayed Extraction DE mode use Continuous Extraction mode for diagnostic purposes only Before optimizing a Continuous Extraction BIC file be familiar with the following information e The Data Explorer Software User s Guide Appendix B Overview of Isotopes e Opening and Viewing Instrument Settings on page 5 7 e List of standard instrument settings files on page H 3 e Saving and Printing Instrument Settings on page 5 11 e Acquiring in Manual Mode from the Instrument Control Panel on page 6 11 e Section H 2 Obtaining Good Spectra in Continuous Extraction Mode e Determining Laser Threshold on page H 12 e Section H 2 3 Checking Resolution e Section H 2 4 Fine Tuning the Laser Setting e Section 5 1 Loading Modifying and Saving Instrument Settings Most instrument settings parameters are optimized for your system and should not require adjustment Change only the parameters listed in the following procedure For more information on remaining instrument settings see Section 5 2 Instrument Settings Parameter Descriptions Optimizing a Continuous Extraction Standard Instrument BIC Setting List of standard This section lists standard instrument setti
363. ion 5 1 Loading Modifying and Saving Instrument Settings for more information Adjusting In general e A higher Grid Voltage with a lower potential difference decreases ion fragmentation e Grid Voltage has a large impact on the quality of data obtained For each Grid Voltage setting there is an optimum Delay Time Voyager Biospectrometry Workstation User s Guide 5 53 5 54 Chapter 5 Optimizing Instrument Settings 5 3 3 Understanding Delay Time Applied Biosystems Delay Time is the time in nanoseconds after the laser ionizes the sample at which full Accelerating Voltage is applied This creates the potential gradient that accelerates ions Delay Time corrects the dependence of ion flight time on initial velocity Observed mass resolution increases in proportion to the effective length of the ion flight path NOTE There is an inherent 180 5 nsec delay between the time the laser fires and the time the voltage is applied The actual time that voltage is applied is equal to the Delay Time you enter plus the inherent delay on your system Changing the Delay Time in increments smaller than 20 nsec may have no significant impact on resolution Figure 5 15 illustrates Delay Time Full Accelerating Delay Time Voltage applied nsec psmmnnnns a 5 a i k z extracting r ASer field 3 pulse a 4 a a Re EE A gt time Figure 5 15 Delay Time Impact of Changing
364. ions are displayed For more information see Color coding on page 2 69 If Use Mass Accuracy Optimizations was selected when you loaded the specified Plate ID a message box indicates that this function is disabled for the OptiPlate run NOTE For optimum display of results make sure the color palette on your computer is set to more than 256 colors To check select Settings from the Start menu select Control Panel select Display then click Settings If you change the Plate ID in the OptiPlate dialog box make sure the correct physical plate is loaded in the mass spectrometer before you continue Voyager Biospectrometry Workstation User s Guide 2 63 Chapter 2 Installing the Voyager Biospectrometry Workstations OptiPlate Plate ID Instrument Settings BIC Data File Path and Root Name OptiPlate x peptide_reflector negative bic es Extraction Correction mm eset gt 0 15 ramman 0 10 oe z oo Include Al Positions O r rite Sat Postion A OOOOOMOOOO Ex Paten os OOOOGOOOOOO aayi ae OOOO OQ OOOO eus 000000000 Mo AE Start Acquisition ay fe SC 289875 v 219075 Average enor ppm Extraction conection 0 0000 rm Position Time sec Position Time sec
365. ions during servicing when interlocks have been circumvented the lasers fall into the following categories can cause permanent eye damage Nitrogen Class IIIb e Nd YAG Class IV The Voyager Biospectrometry Workstation complies with Title 21 U S Government DHEW BRH Performance Standards Chapter 1 Subchapter J Section 1040 as applicable Laser safety The following safety features are included on the Voyager features Biospectrometry Workstation e Cabinet is designed to prevent access to collateral laser radiation exceeding the accessible emission limits in Performance Standards for Laser Products 21 CFR 1040 10 Front and side panels have interlock switches that disable the laser when panels are removed e Safety labels for Class standards are affixed to the unit Laser safety To ensure safe laser operation note the following requirements The system must be installed and maintained by an Applied Biosystems Technical Representative All panels must be installed during operation When all panels are installed there should be no detectable radiation present If any panels are removed when the laser is operational you may be exposed to laser emissions in excess of Class 1 rating Do not remove labels or disable safety interlocks Additional safety Refer to the users manual provided with the laser for information additional information on government and industry safety regulations xxvi Applied Bio
366. iosystems Cleaning Sample Plates Examine the plate If you see any sample or matrix residue oil or fingerprints on the plate soak the sample plate in a working solution of laboratory detergent in water for example RBS 35 detergent from Pierce or LIQUI NOX from VWR for 5 to 10 minutes IMPORTANT RBS LIQUI NOX Please read the manufacturer s MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CAUTION Do not leave the sample plate in detergent for longer than 10 minutes Longer exposure can cause the bottom holders on the sample plate to corrode Do not sonicate sample plates or use acid to clean sample plates Both can alter the surface of the sample plate and reduce the quality of the data obtained If residue remains wipe the plate with a lint free tissue or cotton swab A soft toothbrush also works well Rinse the plate thoroughly in deionized water To speed drying rinse the plate in acetone WARNING CHEMICAL HAZARD Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Allow the plate to dry in an area where it will not
367. iption System Status Instrument Possible states are State e ON Instrument is initialized and high voltage is on e OFF Instrument is not initialized and high voltage is off e FAULT Indicates a fault condition Follow the instructions displayed to correct the fault High Voltage Possible states are e RAMPING Voltage is turning on e ON High voltage is on e OFF High voltage is off e FAULT Indicates a fault condition Follow the instructions displayed to correct the fault Source Displays the source chamber pressure Operating pressure ranges Chamber are BA1 e Voyager DE Voyager DE PRO Less than 10 Pressure Voyager DE STR Less than 5x107 continued Voyager Biospectrometry Workstation User s Guide 2 77 Chapter 2 Installing the Voyager Biospectrometry Workstations Table 2 3 System Status Parameters Continued Parameter Description Mirror Displays the mirror chamber pressure Mirror chamber pressure Chamber ranges are BA2 Voyager DE PRO Less than 5x10 7 Pressure Voyager DE STR Less than 5x10 NOTE This status field is not displayed on Voyager DE systems Acquisition Displays acquisition status e ON lInstrument is currently acquiring a spectrum e OFF Instrument is not acquiring Data Storage Displays data storage status e ON Software is currently storing a data file e OFF Software is currently not storing a data fi
368. iratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Voyager Biospectrometry Workstation User s Guide 3 41 Chapter 3 Preparing Samples WARNING CHEMICAL HAZARD Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Tetrahydrofuran is a flammable liquid and vapor It may be harmful if swallowed Exposure may cause eye and respiratory tract irritation central nervous system depression and liver and kidney damage Loading samples 1 Create a log sheet listing sample position and sample name Appendix D Log Sheets contains a master sheet that you can copy and use NOTE For optimum mass accuracy do not use the outer rows of the sample plate unless you are using internal standards 3 42 Applied Biosystems Loading Samples on Sample Plates 2 Using a clean pipette tip for each new sample load the following e If you premixed samples Load 0 5 to 2 ul of sample matrix solution on the appropriate positio
369. ire Voltage with lower Mirror Ratio Voltage settings may or may not improve the quality of PSD data e Avoid using Guide Wire Voltage settings above 0 02 Higher settings may compromise the selectivity of the Precursor lon Selector NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later 8 76 Applied Biosystems Viewing PSD Data 8 7 Viewing PSD Data For information on viewing PSD data see the Data Explorer Software User s Guide Chapter 8 Viewing Voyager PSD Data Voyager Biospectrometry Workstation User s Guide 8 77 Chapter 8 PSD Analysis 8 78 Applied Biosystems Chapter 9 9 Maintenance and Troubleshooting This chapter contains the following sections 9 1 Maintenance 9 2 9 1 1 Maintenance Schedule 005 9 2 9 1 2 Hardware Maintenance 9 3 9 1 3 Backing Up and Archiving Data 9 6 9 2 Troubleshooting 9 7 9 2 1 Spectrum Troubleshooting 9 7 9 2 2 Software Troubleshooting 9 19 9 2 3 Hardware Troubleshooting 9 23 Voyager Biospectrometry Workstation User s Guide 9 1 Chapter 9 Maintenance and Troubleshooting 9 1 Maintenance This section describes Maintenance schedule Hardware maintenance e Backing up and archiving data 9 1 1 Mainte
370. is performed on the positions you manually select instead of search pattern positions The number of search pattern positions analyzed and therefore the number of spectra accumulated is equal to the Number of Spectra to Acquire even if the number of positions in the search pattern is larger All search pattern positions may not be analyzed Acceptance criteria are not used All spectra acquired are accumulated into one DAT file that contains one spectrum gt Spectrum Accumulation Acquire E spectra under conditions Accumulate all spectra x 1 spectrum from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 250 shots 50 shots 5 spectra Number of DAT files 1 Figure 5 12 Accumulate All Spectra Mode Automatic Control Dialog Box Voyager Biospectrometry Workstation User s Guide 5 45 Chapter 5 Optimizing Instrument Settings Examples Accumulate all spectra that pass 5 46 acceptance criteria Applied Biosystems If Random search pattern is selected and the Number to Acquire is 5 five positions are analyzed and one DAT file containing one accumulated spectrum is created If an SP file with 20 positions is selected and Number to Acquire is 25 20 positions are analyzed and one DAT file containing one accumulated spectrum is created For more information see Section 6 6 7 Process that Oc
371. isition e Stop When acquisition is complete CAUTION The oscilloscope does not save spectra If you acquire a new spectrum before downloading to the Voyager processing software you lose the previous spectrum To adjust the display e Center the signal vertically on the screen using the Vertical Position knob e Adjust the amplitude of the signal using the Vertical Scale knob Make sure the peak of interest is on scale e Widen peaks using the Horizontal Scale knob I 3 Using the Control Stick Starting acquisition Using the Control Stick After you load samples on the sample plate and load the plate into the system start acquiring To start acquiring press the left button on the base of the control stick Figure l 4 Stop data acquisition save spectrum Start stop data acquisition MILA a Move stick up and down left and right to adjust sample position under laser beam PB100474 Figure 1 4 Control Stick NOTE You can also start acquisition by clicking in the toolbar Acquisition continues until the number of Shots Spectrum in the instrument setting is acquired or until you press the start stop left button on the control stick or the toolbar button again To stop acquiring press the left button on the base of the control stick Figure l 4 Voyager Biospectrometry Workstation User s Guide 1 7 Appendix l 8 Applied Bio
372. isition functions in the other control panel are inactive e If you are acquiring data in the Instrument Control Panel Sequence Control Panel functions are disabled until acquisition is complete e If you are acquiring data in the Sequence Control Panel Instrument Control Panel acquisition functions are disabled until acquisition is complete NOTE Other Instrument Control Panel functions are available while acquisition is running in the Sequence Control Panel However prompts and error messages are suppressed to prevent the sequence from being interrupted How the Instrument and Sequence Control Panels Interact Keeping both You can keep the Sequence Control Panel and the Instrument control panels Control Panel open at the same time However if you do not open need Sequence Control Panel functions close the Sequence Control Panel to improve system performance CAUTION If you started the Instrument Control Panel by double clicking the Sequence Control Panel icon the Instrument Control Panel closes when you close the Sequence Control Panel Organizing the You can organize the display to suit your needs desktop To rearrange the Sequence Control Panel and the Instrument and windows Control Panel select commands from the Desktop menu e Tile Horizontal Places the Sequence Control Panel at the top or bottom of the desktop and the Instrument Control Panel in the opposite location e Tile Vertical Places the Seq
373. itate at the bottom of the tube When applying matrix use the supernatant not the precipitate 3 1 3 Matrix Information Refer to the tables on the following pages to determine the requirements for the matrix you are using This section includes information for Sinapinic acid a cyano 4 hydroxycinnamic acid CHCA e THAP e 3 HPA e DHB e DHBs e Synthetic polymer matrixes WARNING CHEMICAL HAZARD Refer to the Material Safety Data Sheet MSDS provided by the chemical manufacturer before handling solvents or matrixes 3 6 Applied Biosystems Preparing Samples Sinapinic acid Use sinapinic acid for peptides and proteins gt 10 000 Da Table 3 1 Sinapinic Acid Matrix Information WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Sinapinic acid may cause eye skin and respiratory tract irritation Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Trifluoroacetic acid causes severe burns to the eyes skin and respiratory tract Matrix concentration 10 mg ml Final sample concentration 0 1 5 pmol ul Solvents Acetonitrile 0 1 TFA in deionized water Preparation Follow the procedure in
374. itizer Dialog Box Click Mode Digitizer in the Instrument Settings control page see Figure 5 3 on page 5 15 to display the Mode Digitizer dialog box Figure 5 4 Mode Diaitizer x Reflector Digitizer Advanced Instrument Mode Linear Digitizer m Operation Mode Linear Reflector C PSD Extraction Type Delayed C Continuous Polarity Type Positive C Negative Laser Type Internal C External Laser Rate Type C Default Optimized Figure 5 4 Mode Digitizer Dialog Box with Instrument Mode Tab Displayed The Mode Digitizer settings dialog box includes tabs for the the following instrument settings parameters Instrument Mode e Linear Reflector Digitizer e Advanced 5 24 Applied Biosystems Instrument Settings Parameter Descriptions Instrument Mode Click the Instrument Mode tab to display the Instrument parameters Mode page Figure 5 4 Instrument Mode parameters are described in Table 5 6 Table 5 6 Instrument Mode Parameters Parameter Description Operation Mode Specifies the Operation Mode e Linear Uses Linear detector only Most sensitive mode due to shorter flight path Also more sensitive because fragments neutrals and molecular ions arrive at the detector at the same time Less sample fragmentation is observed e Reflector Uses Reflector detector only Higher resolution greater mass accuracy due to longer flight path and action of reflector
375. ity used to acquire the reflector mode precursor spectrum and increases with each subsequent segment Examining and 6 Examine the spectrum to ensure that fragments are saving the segment produced Figure 8 2 Voyager Spec 1 MC BP 1296 4 65535 Precursor 1005 _6 6E Fragments Intensity Figure 8 2 Segment Spectrum 8 10 Applied Biosystems Selecting and 8 acquiring remaining segments Stopping the 9 experiment PSD Quick Start If fragments are present and data is acceptable Figure 8 2 click H in the toolbar to save the segment to the DAT file After you save the segment the Saved check box in the segment list is checked If the current spectrum does not contain significant fragment ion signal and you do not want to save the spectrum reselect the row and reacquire the spectrum or select a new row CAUTION Save the current segment if the data is acceptable before starting to acquire the next segment If you do not you will lose the data for the current segment Repeat step 3 through step 7 to collect remaining segments NOTE Segments are listed in the Data Explorer software in the order in which they are acquired If segments with duplicate Mirror Ratios are contained in the file the software uses the last acquired segment when it generates the composite spectrum After you acquire all necessary segments select Stop Experimen
376. ix on page 3 5 Dry down quickly under vacuum for even response If you allow to air dry you will see a less even response during analysis Crystals Needle like crystals arranged in a ring if air dried see Figure 3 3 on page 3 46 Stability Prepare weekly Voyager Biospectrometry Workstation User s Guide 3 17 Chapter 3 Preparing Samples DHBS Use DHBs for peptides and proteins gt 10 000 Da and glycosylated proteins WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Table 3 8 DHBs Matrix Information Matrix concentration 10 mg ml Additive concentration 10 mg ml 5 methoxysalicylic acid Final sample concentration 10 pmol ul to 100 fmol ul Solvents 80 0 1 TFA in deionized water 20 acetonitrile for DHB 50 acetonitrile 50 deionized water for 5 methoxysalicylic acid Preparation Follow the procedure in Preparing matrix on page 3 5 and combine 9 1 DHB 5 methoxysalicylic acid solutions Air dry sample plate after loading sample and matrix Crystals Needle like crystals arranged in a ring if air dried see Figure 3 3 on page 3 46 No
377. k mass Voyager Biospectrometry Workstation User s Guide H 5 Appendix H Continuous Extraction Mode H 6 Applied Biosystems 6 Set the Grid Voltage appropriate for the matrix and 7 8 mass Table H 4 Grid Voltage Settings for Continuous Extraction Mode Matrix Mass Da Grid Voltage a cyano lt 5 000 50 70 4 hydroxycinnamic acid gt 5 000 70 90 Sinapinic acid lt 5 000 80 85 gt 5 000 85 90 DHB lt 5 000 85 90 3 HPA gt 5 000 In Reflector mode lower Grid Voltage settings may yield greater resolution but may compromise sensitivity Save the instrument settings BIC file Determine the laser threshold for the sample and adjust the laser setting until you obtain the resolution needed for your application Laser position and laser intensity are the primary factors affecting the quality of spectra However you may slightly improve the quality of data by fine tuning e Grid Voltage within the ranges listed above e Guide Wire Voltage within the ranges listed below Optimizing a Continuous Extraction Standard Instrument BIC Setting Table H 5 Guide Wire Voltage Settings for Continuous Extraction Mode Mass Range Da Guide Wire Voltage lt 1 500 0 05 1 500 4 00 0 1 4 000 15 000 0 2 gt 15 000 0 3 Voyager Biospectrometry Workstation User s Guide H 7 Appendix H Continuous Extraction Mode
378. l Netherlands Oct 1996 73 87 94 Timofeev E N I P Smirnov L A Haff E l Tishchenko A D Mirzabekov and V L Florentiev Methidium Intercalator Inserted into Synthetic Oligonucleotides Tetrahedron Letters 1996 Bibliography 6 Applied Biosystems Bibliography Vestal M L P Juhasz and S A Martin Delayed Extraction Matrix assisted laser desorption time of flight mass spectrometry Rapid Commun Mass Spectrom 1995 9 1044 1050 Zhu Y F C N Chung N I Tarenenko S L Allman S A Martin L A Haff and C H Chen The Study of 2 3 4 Trihydroxyacetophenone and 2 4 6 Trihydroxyacetophenone as Matrices for DNA Detection in Matrix assisted Laser Desorption lonization Time of Flight Mass Spectrometry Rapid Comm Mass Spectrometry 1996 10 383 388 Zhu Y F N I Taranenko S L Allman N V Taranenko S A Martin and L A Haff Oligonucleotide Sequencing by Fragmentation in matrix assisted laser desorption ionization time of flight mass spectrometry Rapid Comm Mass Spectrometry 1997 11 897 903 B j B L j 0 G R A P H Y Voyager Biospectrometry Workstation User s Guide Bibliography 7 Bibliography B j B L j 0 G R A P H Y Bibliography 8 Applied Biosystems Index Numerics 100 well plate number of cal standards needed 3 39 PLT file 3 67 types of 3 62 3 63 384 well plate number of cal standards needed 3 39 PLT file 3 67 position r
379. l 6 Ch1 and Ch2 markers l 6 connecting Voyager DE and Voyager DE PRO 2 19 connecting Voyager DE STR 2 28 description 1 19 1 32 displaying full range of data l 3 front panel l 1 grid line increments 5 69 guidelines for acquiring l 3 initialization 2 73 intensity versus time spectrum I 5 Live trace not displayed in Spectrum window 6 16 mass to time conversion for standards F 4 Output window Automatic Control tab 4 5 6 45 closing 4 6 Data Storage tab 6 18 maximum number of lines displayed 4 6 Overview Delayed Extraction technology 1 11 MALDI TOF technology 1 7 parts of Voyager DE 1 17 parts of Voyager DE PRO 1 18 parts of Voyager DE STR 1 31 PSD analysis 8 20 Sequence Control Panel 4 32 7 2 Sequence Control Panel calibration 7 5 7 6 Voyager DE system 1 2 Voyager DE PRO system 1 3 Voyager DE STR system 1 5 P Page control types of 4 9 Parent ion see PSD precursor spectrum Password obtaining from system administrator 2 74 Path length Voyager DE 1 4 Voyager DE PRO 1 4 Voyager DE STR 1 6 PDF files provided 2 31 Peak centroid shift 5 63 Peak detection overview 6 28 setting 6 28 setting Sequence Control Panel 7 17 Peak filtering monoisotopic 7 3 7 12 7 17 Peak labels color changing 4 21 enabling and disabling 6 29 overview 6 28 resolution 6 31 Index Peak shape and accurate mass measurement 6 26 and calibration 6 10 description of acceptable 6 21 H 8 improving H 18 troubleshooting 9 11 9 16
380. l Control mode 8 43 Fill in the segment list and save the BIC file 8 47 Acquire and save PSD segments 8 52 Practicing in PSD This section tells you how to use the software to collect mode spectra in PSD mode However to be successful in PSD analysis you need to understand how spectra behave in PSD mode and how to optimize acquisition conditions Follow the steps in this section to understand how to use the software Then follow the steps in Section 8 6 Exploring PSD Mode to learn how to optimize the parameters that affect PSD analysis NOTE Before you run unknowns we recommend that you perform the steps above using a standard Voyager Biospectrometry Workstation User s Guide 8 37 Chapter 8 PSD Analysis 8 4 1 8 38 Determining the Precursor lon Mass Overview Before beginning an analysis in PSD mode Generate a precursor spectrum in Reflector mode to determine the mass of the precursor ion A Reflector mode analysis provides optimum resolution and mass accuracy Generate an external calibration for the precursor ion to use during the PSD acquisition Generating the To generate the precursor spectrum precursor spectrum Applied Biosystems Open the PSD_Precursor BIC file provided with the software This is a reflector mode instrument settings BIC file All other settings should be identical to the settings in the PSD mode BIC you will use to acquire segments Modify the mas
381. l for enhancing analysis of high mass ions Immonium ion in PSD low molecular weight fragment ions derived from amino acids Initial velocity Tte velocity associated with an ion when it is released from the sample plate before acceleration Voyager software allows correction for matrix dependent initial velocities Intensity signal or ion Amplitude of signal shown on oscilloscope screen or spectrum window Intensity laser Amount of laser generated light reaching the sample spot Controlled by adjusting the laser attenuator using laser step setting lon An isolated electron or proton or an atom or molecule which by loss or gain of one or more electrons has acquired a net electric charge lonization Conversion of sample in solid gaseous or liquid phase to ions lon intensity See intensity lon source Device that produces ions In a TOF instrument it refers to the surface of the sample plate the variable voltage grid above the plate and the grounded grid and aperture above the variable voltage grid Isomers Compounds that have the same molecular weight but different structures Isotopes One of two or more atoms with the same atomic number but a different mass For example carbon isotopes 2C 13C and 4C Laser tnergy source for sample ionization in MALDI technology The Voyager Biospectrometry Workstation uses a Nitrogen laser at 337 nm that provides 3 nanosecond wid
382. layed Extraction technology provides improved resolution and mass accuracy PB100466 Figure 1 2 Voyager DE PRO Biospectrometry Workstation Biospectrometry Biospectrometry is the application of mass spectrometry in the field of the life sciences This field uses fast chromatographic techniques enzymatic chemistries and surface chemistries and combines them with mass spectrometry and advanced software to better enable biomolecular research and facilitate data interpretation Voyager Biospectrometry Workstation User s Guide 1 3 Chapter 1 Introducing the Voyager Biospectrometry Workstations Features Features of the Voyager DE and Voyager DE PRO Biospectrometry Workstations include e High speed digitizer options and laser rates for optimum sample throughput e Positive or negative ion detection e m z range in excess of 300 kDa Sensitivity to less than 5 femtomoles with dried droplet application e lon path length e Voyager DE 1 2 meter e Voyager DE PRO in linear mode 1 3 meter e Voyager DE PRO in reflector mode 2 0 meter e Variable acceleration voltage e Compact benchtop design e Variable two stage ion source e Automated single plate sample loading sy
383. lbar to manually accumulate spectra You can also accumulate spectra by selecting Accumulate Spectrum from the Acquisition menu For more information see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Click in the toolbar to manually clear an accumulated spectrum You can also clear an accumulated spectrum by selecting Clear Accumulated Spectrum from the Acquisition menu For more information see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions Controlling the Workstation 4 5 2 Adjusting Laser Intensity and Selecting Sample Position This section describes e Displaying the Manual Laser Sample Position page e Manually adjusting laser intensity e Selecting the active sample position in Plate view e Displaying coordinates of active position e Switching between Plate view and Sample view e Adjusting sample position in Sample view For information on automatically controlling the laser and sample position see Section 5 2 3 Automatic Control Dialog Box Displaying the Select Manual Laser Sample Position from the View menu Manual Laser to display the Manual Laser Sample Position control page Sample Position Figure 4 8 and Figure 4 9 on page 4 31 control page E Laser setting m Manual Laser Intensity ee Slider laser 1811 a J contro control 4 Manual Sample Positioning Coarse Active position Active Pos fan gt laser 100 well plate pt
384. le 2 78 Applied Biosystems 3 Preparing Samples This chapter contains the following sections 3 1 Preparing Samples c ccceeeeeeeeeeeeeeaeeeeeaees 3 2 3 1 1 Selecting a Matrix ssec 3 3 3 1 2 Preparing Matrix 3 4 3 1 3 Matrix Information 3 6 3 1 4 Preparing Sample 3 22 3 1 5 Sample Cleanup sssi 3 25 3 1 6 Mixing Sample and Matrix Dried Droplet Application 3 33 3 2 Loading Samples on Sample Plates 3 35 3 3 Cleaning Sample Plates 3 47 3 4 Loading Sample Plates in the Mass Spectrometer 3 50 3 5 Sample Plate Types 3 61 Voyager Biospectrometry Workstation User s Guide 3 1 Chapter 3 Preparing Samples 3 1 Preparing Samples 3 2 In this section Overview Applied Biosystems NOTE Sample preparation technique has a direct impact on the quality of the data you obtain in MALDI TOF applications This section describes Selecting matrix e Preparing matrix e Matrix information e Preparing sample e Sample cleanup e Mixing sample and matrix dried droplet application To prepare samples you Select matrix Prepare matrix fo Prepare sample Mix sample and matrix Load sample on clean sample plate w se Preparing Samples 3 1 1 Sele
385. le Cleanup On Voyager DE PRO and Voyager DE STR systems not separating isotopic peaks in reflector mode up to mass 1 000 Adjust Accelerating Voltage to 10 000 V and reacquire spectrum See Section 5 4 4 2 Setting Accelerating Voltage Voyager Biospectrometry Workstation User s Guide H 21 Appendix H Continuous Extraction Mode Table H 8 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor resolution in Continuous Extraction mode continued Beam guide wire malfunction Call Applied Biosystems Technical Support Accelerating Voltage malfunction Call Applied Biosystems Technical Support Microchannel plate detector voltage set incorrectly Call Applied Biosystems Technical Support Variable voltage grid contaminated with matrix Call Applied Biosystems Technical Support Poor mass accuracy in Continuous Extraction mode continued on next page Mass scale not accurately calibrated Recalibrate See the Data Explorer Software User s Guide Chapter 5 3 2 Manually Calibrating Used different voltages when acquiring sample and calibration standard Use same voltages when acquiring sample and calibration standards In Linear mode used different laser intensity when acquiring sample and calibration standard Use same laser intensity when acquiring sample and calibration standards Incorrect
386. le bar at the left of the toolbar section To move the toolbar section back to the top of the window click drag the toolbar back to the original position See the Data Explorer Software User s Guide Customizing toolbars on page 1 21 for information on customizing the toolbar To customize the display use the Graph and Plot Options dialog box Figure 4 6 to e Change colors of the trace and trace background e Change color of peak labels e Change the appearance of annotation text e Turn axes on and off and change axis appearance e Turn cursor and grids on and off Hint You can use the same settings for all graphs Before making individual graph selections select the View Setup Tab Select Use same settings for all graphs Select any Graph Setup tab and make selections The selections will be applied to the remaining Graphs Setup tabs Voyager Biospectrometry Workstation User s Guide 4 21 Chapter 4 Voyager Instrument Control Panel Basics Accessing To access the graphic options graphic options 4 Display the trace of interest 2 From the Display menu select Graphic Options then click a Graph Setup tab in the Graph and Plot Options dialog box see Figure 4 6 on page 4 23 3 Set Setup parameters 4 Setcolors line widths data cursors and graphic compression See the Data Explorer Software User s Guide Section 1 5 Setting Graphic Options for more information NOTE Line Widths of
387. le is applied to all spectra in the DAT file Figure 7 2 How Settings Macros and Calibration Are Applied During a Sequence Run Part 1 Voyager Biospectrometry Workstation User s Guide 7 5 Chapter 7 Acquiring Spectra from the Sequence Control Panel If you specify this These SET CAL and macros And a data file Calibration Type are applied if specified is created with in the order shown Internal Update DAT file with Calibration Peak detection settings from SET Calibration constants SET S CAL SET generated after peak calibration reference calibration constants A masses from CAL applied ha a eas Autocal i then calibration with reference masses in we Es ie ww y SET If CAL specified New or updated does not exist no CAL file with constants applied before calibration generated after but CAL created calibration constants after calibration from CAL applied then calibration with reference masses in SET e Calibration constants NOTE If the DAT file contains multiple spectra generated using the Save All or the Save All Passing option in Automatic Control the CAL is applied to all spectra in the DAT file Figure 7 3 How Settings Macros and Calibration are Applied During a Sequence Run Part 2 7 6 Applied Biosystems Before Creating a Sequence 7 3 Before Creating a Sequen
388. lications If you are examining a sample mixture it may be necessary to prepare the mixture with several different matrixes For specialized applications you may need to investigate other matrixes for example Matrix Application 1 4 hydroxyphenylazo Proteins polar and nonpolar benzoic acid HABA synthetic polymers Additional information Appendix C Matrixes contains additional information on matrix structure appearance and matrix solutions For additional information on other matrixes and their applications refer to the bibliography 3 1 2 Preparing Matrix 3 4 Matrix stability What you need Applied Biosystems Prepare fresh matrix as needed Some matrixes degrade upon exposure to light or humidity Some matrixes require daily preparation Other matrixes can be stored for up to one week at room temperature Follow the matrix manufacturer s instructions for storage conditions of chemicals and refer to individual matrix descriptions in the following section for additional stability information Materials required e Balance e 1 5 ml microcentrifuge tubes e Micropipettor and disposable tips e Centrifuge e Vortex mixer e Matrix e Deionized water e Solvents Preparing Samples NOTE HPLC grade water may vary in salt concentration and may produce adduct ions in mass spectra A high salt concentration may interfere with some applications particularly oligonucleotide analysi
389. lick Close Save the calibration file as ANGIO_PSD CAL by selecting Export from the File menu then selecting Calibration Open the Angiotensin_PSD BIC file that you renamed in Setting PSD acquisition parameters on page 8 7 In the PSD calibration section of the PSD Acquisition settings control page see Figure 8 1 on page 8 8 select the ANGIO_PSD CAL file you created in the previous section Select Save Instrument Settings from the File menu Reacquire PSD segments with PSD calibration as described in Acquiring PSD segments on page 8 9 Check the fragment ion masses to make sure they are within acceptable error PSD Quick Start 8 1 2 PSD Analysis of an Unknown Generating the Reflector mode precursor spectrum Generating an external calibration for the unknown precursor ion This step assumes that you have already determined an accurate mass for the precursor ion using reflector mode high resolution analysis with internal or external calibration this analysis requires conditions that differ from PSD analysis conditions You acquire the precursor ion again using the same conditions you will use for PSD analysis to generate a spectrum from which you can generate an external calibration The external calibration you generate from the precursor ion is used to obtain maximum mass accuracy for the precursor ion during the PSD analysis which helps ensure maximum mass accuracy for the fragment ions 1 Open th
390. lider controls on the Manual Laser Sample Positioning control page Laser beam is not irradiating sample in the sample position Adjust position of sample position using the Manual Laser Sample Positioning controls Signal is offscale Adjust Vertical Offset See Section 5 3 5 Understanding Digitizer Settings Problem with electronics Call Applied Biosystems Technical Support Saturated signal in sample and matrix region Laser intensity too high Adjust laser by using the slider controls on the Manual Laser Sample Positioning control page Poor crystallization on the sample plate Sample contaminated Clean up sample See Section 3 1 5 Sample Cleanup Voyager Biospectrometry Workstation User s Guide continued 9 9 Chapter 9 Maintenance and Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action in Delayed Extraction mode Poor resolution sensitivity Delay Time and Grid Voltage not optimized Optimize See Section 5 4 Optimizing Instrument Settings Parameters Guide Wire Voltage not optimized Optimize See Section 5 4 Optimizing Instrument Settings Parameters Wrong Bin size and Vertical Scale selected for components below 15 000 Da In the Mode Digitizer dialog box set Bin size to 2 nsec In the Mode Digitizer Options dialog box select a Vertical Scale of 200 mV
391. lization for sinapinic acid cyano and sinapinic acid matrixes a cyano Sinapinic acid Rounded Rhomboid shaped PB100265 Figure 3 1 Microscopic View of Sample Plate with a Cyano or Sinapinic Acid Matrixes With good crystallization you see small equally sized crystals that are evenly distributed on the plate Clumping is not desirable If you see clumped crystals with a cyano or sinapinic acid matrixes it may indicate e Matrix concentration is too high e Organic concentration is too high e Sample plate was dried too quickly e Sample contains contaminants Voyager Biospectrometry Workstation User s Guide 3 45 Chapter 3 Preparing Samples 3 46 You can acquire data from a well that does not have an ideal crystallization pattern However when sample contains unevenly distributed crystalllization it may be difficult to analyze This can cause a problem in Automatic mode Other matrixes Typical appearance of other matrixes under magnification are shown in Figure 3 2 and Figure 3 3 When analyzing 3 HPA crystals aim the laser at the base of the fan like crystals for best response Each matrix sample class may require different laser intensities for analysis PB100509 3 HPA THAP Needles in fan like arrangement Overlapping shingles around outer edge of sample well non uniform shape PB100508 Figure 3 2 Microscopic View of Sample Plate with 3 HPA and THAP Matrixes PB10
392. lled Timed lon Selector in Reflector mode and observe the spectrum e Distinguish between the two types of fragments prompt and PSD e Understand the impact of the Precursor lon Selector on prompt fragments Turning off To observe the effects of the Precursor lon Selector called Precursor lon Timed lon Selector in Reflector mode Selector 1 Inthe PSD Acquisition Settings control page deselect Precursor lon Selector 2 Acquire a spectrum 3 Observe the 1 180 to 1 190 Da mass region Note the additional peaks that appear below 1 185 Da when the Precursor lon Selector is turned off Figure 8 16 Additional peaks appear when Precursor lon Selector is turned off Spec 1 P 1296 9 60852 2341 4 Intensity O 0 T T T 7 1164 1170 11 1182 1188 1194 Mass m z Figure 8 16 Angiotensin I Fragment lons Precursor lon Selector Off Voyager Biospectrometry Workstation User s Guide 8 69 Chapter 8 PSD Analysis Figure 8 17 compares the spectrum above with the spectrum acquired in the previous section so you can more easily see the peaks that appear when the Precursor lon Selector is turned off Figure 8 17 also identifies the two types of fragments seen when the Precursor lon Selector is turned off 100 90 80 70 60 50 Intensity 40 30 20 10 Precursor lon Selector off Prompt fragments Spec 1IBP 1296 9 608521 2341 4
393. llow the procedure in Section 5 4 3 5 Optimizing Grid Voltage However optimizing Delay Time is a simpler procedure For more For more information see Section 5 3 3 Understanding information Delay Time Inherent Delay There is an inherent 180 5 nsec delay between the time the Time offset laser fires and the time the voltage is applied The actual time that voltage is applied is equal to the Delay Time you enter plus the inherent delay on your system Changing the Delay Time in increments smaller than 20 nsec may have no significant impact on resolution Voyager Biospectrometry Workstation User s Guide 5 77 Chapter 5 Optimizing Instrument Settings Optimizing Delay NOTE If you are analyzing a broad mass range and need Time optimum resolution in all mass regions you may need to acquire smaller portions of the mass range and set the Delay Time to optimize resolution for each mass range To optimize Delay Time 1 Open a standard BIC file for the mass range you are acquiring For more information see Selecting a BIC file on page 5 8 2 Acquire a spectrum and observe the resolution For information see Section 6 5 2 Calculating Mass Resolution If the resolution is not acceptable increase the Delay Time by 100 nsec Linear mode or 50 nsec Reflector mode acquire a spectrum and observe Leave the Grid Voltage setting unchanged If the signal is saturated at the higher Delay Time setting dec
394. lons ccsceeeeeeeeeeeeeeeeeees 8 28 8 2 6 Optimizing the Precursor lon Selector ccsecceeeeseeeeeeeees 8 29 Applied Biosystems 8 3 8 4 8 5 8 6 8 7 Table of Contents Enhancing Fragmentation with CID 0 2 ccc eeeee eee ee teen eee eee eee 8 31 Acquiring PSD Data with Standard BIC Files in Manual Control MO06 4 42 iuse sis der dvesteneressteneesctenescetns 8 37 8 4 1 Determining the Precursor lon Mass 8 38 8 4 2 Determining the Number of Segments to Acquire for a Complete Composite Spectrum ccceeeeeeeeeee eee eeeteeneenteeeees 8 40 8 4 3 Setting PSD Acquisition Parameters for Manual Mode 8 43 8 4 4 Filling in the Segment List and Saving the BIC File 8 47 8 4 5 Acquiring and Saving PSD Segments in Manual Mode 8 52 Acquiring PSD Data with Standard BIC Files in Automatic Control Mode eeeeeeeaeeeeee 8 57 8 5 1 Setting PSD Acquisition Parameters for Automatic Control Mode ccceeeeeeeeeeeeeeeeeeeeaeaaaeaeeneeeeees 8 58 8 5 2 Setting Laser Increment and Saving the BIC File 8 62 8 5 3 Acquiring PSD Segments in Automatic Control Mode 8 63 Exploring PSD Mode suis era ea kerna dans mean aneenne a iat 8 65 8 6 1 Observing the Effects of Laser Intensity 0 cceccesseeeeeees 8 66 8 6 2 Observing the Effects of Precursor lon S
395. lows you to assign a unique Plate ID to each plate that allows customized alignment of more than one plate that uses the sample PLT file Figure 2 21 Plate A Plate ID 1A Plate B Plate C Plate ID 1B Plate ID 1C 100well PLT 100well PLT 100well PLT Aligned Aligned 6 15 00 6 30 00 Aligned 6 1 00 Figure 2 21 Multiple Alignments Using Sample PLT File How the system When you load a sample plate the system aligns the plate as aligns a plate described below e The software looks for alignment information associated with the Plate ID you load and adjusts accordingly e If there is no alignment information available for the Plate ID sample plate has not been aligned the software uses the default alignment Voyager Biospectrometry Workstation User s Guide 2 47 Chapter 2 Installing the Voyager Biospectrometry Workstations Overview of video The following examples show how the sample positions and monitor display laser spot may be displayed on the video monitor during sample plate alignment Example Description Ideal sample position alignment The center of Perimeter of _ sample position the sample position is aligned with respect to the Laser spot laser spot You mark the outline of this alignment on a Figure 2 22 Ideal Sample Position transparency to use to Alignment calibrate other sample positions during alignment Outline of ideal Sample po
396. lude Selected Positions option click Select Pattern then specify the sample positions to optimize To specify Do the following Individual Click on a single position positions Multiple e Click drag positions or positions e Press Shift and click a position to turn on multi select mode then drag positions click again to turn off multi select mode Evenly Select a Fill Increment then click Fill distributed For example select 2 as the fill number of increment to fill every other position in positions every other row Voyager Biospectrometry Workstation User s Guide 2 65 Chapter 2 Installing the Voyager Biospectrometry Workstations NOTE If you change the reference position after you specify the Acquisition Pattern using Select Pattern the original reference position is no longer selected for acquisition You must manually select the position if you want to acquire it For example if you select position 44 as the reference position and select positions 43 and 45 for acquisition then change the reference position to position 42 position 44 is not automatically selected for acquisition Setting acquisition 7 Under Data Acquisition select mode New Data To create a new optimization file for a plate that has not been optimized or to reoptimize a plate Append Data To do either of the following Optimize additional positions on a plate that has been optimiz
397. lution If the resolution is not acceptable increase the Grid Voltage by 0 25 percent for Linear mode or 0 5 percent for Reflector mode acquire a spectrum and observe Leave the Delay Time setting unchanged If the signal is saturated at the higher Grid Voltage setting decrease the laser and acquire a new spectrum If the signal decreases at the higher Grid Voltage setting increase the laser and acquire a new spectrum Hint If you are analyzing a mixture and the resolution on the peak of interest is not acceptable observe the resolution of a higher mass peak and a lower mass peak If resolution is better on the higher mass peak increase the Grid Voltage setting by 0 25 percent for Linear mode or 0 5 percent for Reflector mode If resolution is better on the lower mass peak decrease the Grid Voltage setting by 0 25 percent for Linear mode or 0 5 percent for Reflector mode Collect a spectrum and observe Leave the Delay Time setting unchanged If the resolution improves by at least 20 percent you can see fluctuations in resolution of up to 10 percent with the same settings continue increasing the Grid Voltage in 0 25 percent for Linear mode or 0 5 percent for Reflector mode increments Table 5 12 on page 5 79 through Table 5 14 on page 5 80 list valid Grid Voltage settings for different systems and mass ranges Voyager Biospectrometry Workstation User s Guide 5 83 Chapter 5 Optimizing Instru
398. ly 4 28 Laser intensity Automatic Control adjustment criteria automatic mode parameters mode 6 42 adjustment criteria 6 42 displayed in Manual Laser Sample minimum and maximum 5 34 Position control page 4 27 Prescan mode 5 34 displayed in status bar 4 5 step size 5 34 external laser range 5 26 fine coarse control 4 28 guidelines for adjusting 5 67 impact on data 6 6 impact on resolution and signal to noise 5 50 in Continuous Extraction mode H 6 H 11 H 12 Laser position in Sample view 4 31 Laser rate default 5 26 optimized 1 22 1 34 5 26 optimized affected by number of data points A 12 setting 5 26 specifications A 12 H 18 in Delayed Extraction mode 6 24 Laser threshold in PSD mode 8 66 8 76 definition H 11 H 12 no adjustment criteria factors affecting H 12 specified 6 39 in Continuous Extraction optimizing 5 67 mode H 11 H 12 optimizing in Prescan mode 6 58 troubleshooting H 19 relative settings for matrices 5 67 verifying in Continuous Extraction saturating signal 5 69 mode H 16 setting automatically 6 39 LeCroy digitizer see Digitizer setting displayed 4 27 Leucine Enkephalin setting manually 6 14 mass to time conversion F 4 signal intensity adjustment molecular weight F 2 criteria 6 43 Line type 4 22 Voyager Biospectrometry Workstation User s Guide Index 21 Index Linear detector description 1 23 1 35 high current starting serial number 1 23 1 35 5 91 Linear mode BIC files 5 4 path l
399. ly files Open the PLT file that describes the plate type you are customizing edit the file to describe the plate type you need then save the file with a new file name Voyager Biospectrometry Workstation User s Guide 3 65 Chapter 3 Preparing Samples 384 position Sample locations on 384 position Teflon plates are staggered Teflon plate as shown in Figure 3 9 staggered locations Figure 3 9 384 Position Staggered Sample Locations 96 x 2 position Sample locations on 96 x 2 position plates are staggered as plate staggered shown in Figure 3 10 a and b locations Position A1_a 1 2 3 4 5 6 7 8 9 10 11 12 Position A1_b eeeeeeeeeee 060006 00e 00 C606060000000600 Figure 3 10 96 x 2 Position Plate Staggered a and b Sample Locations 3 66 Applied Biosystems Sample Plate Types PLT files The editable configuration plate types provided with the provided Table 3 12 Editable Configuration Plate Types system are described in Table 3 12 Plate Type Number oe Position Position Center Plate Position name of ee of Diameter to Center Description e Arrangement PLT file Positions um Distance um 64 well Disposable 64 8 x 8 subset of 2 540 5 080 x 5 080 disposable with wells 10 x 10 plate PLT 100 well Reusable with 100 10 x 10 2 540 5 080 x 5 080 plate PLT wells 384 well Reusable with 384 16 x 24 990 60 1 803 40 x 2 639
400. m 1997 8 209 217 Takach E J W M Hines D H Patterson P Juhasz A M Falick M L Vestal and S A Martin Accurate Mass Measurements Using MALDI TOF with Delayed Extraction Journal of Protein Chemistry 1997 16 363 Vestal M L and P Juhasz Resolution and Mass Accuracy in Matrix Assisted Laser Desorption lonization Time of Flight J Am Soc Mass Spectrom 1998 9 892 911 Vestal M L P Juhasz and S A Martin Rapid Commun Mass Spectrom 1995 9 1044 1050 Whittal R M and L Li Anal Chem 1995 67 1950 1954 MALDI Applications Brockman A H and Orlando R Anal Chem 1995 67 4581 4585 Dogruel D Williams P and Nelson R W Anal Chem 1995 67 4343 4348 Bibliography 2 Applied Biosystems Bibliography Fitzgerald M C L Zhu and L M Smith The Analysis of Mock DNA Sequencing Reactions Using Matrix assisted Laser Desorption lonization Mass Spectrometry Rapid Commun Mass Spectrom 1993 7 895 897 Hillenkamp F M Karas R C Beavis B T Chait Matrix Assisted Laser Desorption lonization Mass Spectrometry of Biopolymers Anal Chem 1991 63 1193 1203 Huberty M C J E Vath W Yu and S A Martin Site Specific Carbohydrate Identification in Recombinant Proteins Using MALDI TOF MS Anal Chem 1993 65 2791 2800 Hutchens T W and T Yip New Desorption Strategies for the Mass Spectrometric Analysis of Macromolecules Rapid Commun Mass
401. m the Instrument Control Panel 6 To display or suppress numbers and lines displayed in the area click Menu then select Numbers or Lines NOTE If the search pattern contains a large number of spots turn off lines to more easily see the spot positions 7 Click Menu then select Save or Save As Using the Search To automatically generate a search pattern Pattern Generator 4 Click Menu then select Generate Search Pattern to open the Generate Search Pattern dialog box Figure 6 19 Generate Search Pattern x M Generation Area Parameters r Number of Spots Center Width Center Y Height Units Well Width Well Height Pattem Random Spiral r Pattern Type C Uniform C Center Bias C Edge Bias Spot Settings Spot Diameter I Do Not Overlap Spots Cancel Figure 6 19 Generate Search Pattern Dialog Box 6 52 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel 2 Enter the following parameters Table 6 4 Search Pattern Generator Parameters Parameter Description Generation Area Parameters Type values for the parameters that define the center coordinate of the search pattern and the area in which the search pattern is generated generation area e Center X and Center Y Coordinates that define the center of the search pattern The default values of 0 set the center of the generation area to the center of th
402. masses entered in calibration Recalibrate See the Data Explorer Software User s Guide Chapter 5 3 Manual Calibration H 22 Applied Biosystems Table H 8 Troubleshooting in Continuous Extraction Mode Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor mass accuracy in Continuous Extraction mode continued continued on next page Incorrect peaks entered in calibration Recalibrate See the Data Explorer Software User s Guide Chapter 5 3 Manual Calibration When calculating peaks the Centroid value displayed in the Settings dialog box changed Use the same Centroid value for peak calculation and calibration calibration value is displayed when you open the Settings dialog box See the Data Explorer Software User s Guide Chapter 5 5 Centroiding for additional information Voyager Biospectrometry Workstation User s Guide H 23 Appendix H Continuous Extraction Mode Table H 8 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor mass accuracy in Continuous Extraction mode continued Crystals did not form homogeneously on sample spot Prepare new sample spot See Guidelines for good crystallization on page 3 37 Use mass closest to the mean for external calibration only 1 Acquire six averaged scans six DAT files from one sample well 2 Check
403. masses in Voyager processing software 3 Use the file with the mass value closest to the mean High voltage power supplies not warmed up Start high voltages by clicking add icon 30 minutes before calibration Samples and standards not in adjacent wells For optimum mass accuracy place standards in wells adjacent to the samples for which you are calibrating Samples or standards spotted in outer wells on sample plate For optimum mass accuracy do not use outer wells of sample plate H 24 Applied Biosystems Using the Oscilloscope and Control Stick This appendix contains the following sections 1 1 Guidelines for Acquiring l 3 e E ne ty ea l 4 1 3 Using the Control Stick l 7 NOTE If your system includes an internal digitizer board or an external digitizer instead of an external oscilloscope refer to Section 4 3 Using the Spectrum Window Voyager Biospectrometry Workstation User s Guide l 1 Using the Oscilloscope and Control Stick An external oscilloscope instead of the internal digitizer board or the LeCroy digitizer is available as an option on Voyager systems The oscilloscope converts the signal from the mass spectrometer to a signal that the computer can use The oscilloscope has its own screen to display the averaged ion signal in real time For instrument configurations containing an oscilloscope the software does not r
404. matically adjusted Instead the intensity set in the Instrument Control Panel is used for the analysis You can manually adjust the laser intensity by pressing Ctrl PageUp or Ctrl PageDn e If Use Automated Sample Positioning is disabled in the BIC file the sample does not automatically move and the analysis is performed on the center of the sample position unless you manually adjust the position using the control stick 7 3 2 Creating Macros You can specify macros that execute before and after calibration You can use the macros supplied with the system or create your own For information on using the Visual Basic Editor refer to the online help available within the Visual Basic Editor 7 8 Applied Biosystems Before Creating a Sequence Creating a macro To create a macro for the Sequence Control Panel 1 2 Open the Data Explorer software Create a macro as described in the Data Explorer Software User s Guide Section 6 7 2 Recording a Macro NOTE If you create a macro to apply a SET file there are two variables you can use RestorePreferences2 which applies either processing or graphic settings syntax ActiveDocument RestorePreferences2 path and name of SET file deProcessingSettings or deGraphicsSettings RestorePreferences which applies both processing and graphic settings syntax ActiveDocument RestorePreferences2 path and name of SET file If you use RestorePreferences make sure the SET
405. ment Settings 5 84 Applied Biosystems If the resolution does not improve decrease the Grid Voltage by 0 25 percent for Linear mode or 0 5 percent for Reflector mode acquire a new spectrum and observe Continue increasing or decreasing the Grid Voltage in increments or decrements of 0 25 percent Linear mode or 0 5 percent Reflector mode until optimum resolution is obtained Hint If you obtain close to acceptable resolution at one setting but less acceptable resolution at the next setting you may have adjusted the Grid Voltage too far Increase or decrease in small increments until you obtain optimum resolution Adjust the Guide Wire Voltage as needed e Increase the setting to increase sensitivity for higher masses e For Voyager DE PRO and Voyager DE STR models in Reflector mode adjust the Guide Wire between 0 and 0 04 percent NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later If you are analyzing peptides below 2 000 Da you may be able to improve resolution by adjusting the Accelerating Voltage Linear mode Decrease from 20 000 V to 15 000 V to increase flight times Reflector mode Decrease to 15 000 V 10 000 V or 5 000 V However these lower settings will compromise sensitivity Save the BIC file Optimizing Instrument Settings Parameters 5 4 4 Optimizing Signal to Noise Ratio This section includes e Overv
406. mental composition theoretical isotope distributions resolution and signal to noise ratio e Customize windows toolbars and traces Figure 1 23 shows the Data Explorer main window The Data Explorer processing software is described in the Data Explorer Software User s Guide Data Espherer cal_mix20001 dat iol x File Edit View Display Process Peaks Tools Applications Window Help js nja twe ue sss sie ioe S4 Bite le ee r fe IMSPEC cal_mix20001_ dat Spec 1 BP 1314 6 58462 1314 616 2 a 3 3 ES 2117 574 2491 999 3691 346 18527242152643 4473 874 2891 505 3199 147 3599 498 Al SPEC ca For Help press F1 Figure 1 23 Data Explorer Window Voyager Biospectrometry Workstation User s Guide 1 45 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 46 Applied Biosystems 2 Installing the Chapter Voyager 2 Biospectrometry Workstations This chapter contains the following sections 2 1 Installing the System 2 2 2 2 Selecting the Site 2 2 2 3 Connecting Voyager DE and Voyager DE PRO Workstations 2 8 2 4 Connecting the Voyager DE STR Workstation 2 23 2 5 Installing Software 2 29 2 6 Hardware Configuration 2 35 2 7 Aligning the Sample Plate 2 46 2 8 Running OptiPlate to Optimize Mass
407. meter cabinet NOTE The vacuum gauge panel is located behind the front panel of the mass spectrometer cabinet ATM 1 2 1 0 TC 1 1 oo EMIS TORR PB100270 Figure 9 2 Vacuum Gauge Panel Voyager Biospectrometry Workstation User s Guide 9 25 Chapter 9 Maintenance and Troubleshooting CAUTION Do not press any other buttons on the panel Pressing buttons other than the Chan and EMIS buttons can recalibrate the pressure scale of the system You use two buttons on the pressure gauge panel e Chan Toggles through readings for Gauge Measures Expected Pressure BA1 Pressure in main e Voyager DE and Voyager DE PRO source chamber Less than 107 e Voyager DE STR Less than 5 x 1077 BA2 Pressure in mirror e Voyager DE and Voyager DE PRO chamber Less than 2x107 Voyager DE PRO e Voyager DE STR Less than 5 x 10 8 only TC2 Pressure in Less than 5 x 102 during operation Higher sample loading when loading or chamber ejecting sample plate TC1 Not used displays m TC3 E03 indicates TC4 gauge not connected e EMIS Turns BA1 and BA2 on or off Used during troubleshooting only 9 26 Applied Biosystems Troubleshooting Table 9 6 Vacuum Gauge Panel Troubleshooting Symptom Possible Cause Action E02 error message displ
408. microns 5 92 Applied Biosystems information that is converted Converting Version 4 Methods and Search Pattern Files Method Autosampler parameters Data Acquisition parameters Digitizer Laser parameters and PSD parameters are converted from MNU files to BIC files However some version 4 x parameters have been renamed or relocated and some parameters are not supported in Version 5 x Table 5 15 Method Information That Is Not Converted Version 5 x Version 4 x Method Instrument Parameter Changes in Version 5 x Settings Described in Parameter Parameter Location s Location Autosampler Scans per position is replaced by Number of Automatic Section 5 2 3 tab Spectra To Acquire Control Mode Automatic Control dialog box Dialog Box Total Scans is replaced by Shots Spectrum Instrument Section 5 2 1 Settings page Instrument Settings Page Acceptance Criteria Mass Range is replaced by Criteria Evaluation Mass Range Spectrum Acceptance Criteria dialog box Setting spectrum acceptance and laser adjustment criteria on page 6 42 Data Acquisition tab Instrument Range is replaced by Mass Range Data File Range is replaced by Mass Range Total Scans is replaced by Shots Spectrum Method Description is not supported Instrument Settings page Section 5 2 1 Instrument Settings Page Instrument Mode is replaced by Operation Mode Timed lon Selector moved same fu
409. mix2 Angiotensin_line scptest set External Defautt cal Overall Run Status OFF cal_mix2 Angiotensin_line scptest set External Default cal cal_mix2 Angiotensin line soptest set External Default cal Elapsed Time 0 sec Current Entry Index 0 Remaining Entries Figure 7 1 Sequence Control Panel 7 2 Applied Biosystems Understanding Settings Macros and Calibration 7 2 Understanding Settings Macros and Calibration This section includes e Types of files and calibration you specify in the Sequence Control Panel e How files and calibration you specify affect the data during a sequence run Types of files and You select the following types of files and the Calibration Type calibration inthe Sequence Control Panel to specify how data is detected calibrated and processed File Type Description For information see Data Contains peak detection parameters e Creating Processing Explorer monoisotopic peak filtering and the Settings SET Files on SET reference masses needed for Internal page 7 12 or Internal update calibration Optional Data Explorer SET File if you are not calibrating or are on page 7 17 performing External calibration Macro Optional selections that allow you to e Creating Macros on specify processing before and after page 7 8 calibration Pre Macro on page 7 17 e Post Macro on page 7 20 Calibration Specifies External Internal o
410. modate your power source Do not plug in or power up the Voyager DE STR Biospectrometry Workstation unless it has been configured correctly by an Applied Biosystems Technical Representative In addition you need grounded outlets for Computer monitor External digitizer if your system includes one Printer optional 2 3 Connecting Voyager DE and 2 8 Applied Biosystems Voyager DE PRO Workstations This section describes Side panel diagrams for mass spectrometer and computer Connecting the mass spectrometer to the computer Connecting the Signatec 500 MHz digitizer board Connecting the LSA1000 LeCroy digitizer Connecting the Acqiris digitizers Connecting the Tektronix oscilloscope Connecting the video monitor Connecting devices to the computer Before connecting devices to the mass spectrometer power down the mass spectrometer When you plug in cables make sure they are securely connected and screwed in place if connectors include screws Connecting Voyager DE and Voyager DE PRO Workstations 2 3 1 Side Panel Diagrams for Mass Spectrometer and Computer This section includes diagrams for e Mass spectrometer Computer Mass Figure 2 2 shows the connections on the right side panel of spectrometer the Voyager DE and Voyager DE PRO mass spectrometer cabinet VOCTL STP MTR CTL p VAC GAUGE CTL Not used on VIDEO VO POWER Serial Number 1171 N and later DE
411. mode Table H 6 Continuous Extraction Mode Resolution Rating Scale Compounds lt 10 000 Da Compounds 10 000 to 20 000 Da Compounds gt 20 000 Da Resolution Rating Resolution Rating Resolution Rating 250 Acceptable 200 Acceptable 60 Acceptable 350 Fair 250 Fair 100 Good 500 Good 400 Good 600 Very good 600 Very good a You may see better resolution than indicated when analyzing compounds below 1 000 Da due to isotope separation Voyager Biospectrometry Workstation User s Guide H 17 Appendix H Continuous Extraction Mode H 2 4 Fine Tuning the Laser Setting When you find the laser threshold whether you need to fine tune the setting depends on your needs If you are looking for an estimate of molecular weight a laser setting slightly higher than the laser threshold setting may be sufficient If you need good peak shapes you may need to increase the laser setting to improve signal to noise ratio If you need maximum resolution to differentiate between compounds you may need to operate close to the laser threshold setting to improve resolution H 18 Applied Biosystems Troubleshooting in Continuous Extraction Mode H 3 Troubleshooting in Continuous Extraction Mode This section includes e Laser threshold troubleshooting e Spectrum troubleshooting Refer to Table H 7 if you are having trouble determining laser threshold Table H 7 Laser
412. more information see Section 8 4 2 Determining the Number of Segments to Acquire for a Complete Composite Spectrum Rows are added with the default values listed in Table 8 4 for all columns Voyager Biospectrometry Workstation User s Guide 8 47 Chapter 8 PSD Analysis Table 8 4 Default Values for the PSD Segment List Parameter Default Value Segment Sequential number starting at 1 Saved check box Blank until the segment is saved the software automatically places a check mark in this field when you save a segment Mirror Ratio 1 000 Guide Wire 0 020 NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later Vertical Scale Lowest setting for your digitizer type Input Bandwidth Lowest setting for your digitizer type Laser Increment Disabled in Manual Control mode For information see Appendix 8 5 Acquiring PSD Data with Standard BIC Files in Automatic Control Mode Max Stitch Mass Calculated value equal to Precursor Mass x Mirror Ratio Reflects the maximum mass of the segment that will be included in the composite spectrum The mass range included in the segment is approximately 15 percent higher than the Max Stitch Mass 2 Edit the values as needed See e Typing or selecting new values on page 8 49 e Using the Fill Down function on page 8 50 8 48 Applied Biosystems A
413. mpatible computer hardware components Minimum computer configuration of Pentium III 500 MHz CPU 9 GB hard disk and 128 MB RAM random access memory e 3 5 inch HD high density 1 44 MB floppy disk drive e CD ROM drive integrated sound card e 17 inch color monitor e WIN95 Spacesaver Quiet Key 104 key keyboard e Microsoft compatible mouse e Control stick e Optional laser printer Software The Voyager DE and Voyager DE PRO Biospectrometry Workstations include the following software components Microsoft Windows NT version 4 0 or later e Voyager software includes Voyager Instrument Control Panel Voyager Sequence Control Panel and Data Explorer software e Microsoft Office 2000 Problems using Do not enable screen savers on the Voyager computer screen savers Screen savers use computer memory and may decrease system performance or cause other system problems 1 30 Applied Biosystems Parts of the Voyager DE STR System 1 6 Parts of the Voyager DE STR System This section describes System components Mass spectrometer Vacuum system Front panel indicators Computer components 1 6 1 System Components The Voyager DE STR Biospectrometry Workstation is shown in Figure 1 17 Computer Video monitor monitor Mass Control stick spectrometer f 0 Comput
414. mple plate dried droplet application 3 34 premixing with matrix dried droplet application 3 33 preparing 3 2 preparing for dried droplet application 3 22 preparing for thin layer application 3 24 purifying see Sample cleanup volume to load on plate 3 43 when to prepare 3 22 Sample cleanup C18 3 31 cation exchange beads 3 29 drop dialysis 3 27 floating membrane dialysis 3 27 washing 3 26 when to do 3 25 ZipTips 3 31 Voyager Biospectrometry Workstation User s Guide Index 33 I N D E X Index Sample concentration dried droplet application 3 22 general 3 22 in 3 HPA 8 15 in alpha cyano 4 hydroxycinnamic acid dried droplet application 3 8 3 9 in alpha cyano 4 hydroxycinnamic acid thin layer application 3 11 in DHB 8 16 3 17 3 19 in DHBs 3 18 in Dithranol 3 19 in IAA 3 19 in sinapinic acid 3 7 in THAP 3 13 low concentration application technique 3 22 thin layer application 3 22 Sample holder ejecting 3 54 loading 3 54 Sample ionization see lonization sample Sample list Sequence Control Panel saving 7 20 Sample loading dried droplet application 3 41 in mass spectrometer 3 50 techniques 3 35 thin layer application 3 44 Sample loading chamber max load pressure 2 37 wait time 2 37 Sample plate see also PLT files see also Sample plate aligning see also Sample plate loading see also Sample plate types of alignment checking 3 73 Automatic Control mode type required 3 41 Index 34 Applied
415. mple plate and spot or respot positions or modify then save instrument settings 4 Allow the sample plate to dry if necessary 5 Load the sample plate if necessary Running OptiPlate to Optimize Mass Accuracy 6 Select Append Data for Data Acquisition mode Each failed position and each position previously selected for optimization but not yet acquired is indicated with a white dot in the center of the position If you want to overwrite all previously acquired positions click Reset then select New Data for Data Acquisition mode 7 Click Start Acquisition 2 8 5 Evaluating and Saving Results Color coding Positions are marked with the following colors Color Meaning Position selected for optimization e Reference position oO Extraction Correction value exceeded the allowed 1 mm maximum or the Extraction Correction could not be calculated for any of the following reasons e Data was not acquired e Laser range specified in instrument settings does not yield the minimum maximum laser intensity specified in acceptance criteria See Verifying laser intensity on page 2 60 e Required peaks were not detected in the reference position or the acquired position during processing Position not selected for optimization Other colors Colors correspond to the legend for Extraction Correction Voyager Biospectrometry Workstation User s Guide 2 69 Chap
416. mple plate will not load e Sample Loading Chamber TC2 Wait Time Time seconds that the software waits for the instrument to reach the Sample Loading Chamber Maximum Load Pressure after the sample plate is loaded If the wait time is exceeded an error message is displayed which gives you the option of an additional wait time or ejecting the plate Valid range is 0 to 300 seconds Default is 120 seconds 4 Click OK to exit vacuum configuration Voyager Biospectrometry Workstation User s Guide 2 37 Chapter 2 Installing the Voyager Biospectrometry Workstations 2 6 2 High Voltage Configuration To check high voltage configuration 1 Inthe Instrument Control Panel select Hardware Configuration from the Instrument menu 2 Click the High Voltage tab to display the High Voltage page Figure 2 16 Hardware Configuration x Vacuum Laser Timed lon Selector High Voltage Instrument Digitizer Accelerating Voltage Maximum V Polarity Switch Delay sec 20 M Idle Power Off Idle Time min 20 Figure 2 16 High Voltage Configuration 3 Check the following values as needed Maximum Accelerating Voltage Maximum value in volts that the Accelerating Voltage is configured 25 000 V e Polarity Switch Delay Read only Time seconds that the high voltage power supply waits between switching from positive to negative polarity 2 38 Applied Biosystems Hardware Con
417. n If you are mixing sample and matrix on the sample plate See Mixing sample and matrix on the sample plate on page 3 34 If you prepared samples with ZipTips Load 0 5 to 2 ul of sample matrix solution on the appropriate position Do not touch the tip of the pipette to the sample spot Make sure the sample is evenly applied to the spot 3 If you are using DHB matrix for neutral carbohydrates dry the sample plate quickly under vacuum If you are using other matrixes allow the sample plate to dry for at least five minutes Preparations with high water or salt content may require longer to dry 4 Visually examine the sample spots to make sure they are dry NOTE If you load the sample plate into the Voyager Biospectrometry Workstation before the plate is dry the pressure in the sample chamber rises and a TC2 pressure too high error code may be displayed in the Control window Wait a few minutes for the chamber to reach pressure 5 Place a protective cover over the plate until you are ready to load the plate Do not allow the cover to touch the surface of the plate Voyager Biospectrometry Workstation User s Guide 3 43 Chapter 3 Preparing Samples 3 2 4 Loading Samples Thin Layer Application When to use Use the thin layer application technique for increased sensitivity when analyzing peptides with a concentration lt 0 1 pmol ul Loading matrix To load matrix and samples an
418. n half the center to center distance radius Figure 3 12 Area Available for Analysis Voyager Biospectrometry Workstation User s Guide 3 75 Chapter 3 Preparing Samples 3 5 4 Creating and Editing PLT Files 3 76 This section includes e Preparing the sample plate e Creating a PLT file using Create PLT File e Creating or editing a PLT file using Notepad editor e Creating PLT files for SymBiot plates Preparing the Preparing the sample plate involves sample plate Preparing Applied Biosystems e Spotting a sample plate with matrix in specific positions to allow you to see the positions on the video monitor and correctly locate the center of the sample position under the laser e Loading a BLANK PLT file containing one sample position that occupies the entire plate to allow you to navigate anywhere on the plate and determine x y coordinates WARNING CHEMICAL HAZARD Alpha cyano 4 hydroxycinnamic acid CHCA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare the sample plate before creating a PLT file 1 Spot a sample plate with a cyano 4 hydroxycinnamic acid e If you are using the Create PLT File function to create the PLT file spot the first and last positions on the plate Figure 3 13 e If you are using Notepad editor to create the PLT
419. n approximately every 3 minutes calibration update may be required less frequently or not at all depending on your mass accuracy needs To set the run to update the calibration list the standard position in the sequence run list every fifth line or so the precise line on which to list the standard depends on the time required for each line in the sequence to run and specify Internal Update calibration For more information see Section 7 Acquiring Spectra from the Sequence Control Panel Voyager Biospectrometry Workstation User s Guide 3 39 Chapter 3 Preparing Samples With an You can use an unoptimized sample plate and still obtain unoptimized plate better mass accuracy than if you do not use Mass Accuracy Optimization However you must run the OptiPlate software and optimize at least one sample plate on your system before using an unoptimized plate For more information see Section 2 8 Running OptiPlate to Optimize Mass Accuracy To obtain optimum mass accuracy during analysis using the Mass Accuracy Optimizations option and a plate you have not optimized with OptiPlate you can Spot more standards on the plate than if you are using an optimized plate but fewer than if you are not using the Mass Accuracy Optimization option e Locate standards in regular positions on the plate for example every fifth position across the plate e When you analyze set the run to update the calibration approximately every 3 min
420. n by a local agent Secure a written report of the findings to support any claim Do not return damaged goods to Applied Biosystems without first securing an inspection report and contacting Applied Biosystems Technical Support for a Return Authorization RA number After a damage inspection report is secured claims should be processed through Applied Biosystems unless other instructions are provided Please do not return any material without prior notification and authorization If for any reason it becomes necessary to return material to Applied Biosystems please contact Applied Biosystems Technical Support or your nearest Applied Biosystems subsidiary or distributor for e Return Authorization RA number e Forwarding address e Decontamination instructions e Packing and shipping instructions Place the RA number in a prominent location on the outside of the shipping container and return the material to the address designated by the Applied Biosystems representative Voyager Biospectrometry Workstation User s Guide B 5 Appendix B Warranty Service Information B 3 Spare Parts Standards and Tne following compounds are available from the listed matrixes vendors Part numbers are listed for your convenience and may change without our knowledge Compound Vendor hrs Sequazyme Peptide Mass Standards Install Applied 4316866 Kit includes Biosystems e Sequazyme Mass Stand
421. n considerations 7 7 1 Calibration Options in a Sequence The Sequence Control Panel allows three types of automatic calibration Type Function External e Applies calibration constants in a specified CAL file Internal e Applies calibration constants if a CAL file is specified e Generates new calibration constants using standard reference masses specified by a SET file e Applies the constants Internal Update e Applies calibration constants if a CAL file is specified e Generates new calibration constants using standard reference masses specified by a SET file e Applies the constants e Updates the specified CAL file or creates a new CAL file if the specified file does not exist Use this option to perform a close external calibration described in Section 7 7 3 Performing Close External Calibration 7 32 Applied Biosystems Automatic Calibration During a Sequence Run NOTE If the DAT file contains multiple spectra generated using the Save All or the Save All Passing option in Automatic Control the calibration is applied to all spectra in the DAT file For the mass accuracy provided by each calibration type see Types of calibration on page 6 7 7 7 2 External Calibration Standard Requirements Mass calibration The requirements for mass calibration standards are standards determined by your application The following are general guidelines To
422. n ion focusing lens Linear detector A device that detects ions that travel down the flight tube The linear detector measures ion abundance over time and sends a signal to the digitizer for conversion The linear detector is used in Linear mode only It is not used in Reflector or PSD mode Linear detectors are hybrid high current detectors consisting of a single microchannel plate a fast scintillator and a photomultiplier These detectors have superior tolerance for high ion currents NOTE High current detectors are standard on Voyager DE STR systems with serial numbers 4112 4113 and 4116 and later Voyager Biospectrometry Workstation User s Guide 1 35 Chapter 1 Introducing the Voyager Biospectrometry Workstations e Reflector A single stage gridded mirror that focuses energy In reflector mode a uniform electric field is applied to the mirror to reflect ions lon reflection Filters out neutral molecules e Corrects time dispersion due to initial kinetic energy distribution e Provides greater mass accuracy and resolution The single stage design provides high mass resolution across a wide range and highly accurate mass measurements Accurate calibration formulas for the single stage reflector allow the user to vary instrument conditions without degrading mass accuracy Also easy calibration of PSD data is facilitated by single stage reflectors For more information see Chapter 8 PSD A
423. n the BIC is set to alpha cyano Automatic Calibration not supported within Sequence Chapter 7 Acquiring BIC Control Panel Spectra from not in BIC the Sequence Control Panel Data Parameters not supported in instrument ss Processing settings Peak detection parameters set in tab Data Explorer software and stored in SET file PSD tab C1 C3 C4 Calibration Constants not PSD Section 8 4 3 Setting supported Values are calculated using the Acquisition PSD PSD calibration specified in PSD Acquisition Settings page Acquisition Parameters Settings for Manual Mode NOTE All methods are converted to Manual Control mode If you are converting an autosampler method you must set the control mode to automatic and respecify the SP file after you convert the MNU file to a BIC file 5 94 Applied Biosystems Information that is not converted New parameter Before converting Converting Version 4 Methods and Search Pattern Files Control mode is set to Manual for all files The path for search pattern files is not maintained Automatic Calibration parameters are not converted from MNU files to BIC files Calibration mode is automatically set to Default Data Processing parameters are not supported A new parameter called Mirror To Accelerating Voltage Ratio is included on the Advanced tab of the Mode Digitizer Settings dialog box in the BIC file This parameter was previously called HV Tune Ratio and was not part o
424. n the mass spectrometer Allows you to start and stop acquisition and transfer data to the processing software NOTE You can also contro sample position start and stop acquisition and transfer data using the Voyager Instrument Control Panel software Voyager Biospectrometry Workstation User s Guide 1 19 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 1 5 2 Mass Spectrometer Voyager DE The parts of the Voyager DE Biospectrometry Workstation mass spectrometer are shown in Figure 1 13 Linear detector D of gt lon path PARUS gt Laser path Beam e guide wire Laser Video l camera Laser m Aperture grounded attenuator i Ground grid Prism M m4 Variable voltage Sample grid loading chamber Sample plate Main source chamber Figure 1 13 Voyager DE Mass Spectrometer 1 20 Applied Biosystems Parts of the Voyager DE and Voyager DE PRO Systems Voyager DE PRO The parts of the Voyager DE PRO Biospectrometry Workstation mass spectrometer are shown in Figure 1 14 Variable voltage on rid attenuator peter Au Prism l Laser detector l Sample a mirror plate Main source chamber Video tube camera Ground grid Linear Timed lon detector Aperture grounded Selector gt lon path in reflector mode BP Laser path Figure 1 14 Voyager DE P
425. nalysis Reflector detector The reflector detector measures ions reflected by the mirror The reflector detector sends a signal to the digitizer for conversion Two multichannel plate reflector detectors optimized for response time are included Timed lon Selector A device that allows only ions of a selected mass of interest to pass to the detector The Timed lon Selector TIS device is a Bradbury Nielson gate positioned approximately 676 mm from the ion source This prevents ions deflected by the gate from entering the reflector and reduces background noise in the detector in PSD experiments When the Timed lon Selector is turned on voltage is applied to the Time lon Selector to deflect ions At the time that corresponds to the ion of interest voltage is turned off and the ion of interest passes to the detector After the ion passes through the Timed lon Selector voltage is turned on again 1 36 Applied Biosystems 1 6 3 Vacuum System Overview The Voyager DE STR Biospectrometry Workstation provides a Vacuum chambers Vacuum pumps Parts of the Voyager DE STR System high vacuum environment for time of flight analysis The high vacuum environment Allows unobstructed ion drift e Provides conditions needed to maintain the high potential difference between the ion source and ground The Voyager DE STR Biospectrometry Workstation includes three vacuum chambers Figure 1 19 e Main source chamber e
426. name in the Attributes column NOTE If attributes are not displayed select Details from the View menu Saving To save changes to a read only instrument settings file you read only must save the file with a new name Select Save Instrument instrument Settings As from the File menu enter a new name and save settings files M file Voyager Biospectrometry Workstation User s Guide 5 13 Chapter 5 Optimizing Instrument Settings 5 2 Instrument Settings Parameter Descriptions This section describes the parameters on the Instrument Settings control page and associated dialog boxes that are stored in a BIC file The following control pages or dialog boxes are available from the View menu or Instrument menu in the Instrument Control Panel Instrument Settings Page e Mode Digitizer dialog box e Automatic Control dialog box NOTE For a description of PSD acquisition settings see Chapter 8 PSD Analysis 5 14 Applied Biosystems Instrument Settings Parameter Descriptions 5 2 1 Instrument Settings Page Select Instrument Settings from the View menu to display the Instrument Settings page Figure 5 3 Instrument Settings xl m Instrument Mode Linear Positive Mode Digitizer r Control Mode f Manual Automatic Automatic Control Voltages Accelerating fi 0000 Yy Grid jo 0 0 110 0 fo Guide Wire 0 000 0 300 Delay Time 10 nsec Spectrum Acquisiti
427. nance Schedule Maintenance Regular preventative maintenance will help keep your schedule Voyager system functioning properly Perform the following procedures as indicated When to perform Task Page Weekly Back up or archive data page 9 6 Yearly Hardware maintenance performed by page 9 3 Applied Biosystems engineer Maintenance log Appendix G Maintenance Log includes a log sheet that you can copy and use to keep track of maintenance on your Voyager system 9 2 Applied Biosystems Maintenance 9 1 2 Hardware Maintenance Yearly preventative maintenance WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result by operating the instrument without panels in place Do not remove instrument panels High voltage contacts are exposed with panels removed Wear proper eye protection if panels are removed for service WARNING LASER HAZARD The laser emits ultraviolet radiation Lasers can burn the retina and leave permanent blind spots Do not remove instrument panels or look directly into the laser beam or allow a reflection of the beam to enter your eyes Wear proper eye protection if panels are removed for service The Voyager Biospectrometry Workstation requires minimal preventative maintenance Preventative maintenance procedures should be performed by an Applied Biosystems technical representative Please contact Applied Biosystems for information on service co
428. nction as Precursor lon Selector in PSD Acquisition Settings Polarity moved Extraction mode moved Mode Digitizer Settings dialog box Section 5 2 2 Mode Digitizer Dialog Box Precursor lon Selector now present same function as Timed lon Selector in Mode Digitizer Settings Precursor lon Mass moved PSD Acquisition Settings page Section 8 4 3 Setting PSD Acquisition Parameters for Manual Mode Voyager Biospectrometry Workstation User s Guide continued 5 93 Chapter 5 Optimizing Instrument Settings Table 5 15 Method Information That Is Not Converted Continued Version 5 x Version 4 x Method Instrument Parameter Changes in Version 5 x Settings Described in Parameter Parameter Location Location Digitizer All parameters moved Mode Digitizer Section 5 2 2 Laser tab Settings dialog Mode Digitizer Dialog box Box Step size is replaced by Small Manual Intensity Hardware Section 2 6 Hardware Adjustment This parameter is not part of Configuration Configuration Instrument Settings Calibration Automatically set to Default Instrument Section 5 2 1 tab If a matrix is specified in the MNU checks the Settings page Instrument Settings matrix file during conversion and if itis present Page in the matrix file retains the matrix in the BIC If the matrix specified in the MNU is not present in the matrix file the matrix i
429. ndwidth e Low Mass Gate NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later For more information on optimizing signal to noise see Section 5 4 4 Optimizing Signal to Noise Ratio Voyager Biospectrometry Workstation User s Guide 6 25 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 4 Making Accurate Mass Measurements Overview Accurate mass measurements are typically made by including reference compounds internal standards in the same spectrum as the analyte and then recalibrating the spectrum In Reflector mode Voyager DE PRO and Voyager DE STR internal calibration provides mass spectra with a mass accuracy of 10 to 20 ppm routinely This section describes the factors that influence mass accuracy at this level During When acquiring data the following factors contribute to the acquisition accuracy of mass measurements e Symmetrical peak shape Depending on the m z in question the spacing between data points can represent a change in mass from 10 or 20 ppm to more than 100 ppm When determining the position of an ion in terms of m z the software interpolates between data points to make a mass assignment at the 10 ppm level Therefore a skewed peak shape can have a strong impact on the observed mass accuracy e Signal to noise ratio as high as possible without skewing peak shape Peak position can be affected by noise On Voyager ins
430. ng Good Spectra in Delayed Extraction Mode 6 3 Obtaining Good Spectra in Delayed Extraction Mode This section describes e What is a good spectrum e Laser intensity e Parameters affecting resolution and signal to noise ratio 6 3 1 What Is a Good Spectrum A good spectrum is one that is acceptable for your analysis In general it e Contains sharp symmetrical well defined peaks e Has acceptable resolution e Has acceptable signal to noise ratio For some applications for example when you are looking for detailed structural information you may require very well separated isotope peaks and the maximum resolution possible For other applications for example when looking for an estimate of molecular weight your requirements may be less strict Figure 6 6 through Figure 6 9 are examples of poor and good spectra NOTE Masses are typically accurate in Delayed Extraction mode even at high laser power NOTE Dimers in a spectrum may indicate that the molecular ion is saturated or that sample is too concentrated Decrease laser intensity or sample concentration to minimize the dimer Voyager Biospectrometry Workstation User s Guide 6 21 Chapter 6 Acquiring Spectra from the Instrument Control Panel High laser power causing the following Poor separation a between molecular Signal near Mass is fairly ion and matrix saturation accurate _ i point NT adduct ion 6 3E 4 Baseline n
431. ng instrument settings see Section 5 1 4 Modifying an Instrument Settings File BIC Standard BIC File i To Optimize Signal intensity Resolution Signal to Noise Ratio LN Adjust Laser intensity Delay Time Guide Wire Voltage Grid Voltage Accelerating Voltage Guide Wire Voltage Shots Spectrum Low Mass Gate Figure 5 20 Strategy for Optimizing Instrument Settings Voyager Biospectrometry Workstation User s Guide 5 65 Chapter 5 Optimizing Instrument Settings Strategy To optimize instrument settings do the following 1 Open a BIC file for the mass range you are analyzing For information on mass ranges in BIC files see Section 5 1 2 Standard Instrument Settings BIC Files Provided If a BIC file for the mass range you are analyzing does not exist open a standard BIC file with the closest higher mass Hint To optimize a wide mass range select a BIC file with the mass of the highest component in the range 2 Find an adequate laser setting See Section 5 4 2 Determining the Laser Setting 3 Optimize resolution See Section 5 4 3 Optimizing Resolution 4 Optimize signal to noise ratio See Section 5 4 4 Optimizing Signal to Noise Ratio Remaining parameters in the standard instrument settings provided are optimized for your workstation and should not require adjustment
432. ngs BIC files for instrument Continuous Extraction in the following modes settings files Linear mode Reflector mode PSD mode Standard instrument settings files are located in the C VOYAGER DATA FACTORY directory Table H 1 Continuous Extraction Linear Mode Standard Instrument Settings BIC Files BIC File Sample Test Mass Range Da L1000 Low mass peptide mix Calibration and resolution 100 2 000 L1001 Insulin Resolution 5 000 7 000 L1002 Myoglobin Resolution 10 000 20 000 L1003 BSA Resolution 60 000 70 000 L1004 High mass peptide mix Calibration ACTH 7 38 2 000 6 000 L1005 Neurotensin in mix Sensitivity 1 000 2 000 L1008 Low mass peptide mix Negative ion mode 100 2 000 Voyager Biospectrometry Workstation User s Guide H 3 Appendix H Continuous Extraction Mode Table H 2 Continuous Extraction Reflector Mode Standard Instrument Settings BIC Files BIC File Sample Test Mass Range Da R1000 Angiotensin Resolution at 20 000 V 100 2 000 R1001 Angiotensin Resolution at 10 000 V 100 2 000 R1002 Insulin Resolution 5 000 7 000 R1003 E coli Thioredoxin Resolution 10 000 14 000 R1004 Low mass peptide mix Calibration angiotensin l 100 2 000 R1005 Neurotensin in mix Sensitivity 1 000 2 000 R1006 High mass peptide mix Calibration ACTH 7 38 2 000 6 000 R1008 Low mass peptide mix Negative ion mode 100 2 000 R1
433. nitial velocity setting 5 22 laser intensity relative 5 67 mass spectrum C 2 organic concentration 3 7 3 8 preparing 3 5 3 7 sample concentration 3 7 stability 3 8 when to use 3 3 Single shot mode of acquisition setting 2 45 when to use 2 45 Singly charged ions drift time 1 10 Slow fragments see PSD fragments Small molecules matrix for C 8 Smoothing before calibration 6 10 Sodium adduct ion effect on masses 9 17 from buffer 3 24 Software Control Panels 4 1 Data Explorer 1 45 disk space required 2 29 exiting 2 34 included with system 1 30 1 41 installing 2 29 2 30 Instrument Control Panel 1 42 interaction Instrument and Sequence Control Panels 4 33 overview 1 42 Sequence Control Panel 1 44 4 32 starting 2 32 Source chamber maximum operating pressure 2 36 wait time 2 37 Source see lon source Index SP file see also Search pattern file converting version 4 to version 5 5 92 definition 6 46 difference between version 4 and version 5 5 92 location 6 46 Space required Voyager DE and Voyager DE PRO 2 2 Voyager DE STR 2 7 A 10 Spare parts B 6 Specifications A 1 Voyager DE workstation A 2 Voyager DE PRO workstation A 5 Voyager DE STR workstation A 8 Spectra acceptable 6 6 dimers or trimers 9 15 examples of good and bad in Continuous Extraction mode H 9 H 10 examples of good and bad in Delayed Extraction mode 6 22 6 23 saturated H 13 starting acquisition l 7 stopping acquisition l 7 troubleshooting 9 7
434. nt Settings Table 5 7 Linear and Reflector Digitizer Parameters Continued Parameter Description Vertical Scale Specifies the input range of the digitizer in millivolts To take full advantage of the dynamic range of the digitizer set the Vertical Scale high enough to allow full vertical resolution of the signal intensity Choices depend on the digitizer model For more information see Section 5 3 5 Understanding Digitizer Settings Vertical Offset Applies a y offset to the signal to bring the baseline closer to zero counts on the y axis This makes data processing easier Vertical Offset is a percentage of the selected Vertical Scale For example with a 200 mV Vertical Scale a 1 offset is 2 mV Enter or select an offset Valid entries depend on digitizer model For more information see Section 5 3 5 Understanding Digitizer Settings Input Bandwidth Reduces higher frequency noise when analyzing wide not available with high mass peaks Signatec digitizers Choices depend on the digitizer model In general use FullFor no filtering Higher settings For high resolution data such as small and large peptides Lower settings For high mass proteins for noisy peaks or to apply more filtering For more information see Section 5 3 5 Understanding Digitizer Settings 5 30 Applied Biosystems Instrument Settings Parameter Descriptions Advanced Click the Advanced tab to dis
435. nt blind spots Do not remove any instrument panels or look directly into the laser beam or allow a reflection of the beam to enter your eyes Wear proper eye protection if any panels are removed for service AVERTISSEMENT DANGER LASER Le laser met des radiations ultraviolettes Les lasers peuvent br ler la r tine et laisser des points aveugles permanents Il convient de ne pas retirer le panneau avant ou les panneaux lat raux de l appareil et de ne pas regarder directement dans le faisceau laser ou laisser une r flexion du faisceau entrer dans les yeux Portez des protections ad quates pour les yeux si le panneau avant ou les panneaux lat raux ont t retir s afin d effectuer l entretien Voyager Biospectrometry Workstation User s Guide xxi Safety and Compliance Information xxii Applied Biosystems WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result by operating the instrument without the panels in place Do not remove instrument panels High voltage contacts are exposed with panels removed AVERTISSEMENT RISQUE DE D CHARGE LECTRIQUE Des d charges lectriques s rieuses peuvent r sulter du fonctionnement de l appareil lorsque le panneau avant et les panneaux lat raux sont retir s Ne pas retirer le panneau avant ou les panneaux lat raux Des contacts haute tension sont expos s lorsque les panneaux sont retir s WARNING CHEMI
436. nt settings BIC file Selecting To select instrument settings to manually acquire data instrument 4 inthe Instrument Control Panel open or create an settings instrument settings BIC file with the appropriate parameters See Section 5 4 Optimizing Instrument Settings Parameters for information Voyager Biospectrometry Workstation User s Guide 6 11 Data Storage Laser Sample Positioning 6 12 Chapter 6 Acquiring Spectra from the Instrument Control Panel The parameters from the selected instrument settings BIC file are displayed in the Instrument Settings Control page Figure 6 2 Name of BIC file Sur VYovager instrument Control Panel SPEC Angio tnsin_linear bic l Eile Edit View Instrument Acquisition SamplePlate Display Tools Window Instrument Settings e 2al A ele elsi TE K Aea ele zlea r Data Storage Directory E Woyage Data zl Filename Fs M Autosequence Filenames Sample Description Comment r Manual Laser Intensity 2 _ 4 4 1 Manual Sample Positioning Active Well es z 100 well plate Cromer OE OmOm mong gy ooooooooo CO OC G 0 6 DO EO ter cS Ta es ay a Cae o GOO EI ONCE SOY CS Mee ee CSP wh es Nop Ley KSC nea O TO OG yd EE GS Gy ey tn ee te a eRe ite te ca toy ey a O eaaa enti n Last Acquired Spec 100 XX 505 Intensity or d
437. nt to type in text Type in text as needed The text is added to the trace NOTE The text is associated with the x coordinate If you zoom on a different region of the trace and the x coordinate moves out of view the annotated text also moves out of view Hint To move the text left click and hold on the text then drag to the desired position 4 16 Applied Biosystems Using the Spectrum Window 3 To customize the appearance of the annotated text see Section 4 4 Customizing the Instrument Control Panel NOTE Text annotations are associated with the Spectrum window not the trace Text annotations remain in the window after the trace is overwritten by a new trace Text annotations are not saved in the data file 4 To delete the annotation do either of the following e Select the text and right click Select the appropriate delete or cut option e Right click on the trace and select Delete All Text Voyager Biospectrometry Workstation User s Guide 4 17 Chapter 4 Voyager Instrument Control Panel Basics 4 3 5 Previewing and Printing Traces This section includes e Previewing and printing traces e Dedicating a printer to landscape orientation e Print Setup Previewing and To preview and print traces printing Setting trace colors 1 Display the traces as desired manually For a clear printout you can set the Trace Color and other attributes to black before printing traces
438. ntation when properly installed on the Product Applied Biosystems does not warrant that the operation of the instrument or software will be uninterrupted or error free Applied Biosystems will provide any software corrections or bug fixes if and when they become available for a period of ninety 90 days after installation Any applicable warranty period under these sections will begin on the date of installation for hardware and software installed by Applied Biosystems personnel unless that date has been delayed at the buyer s request but in no event later than thirty 30 days after shipment In that case and for all hardware and software installed by the buyer and for all other products the applicable warranty period begins the date the product or component is delivered to the buyer Warranty claims must be made within the applicable warranty period or for chemicals or other consumable products within thirty 30 days after receipt by the buyer The foregoing warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation with incompatible solvents or samples in the system operation outside of the environmental or use specifications or not in conformance with the instructions for the product or accessories performance of improper or inadequate maintenance by the user installation of software or interfacing not supplied by Applied Biosystems and modification or repair
439. nterference received including interference that may cause undesired operation WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense NOTE Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits Canadian Safety and EMC Standards European Safety and EMC Standards CE Safety and Compliance Information Safety This instrument has been tested to and complies with standard CSA 1010 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements It is an ETL Testing Laboratories listed product S curit Cet instrument a t v rif
440. ntracts for yearly preventative maintenance The following preventative maintenance is needed on a yearly basis e Replace rotary pump oil e Inspect molecular sieve oil mist filter and vent line dryer filter e Clean filters and fans e Lubricate X Y feedthroughs e Inspect flap valve 1 flap valve 2 linear actuator o rings Voyager Biospectrometry Workstation User s Guide 9 3 Chapter 9 Maintenance and Troubleshooting e Clean optics and air lines e Inspect grids compressor air pressure turbo pump and laser power e Adjust laser flash rate load offsets sample offsets detector gain and voltage and instrument covers e Inspect load and eject cycles high voltage power supplies and computer e Calibrate thermocouple gauges e Check that the instrument meets specifications Changing fuses This procedure is required for Voyager DE and Voyager DE PRO systems only the Voyager DE STR does not require power fuses Extra fuses for different voltage settings are supplied with the system To change the main power fuse 1 Exit the Voyager software 2 Power down the mass spectrometer 3 Remove the power cord from the mass spectrometer 4 Carefully remove the voltage selector fuse holder from the system Figure 9 1 using a small flat blade screw driver 5 Carefully remove the voltage selector from the holder and insert the selector with the proper voltage displayed in the window of the holder CAUTI
441. ntrol page is not displayed select Manual Laser Sample Positioning from the View menu 2 Select the sample to analyze by doing any of the following in the Active Position box Type a position number e Select a position number from the drop down list e Single click on a sample position Hint You can also move to a sample position using the control stick by selecting Move to Sample Position from the Sample Plate menu or by clicking To zoom on the Active Sample Position you can do any of the following e Double click on the sample position in the Plate view e Select Sample View from the Sample Plate menu e Right click the mouse and select Toggle Sample Plate View Voyager Biospectrometry Workstation User s Guide 6 13 Chapter 6 Acquiring Spectra from the Instrument Control Panel Hint You can double click on the control page to undock it and click drag it to any location on the screen Double click again to re dock the page 3 Set the laser intensity by click dragging the slider or clicking the arrows For more information see Section 4 5 2 Adjusting Laser Intensity and Selecting Sample Position and Section 5 4 2 Determining the Laser Setting Hint You can also set laser intensity by pressing Cirl PgUp and Cirl PgDn on the keyboard Setting Data You can specify Data Storage parameters before or after Storage acquiring in Manual Control mode To specify Data Storage 1
442. o Noise is enabled in the Spectrum Acceptance Criteria dialog box the system evaluates the signal to noise ratio for each spectrum If the signal to noise ratio is Equal to or greater than the ratio set in the Spectrum Acceptance Criteria dialog box The system checks resolution as described in step 3 if it is enabled If resolution is not enabled the system saves the data file e Less than the ratio set in the Spectrum Acceptance Criteria dialog box The system increases the laser setting one laser step as specified in instrument settings and acquires a new spectrum 3 If Resolution is enabled in the Spectrum Acceptance Criteria dialog box the system evaluates the resolution for each spectrum If the resolution is Equal to or greater than the resolution set in the Spectrum Acceptance Criteria dialog box The system saves the data file e Less than the resolution set in the Spectrum Acceptance Criteria dialog box The system acquires a new spectrum 6 62 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel If Save All or Accumulate All is selected for accumulation the system moves to the next search pattern position For all other accumulation conditions the system repeats step 1 through step 3 until one of the following is true e Acceptance criteria selected are met the signal is in range the minimum Signal to Noise ratio and resolution are achieved e The spec
443. o the computer Connection on right Connection on Connection on rear side panel of mass oscilloscope panel of computer Cable see Figure 2 6 PES Omer see Figure 2 3 g see Figure 2 2 g AUX1 or CH3 TRIG None Trig BNC CH1 CH 1 None Ch 1 BNC with oscilloscope end CH2 CH2 None Ch 2 BNC Voyager DE PRO with only oscilloscope end IEEE 488 GPIB None IEEE 488 GPIB IEEE 488 on rear of oscilloscope not shown 2 20 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations 2 3 7 Connecting the Video Monitor Figure 2 7 shows the rear panel of the video monitor PB100844 To Video on side panel Power cor of mass spectrometer ower cord Figure 2 7 Rear Panel of Video Monitor Refer to the following table when connecting the video monitor to the Voyager DE and Voyager DE PRO workstations Connection on Connection on side rear panel of panel of Mass a Cable video monitor Spectrometer see Figure 2 7 see Figure 2 2 Video In VIDEO BNC with video adapter Power receptacle VIDEO MONITOR Power ONLY Voyager Biospectrometry Workstation User s Guide 2 21 Chapter 2 2 22 Installing the Voyager Biospectrometry Workstations 2 3 8 Connecting Devices to the Computer Refer to the following table when connecting devices to the Voyager DE and Voyager DE PRO computers Connection
444. obtained when masses are in the same range For example reference masses of 904 and 1 570 are adequate when evaluating an analyte mass of 1 296 Da When calibrating mass described in the Data Explorer Software User s Guide and detecting peaks described in the Data Explorer Software User s Guide the following factors contribute to the accuracy of mass measurements e Use Centroid mass instead of Apex mass Before calibrating set peak labels to Centroid Centroid mass is the best representation of peak position Apex mass represents the highest data point e Set Centroid Percent to 50 Lower settings can include noise or contaminant peaks Higher settings may not include adequate data to ensure accurate mass assignment Voyager Biospectrometry Workstation User s Guide 6 27 Chapter 6 Acquiring Spectra from the Instrument Control Panel 6 5 Evaluating Data in the Instrument Control Panel This section describes e Detecting integrating and labeling peaks Calculating mass resolution e Calculating signal to noise ratio 6 5 1 Detecting Integrating and Labeling Peaks Detecting peaks Peaks are not detected in the Spectrum window until acquisition is complete To adjust peak detection when acquisition is complete 1 From the Tools menu select Peak Detection The Peak Detection dialog box is displayed Figure 6 11 Spectrum Peak Detection Setup Trace 1 x m Peak Detection Settings Detecti
445. oc Mass Spectrom 1996 8 209 217 2 Juhasz P M Vestal and S A Martin Novel Method for the Measurement of the Initial Velocity of lons Generated by MALDI presented at the 44th ASMS Conference on Mass Spectrometry and Allied Topics May 12 16 1996 Portland OR p 730 Instrument Settings Parameter Descriptions Modifying the Matrix options are located in the Matrix field in the Instrument matrix reference Settings control page You can add or delete information in the file Matrix reference file using Microsoft Notepad Editor You can add information to the matrix reference file by doing the following 1 Open the Microsoft Windows NT Notepad text editor See the Microsoft Windows NT User s Guide if you need help using Notepad 2 Navigate to the C VOYAGER directory 3 Open the file named MATRIX TXT The following text file appears Matrix Type1 a Cyano 4 hydroxycinnamic acid Matrix Welocity1 366 Matrix Type2 Sinapinic acid Matrix Welocity2 356 Matrix Type3 2 5 Dihydroxybenzoic acid Matrix Welocity3 5 66 Matrix Type4 3 Hydroxypicolinic acid Matrix Welocity4 556 4 Type new matrix and velocity information by using the format above If you do not have a velocity value for a matrix use 300 m sec a cyano 4 hydroxycinnamic acid value 5 Save the modified TXT file Voyager Biospectrometry Workstation User s Guide 5 23 Chapter 5 Optimizing Instrument Settings 5 2 2 Mode Dig
446. of crystals lt 212 032 spectrum on solution 294 076 page C 2 Yellow MW 189 04 Da CH C CN COOH SS A si PB100252 2 5 Applications 10 mg ml in water e 155 034 dihydroxybenzoic Peptides e 154 027 see mass e Carbohydrates A EaD spectrum on GI ed ycolipids page C 3 negative ion MW 154 03 Da mode zoh e Polar synthetic OH HO PB100253 polymers Color of crystals solution White Voyager Biospectrometry Workstation User s Guide C 7 Appendix C Matrixes Table C 1 Matrix Information Matrix Applications Color Matrix Solution Concentration Characteristic Matrix lons monoisotopic 2 5 dihydroxybenzoic acid 2 5 DHB see mass spectrum on page C 3 MW 154 03 Da COOH OH ye HO PB100253 Applications Small molecules Color of crystals solution White 10 mg ml in solvent in which sample and matrix are soluble 155 034 154 027 137 024 273 040 C 8 Applied Biosystems Matrixes Table C 1 Matrix Information Matrix Solution Characteristic Matrix Applications Color Matrix lons Concentration monoisotopic Mixture of 2 5 Applications 10 mg ml in solvent in e 155 034 dihydroxybenzoic Large proteins Which sample and e 154 027 acid and Color of crystals matrix are soluble 137 024 5 methoxysalicylic solution e 273 040 acid DHBs Whit e 151 040 Geama oy 168 042 e 169 050
447. oise 100 5741 5 90 80 70 60 50 40 30 20 10 11670 7 Intensity 0 T T F 0 1000 8800 16600 24400 32200 40000 Mass m z Figure 6 6 Example of Poor Mass Spectrum for Myoglobin Using Sinapinic Acid Lower laser power producing Correct intensity ratio between single charged ion Good separation M H and double charged between molecular ion M 2H 2 ion and matrix 100 16951 16 adduct ion 27E 4 904 80 2 m 5735 00 11674 52 Resolved Diri r S 50 to over 1 000 minimized o resolution 161 101 1709 07 68326 A 23343 82 2862516 33890 44 1000 8800 16600 24400 32200 40000 Minimal Mass m z noise Figure 6 7 Example of Good Mass Spectrum for Myoglobin Using Sinapinic Acid 6 22 Applied Biosystems Obtaining Good Spectra in Delayed Extraction Mode High laser power causing the following Matrix peaks and sample saturated 379 2 Intensity Minor components or fragments of major components ionized Massis Signal is y accurate saturated 1297 5 N 6 5E 4 Broad peak Poor resolution Baseline noise 0 2000 Mass m z Figure 6 8 Example of Poor Mass Spectrum for Angiotensin Lower laser power producing 1296 6 3 9E 4 ene Peak not saturated 80 Sharp narrow peak 70 Matrix peaks minimized resolved to isotopes 4 304 1721 3794 No minor 304 components 204 or fragments 1
448. olecular weight F 3 Cation exchange sample cleanup 3 29 Cautions Deflector Gate Width in PSD changing the setting 8 29 exiting Voyager Control software 2 34 Flight Length to Deflector do not change 8 30 internal jumpers setting in STR models 2 8 vacuum disruption in CID 8 34 voltage selector plastic tabs fragile 2 4 Center bias search pattern 5 37 Centroid mass shift caused by Input Bandwidth setting 5 63 Centroid for maximum mass accuracy 6 6 Ch1 and Ch2 on oscilloscope l 6 CHCA matrix see Alpha cyano 4 hyroxycinnamic acid Chemical hazards 2 49 Chemical structure of matrices C 1 Child ion see PSD precursor spectrum CID adjusting 8 36 benefits 8 32 description 8 31 enhancing fragmentation with 8 31 gas pressure 8 36 gas too high or too low 8 36 Index 8 Applied Biosystems CID continued Guide Wire Voltage 8 36 interlock error 8 34 plumbing 8 31 purging 8 34 sample spectra 8 32 8 33 troubleshooting 8 36 turning off 8 36 turning on 8 35 vacuum gauges pressure 8 34 Cleaning sample plates 3 47 samples see Sample cleanup Clearing accumulated spectrum 4 26 Close external calibration see calibration external Sequence Control Panel Collision induced dissociation see CID Colors changing Instrument Control Panel 4 21 changing to black before printing 4 18 OptiPlate results 2 69 Comment Sequence Control Panel 4 32 Composite spectrum see PSD composite spectrum Computer archiving 9 6 configuration re
449. oltage and Delay Time e Ata fixed Grid Voltage higher masses require a higher Delay Time See Appendix E Grid Voltage and Delay Time Settings for a graphic representation of the relationship between mass Grid Voltage and Delay Time e Optimum tuning of Delay Time and Grid Voltage is slightly dependent on matrix due to the different initial velocities associated with different matrixes The optimum Delay Time may be different within 100 nsec at the same Grid Voltage for the same sample in different matrixes NOTE The Grid Voltage setting in Reflector mode is typically significantly lower than the setting in Linear mode for the same mass range For example if Grid Voltage in Linear mode is 94 Grid Voltage in Reflector mode may be 70 These values are used as an example they may not be optimal for your system Readjusting the After you have optimized Guide Wire Voltage Delay Time laser after and Grid Voltage for optimum resolution you can further optimization fine tune by adjusting the laser intensity For information on adjusting the laser intensity see Adjusting laser intensity on page 5 68 5 74 Applied Biosystems Optimizing Instrument Settings Parameters 5 4 3 2 Acceptable Resolution in Delayed Extraction Mode Isotope resolution You should be able to partially resolve isotopes up to the following masses Linear mode Approximately 2 000 Da Reflector mode Approximately 6 000
450. omatic Control mode Applied Biosystems 3 Select Manual Control mode For parameter descriptions see Section 5 2 1 Instrument Settings Page 4 Adjust the mass range if needed 5 To include matrix peaks in the spectrum for calibration deselect Low Mass Gate and set the mass to a mass below the matrix peak mass For matrix masses see Appendix C Matrixes 6 Select a calibration file CAL or if you are screening samples use the default calibration For better mass accuracy select a CAL file you recently same day generated in the Data Explorer software For more information see the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating 7 Save the BIC file For more information see Section 5 1 5 Saving and Printing Instrument Settings For suggested strategies for optimizing parameters see e Section 5 3 Impact of Changing Instrument Settings Parameters e Section 5 4 Optimizing Instrument Settings Parameters After you optimize the instrument settings file for Manual Control mode and ensure that parameters are adjusted to yield optimum performance you can adjust the instrument settings for Automatic Control mode if desired For more information see Section 6 6 2 Setting Instrument Settings for Automatic Control Mode Loading Modifying and Saving Instrument Settings 5 1 5 Saving and Printing Instrument Settings Saving To save instrument settings 1 Set all parameters as need
451. omatic mode 8 63 preparing thin layer PSD manual mode 8 53 application 3 3 3 5 3 11 single shot mode 2 45 sample concentration dried droplet starting 4 25 6 16 application 3 3 3 9 status Sequence Control sample concentration thin layer Panel 7 20 7 29 application 3 3 3 11 stopping 4 25 6 16 1 7 stability 3 10 3 12 with low number of Scans To when to use 3 3 Average l 8 Amino acids immonium ions 8 23 Acquisition mode description 6 61 Angiotensin Acrobat see Adobe Acrobat BIC file 5 4 5 5 ACTH BIC files 5 6 BIC file 5 4 5 5 expected masses and ion molecular weight F 2 F 3 types 8 13 Active position mass to time conversion F 4 displayed in status bar 4 5 molecular weight F 2 incorrect number 9 20 PSD analysis of 8 3 selecting automatically 6 41 PSD fragments observed F 7 selecting manually 4 25 4 29 theoretical cleavages F 6 4 30 6 13 troubleshooting 9 20 Voyager Biospectrometry Workstation User s Guide Index 3 Index Annotating traces adding text 4 16 changing font and color 4 21 deleting text 4 17 text from previous trace displayed 4 16 with ASCII text 4 16 with results 4 16 Applied Biosystems Technical Support 9 7 Archiving definition 9 6 when to perform 9 6 Arcing 9 24 ASCII text annotating traces with 4 16 Attenuator see Laser Automated Laser Intensity Adjustment enabling 5 34 Automated Sample Positioning enabling 5 37 Search Pattern file 5 38 Automatic Control mode see also Automatic Cont
452. on Shots Spectrum fico Mass Range Da jo to 2000 V Low Mass Gate Da 500 r Calibration Matrix a Cyano 4 hydroxycinnamic acid x Default C External File al Figure 5 3 Instrument Settings Page Voyager Biospectrometry Workstation User s Guide 5 15 Chapter 5 Optimizing Instrument Settings Instrument settings parameters are described in Table 5 4 Table 5 4 Instrument Settings Parameters Parameter Description Instrument Mode Displays e Reflector Linear or PSD operating mode e Positive or Negative polarity For more information see Section 1 4 Voyager DE Delayed Extraction Technology Mode Digitizer settings Click to display Instrument Mode Digitizer dialog box See Section 5 2 2 Mode Digitizer Dialog Box Control Mode Select control mode e Manual Enables manual control of laser sample positioning data storage and spectrum accumulation Automatic Enables automated control of laser sample positioning data storage spectrum accumulation and spectrum acceptance See Section 5 2 3 Automatic Control Dialog Box Automatic Control settings Click to display automated laser sample positioning data storage spectrum accumulation parameters and spectrum acceptance criteria See Section 5 2 3 Automatic Control Dialog Box NOTE This button is disabled until you select Automatic Control mode 5 16 Applied Biosys
453. on Decrement Ratio PSD correlation with PSD Mirror Ratio 8 41 default settings 8 41 setting 8 50 setting and segment size in PSD 8 41 Default calibration description 6 7 equation 6 9 PSD 8 28 selecting 5 10 Index Default layout Instrument Control Panel 4 8 DEFAULT SP 6 48 Deflector Gate Width impact on system performance 2 40 setting 2 40 Deflector Gate Width setting 8 30 Deisotoping before calibration to ensure monoisotopic peak is identified 7 10 Delay Time affected by matrix 5 55 5 74 and Grid Voltage 5 55 5 73 E 1 description 5 18 5 54 effect on resolution 5 49 impact on fragmentation 5 49 impact on sensitivity 5 49 isotope resolution 5 81 minimum increments needed 5 54 5 77 5 80 optimizing 5 77 settings for mass ranges 5 79 Delayed Extraction acceptable spectra 6 21 advantages 1 14 comparison to Continuous Extraction 1 13 Delay Time setting 5 79 E 1 description 1 12 determining if hardware installed 2 41 Grid Voltage and Delay Time relationship between 5 55 5 73 Grid Voltage setting E 1 Guide Wire Voltage 5 84 Instrument Settings parameters 5 73 isotope resolution 1 13 5 75 laser intensity 6 24 Voyager Biospectrometry Workstation User s Guide Index 11 I N D E X Index Delayed Extraction continued mass accuracy troubleshooting 9 11 9 12 optimizing parameters 5 73 overview 1 11 parameters with primary impact 5 73 6 25 resolution 6 32 resolution acceptabl
454. on Selector voltage is turned off is determined by the Deflector Gate Width parameter The Deflector Gate Width is optimized for your system when your system is installed However you can increase or decrease the selectivity of the Precursor lon Selector by decreasing or increasing the Deflector Gate Width setting CAUTION Do not alter the Timed lon Selector parameters unless instructed to do so by an Applied Biosystems Technical Representative Altering these settings may cause your Voyager Biospectrometry Workstation to function improperly Before changing the Deflector Gate Width setting write down the current setting This setting has been optimized for your system and you may want to reset to the optimized value Smaller width values provide narrower Precursor lon Selector resolution If you set the width too low fragment ion yield is compromised due to product ion dispersion that occurs as ions travel down the flight tube Voyager Biospectrometry Workstation User s Guide 8 29 Chapter 8 PSD Analysis 8 30 Applied Biosystems To change the width setting select Hardware Configuration from the Instrument menu in the Instrument Control Panel then click the Timed lon Selector tab Figure 8 8 Hardware Configuration xj High Voltage Instrument Digitizer Vacuum Laser Timed lon Selector Flight Length to Deflector mm 1135 Deflector Gate Width mm fo OK Cancel Apply Help
455. on Range s Full 55 x Range s Lower Bound Upper Bound Range 1 Start End Active Local Range Thresholds BP Relative 1 j MinIntensity 0 a BP Area fe Min Area 0 a Noise Threshold Filter Settings pY Cursor Mode Width E a ZBP Intensity Increment fi a An Meme I Use same settings for all traces in view OK Cancel Apply Advanced Figure 6 11 Peak Detection Dialog Box 6 28 Applied Biosystems Labeling spectrum peaks Disabling spectrum peak labels Evaluating Data in the Instrument Control Panel NOTE The parameters in this dialog box correspond to the Advanced Settings tab in the Peak Detection dialog box in the Data Explorer software 2 Select a detection range and set parameters as described in the Data Explorer Software User s Guide 3 Click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box If peaks are not labeled enable labels as described in the next section NOTE The Instrument Control Panel uses many of the same parameters detection and integration algorithms as the Data Explorer software For information see the Data Explorer Software User s Guide Labeling spectrum peaks in the Instrument Control Panel is similar to labeling spectrum peaks in the Data Explorer software Activate the trace to label select Peak Label from the Display menu enable and sele
456. on analyzed Signal to Noise Ratio 2200 1 for IgG 0 6 pmol ul 2100 1 for BSA 4 pmol ul Mass Accuracy in Delayed Extraction mode difference between the mean of six measurements and the theoretical mass of the sample Linear mode External Calibration 0 05 Internal Calibration 0 02 Reflector mode External Calibration 0 01 Internal Calibration 0 002 Sensitivity Routine detection of 5 fmol of neurotensin with signal to noise ratio 220 1 in Linear mode 2 210 1 in Reflector mode Timed lon Selection Mass Accuracy of Fragment 1 Da lons Resolution of PSD Precursor 80 Voyager Biospectrometry Workstation User s Guide A 5 Appendix A Specifications Table A 5 Voyager DE PRO Mass Spectrometer Specifications Condition Specification Mass range 2300 000 Da Upper limit set by suitably ionized biomolecule Reflector Single stage with optimized optics for PSD Analysis Flight tube horizontal e Linear mode 1 3 m e Reflector mode 2 0 m lon source Two stage lon source voltages Tunable Accelerating Voltage Up to 25 000 V Grid Voltage Range determined by Accelerating Voltage Laser Nitrogen 337 nm 3 ns pulse 20 Hz maximum firing rate Actual laser firing rate dependant on digitizer See Section A 4 Digitizer Specifications Digitizer 2 GHz digitization for enhanced resolution
457. on is obtained Hint If you obtain close to acceptable resolution at one setting but less acceptable resolution at the next setting you may have adjusted the Delay Time too far Increase or decrease in smaller increments until you obtain optimum resolution 5 If resolution is still not optimized after you adjust the Delay Time setting adjust the Grid Voltage as described in Section 5 4 3 5 Optimizing Grid Voltage Reload the standard BIC file you started with and use the Delay Time value provided in the BIC file 6 If you are analyzing samples below 2 000 Da you may be able to improve resolution by adjusting the Accelerating Voltage Linear mode Decrease from 20 000 V to 15 000 V to increase flight times Reflector mode Decrease to 15 000 V 10 000 V or 5 000 V However these lower settings will compromise sensitivity 7 Save the BIC file Setting Delay To resolve isotopes across a broad mass range select a Time to resolve Delay Time setting that yields optimum resolution at the higher isotopes across a end of the mass range Isotopes at lower masses can be resolved at lower resolution Selecting a Delay Time that broad mass range maximizes resolution at lower masses may not resolve isotopes at higher masses When selecting a Delay Time to resolve isotopes across a broad mass range resolution and signal intensity at the lower end of the mass range may be compromised but you should observe isotopi
458. on of Spectrum Accumulation Options Spectrum Acceptance Criteria Click to display Spectrum Acceptance Criteria parameters See Setting spectrum acceptance and laser adjustment criteria on page 6 42 5 36 Applied Biosystems Instrument Settings Parameter Descriptions Table 5 11 Automatic Control Parameters Sample Positioning Parameter Description Sample Positioning Use Automated Sample Positioning Enables or disables automated sample positioning For more information see Sample positioning on page 6 41 Random Search Pattern Sets the software to randomly determine search pattern positions as data is acquired Options include Uniform Samples from positions evenly distributed across a sample position e Edge Bias Samples from the perimeter of a sample position e Center Bias Samples from the center of a sample position Uniform Edge Bias Center Bias If you specify Random Search Pattern and specify Acceptance Criteria you must specify a Stop Condition Each time you acquire data using the same BIC file with Random Search Pattern selected the software starts random sampling at an x y position different from the last acquisition continued Voyager Biospectrometry Workstation User s Guide 5 37 Chapter 5 Optimizing Instrument Settings Table 5 11 Automatic Control Parameters Sample Positioning Continued Parameter
459. on rear panel of Device computer see Figure 2 3 on page 2 10 Keyboard 5 pin round connector VGA monitor 12 pin connector 3 rows of pins Control stick Top 15 pin connector NOTE There are two 15 pin connectors You must connect the control stick to the top connector Mouse 5 pin round connector Printer LPT1 or Parallel Applied Biosystems Connecting the Voyager DE STR Workstation 2 4 Connecting the Voyager DE STR Workstation This section includes e Rear panel connections e Keyboard mouse and control stick e Digitizer e Video monitor e Oscilloscope video monitor and computer monitor Rear panel Figure 2 8 shows the connections and the on off switch main connections power circuit breaker on the rear panel of the mass spectrometer cabinet Top left rear panel Optional external laser connections NETWORK PRINTER SPARE PB100845 Bottom right rear panel On off olo switch s fy Receptacle 4s for power cord Figure 2 8 Rear Panel of Voyager DE STR Mass Spectrometer Component Connections and On Off Switch Voyager Biospectrometry Workstation User s Guide 2 23 Chapter 2 Installing the Voyager Biospectrometry Workstations Keyboard mouse Thread cables for the computer keyboard mouse and control and control stick stick through the circular hole in the front panel o
460. onents under warranty does not extend the original warranty period Applied Biosystems warrants that all optional accessories supplied with the Product such as cameras peripherals printers and special monitors will be free of defects in materials and workmanship for a period of ninety 90 days Applied Biosystems will repair or replace at its discretion defective accessories during this warranty period After this warranty period Applied Biosystems will pass on to the buyer to the extent that it is permitted to do so the warranty of the original manufacturer for such accessories With the exception of chemicals and other consumable products replaceable products or components that are obtained from Applied Biosystems and are used on the Product are themselves warranted to be free of defects in materials and workmanship for ninety 90 days Applied Biosystems warrants that chemicals and other consumable products obtained from Applied Biosystems will be free of defects in materials and workmanship when received by the buyer but not thereafter unless otherwise specified in documentation accompanying the product Warranty period effective date Warranty claims Warranty exceptions Limited Product Warranty Applied Biosystems warrants that for a period of ninety 90 days the software designated for use with the Product will perform substantially in accordance with the function and features described in its accompanying docume
461. oning control page select the same sample position from which you acquired the precursor spectrum 2 From the View menu select Data Storage Set parameters as needed See Setting Data Storage parameters on page 6 14 for information Hint Include a_PSD suffix when you name PSD data files to help you distinguish them from non PSD data files For example if you type in Experiment1_PSD as the file name the complete data file name will be Experiment1_PSD DAT or Experiment1_PSD_0001 DAT if Autosequence Filenames is enabled Voyager Biospectrometry Workstation User s Guide 8 53 Chapter 8 PSD Analysis Selecting and 3 Inthe PSD Acquisition Settings control page select click acquiring a on the row that corresponds to the segment you want to 4 To start acquiring select Start Acquisition from the Acquisition menu or click A 5 Adjust laser intensity to optimize signal intensity You typically need a higher laser intensity to optimize signal intensity for segments with lower Mirror Ratio settings During The following occurs acquisition e Mass range for the segment is set to Mass Equivalent to Start Precursor mass 4 which is equal to Precursor flight time 2 End Mirror to Accelerating Voltage Ratio x Precursor mass which is equal to Mirror to Accelerating Voltage Ratio x Precursor flight time e Acquisition starts All instrument settings except Shots S
462. ontinued Voyager Biospectrometry Workstation User s Guide 3 13 Chapter 3 Preparing Samples Table 3 4 THAP Matrix Information Continued Matrix concentration e Oligonucleotides 10 mg ml e Acidic carbohydrates 2 mg ml Additive concentration 50 mg ml diammonium citrate in deionized water Preparation Follow the procedure in Preparing matrix on page 3 5 the for e Small oligonucleotides Combine 8 1 THAP diammonium citrate Air dry sample plate after loading sample and matrix e Acidic carbohydrates and glycopeptides Combine 20 1 THAP diammonium citrate Dry sample plate under vacuum after loading sample and matrix Wait a few minutes after drying until the sample absorbs ambient humidity and turns faint white Crystals Larger than sinapinic overlapping shingles non uniform shape see Figure 3 2 on page 3 46 Stability Prepare weekly 1 Other special sample preparation techniques are described in Papac D I A Wong A J S Jones Anal Chem 1996 68 3215 3223 3 14 Applied Biosystems 3 HPA Table 3 5 3 HPA Matrix Information Preparing Samples Use 3 HPA for oligonucleotides gt 3 500 Da WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 3 Hydroxypicolinic acid 3 HPA may cause eye skin and respiratory tract ir
463. ontrol page click resolution Mode Digitizer 2 Click Linear or Reflector Digitizer Voyager Biospectrometry Workstation User s Guide 5 57 Chapter 5 Optimizing Instrument Settings 3 Change the Bin Size to improve resolution as needed Mode Digitizer Bin Size Linear 1 nsec NOTE A Bin Size setting below 1 nsec does not typically improve observed mass resolution in Linear mode Reflector 0 5 or 1 nsec NOTE Setting the Bin Size lower than the default calculated by the system may result in a larger data file size 5 58 Applied Biosystems Impact of Changing Instrument Settings Parameters Effects of A smaller Bin Size lower number of nanoseconds adjusting Bin Size increases resolution between peaks when peaks are narrow in time width see Figure 5 17 e Increases the size of the data file because the mass range is recorded with finer sampling and therefore increases the number of data points collected Data collected at Baseline resolution 2 nanoseconds between 2 adjacent peaks Data collected at Partial resolution peaks Figure 5 17 Effect of Bin Size nsec on Peak Resolution and Data Quality A larger Bin Size higher number of nanoseconds e Decreases resolution between peaks when peaks are narrow in time width see Figure 5 17 e Decreases the size of the data file because the mass range is recorded with coarser sampling decreases the n
464. orrelated with their initial velocity When the extraction voltage pulse is applied initially slower ions acquire slightly higher energy from the accelerating field than initially faster ions By careful tuning of variable voltage grid in the source and the delay time applied to acceleration slow and fast ions of the same mass reach the detector plane at the same time This is referred to as velocity focusing Figure 1 9 and Figure 1 10 illustrate velocity focusing in linear mode lons are focused at the detector Broad velocity distribution Field free expansion no extraction of laser desorbed ions expense meen Numbe ofions lons generated by the laser pulse drift into a field free region of the ion source and separate based on their initial velocity gained from the plume expansion v slow fast 0 Vv Voltage U Detector gt lon source Flight tube Extraction After a well defined delay time an extraction pulse is applied in the ion source so the initially slower ions will be accelerated to slightly higher energy than the initially faster ones U gt U slow fast Voltage U Detector PB100791 Figure 1 9 Velocity Focusing of lons in Linear Mode Field Free Expansion and Extraction Phases Voyager Biospectrometry Workstation User s Guide 1 15 Chapter 1 1 16 Reflector mode Applied Biosystems Introducing the Voyager Biospectrom
465. ortion to the effective length of the ion flight path 1 W C Wiley and H McLaren Rev Sci Instrum 1953 26 1150 1157 W C Wiley U S Patent 2 685 035 Voyager Biospectrometry Workstation User s Guide 1 11 Chapter 1 Introducing the Voyager Biospectrometry Workstations Delayed With Voyager DE Delayed Extraction technology ions Extraction form ina field free region and then are extracted by applying a high voltage pulse to the accelerating voltage after a predetermined time delay Figure 1 6 compares Delayed and Continuous Extraction Delayed Extraction Continuous Extraction laser pulse laser pulse U Accelerating si Accelerating voltage UT voltage Grid voltage voltage time time PB100772 Figure 1 6 Delayed Versus Continuous Extraction Delayed The Voyager DE systems can be operated in Delayed Extraction versus Extraction DE or Continuous Extraction mode Continuous Extraction NOTE Due to the superior results obtained with Delayed Extraction DE mode use Continuous Extraction mode for diagnostic purposes only See Appendix H Continuous Extraction Mode for more information on optimizing methods and obtaining good spectra In Delayed Extraction mode e Potential gradient does not exist when sample is ionized sample plate and grid are at similar potentials e Accelerating voltage is pulsed after a user set time delay po
466. ory They can be saved to any directory on your system Voyager Biospectrometry Workstation User s Guide 5 3 Chapter 5 Optimizing Instrument Settings BIC files and The default control mode for all BIC files is Manual You can modify any BIC file for use in Automatic Control mode and then save it as a new BIC file For information on modifying instrument settings BIC files see Section 5 1 4 Modifying an Instrument Settings File BIC Table 5 1 through Table 5 3 list the standard BIC files provided on your system for the following modes control mode List of BIC files Linear mode Reflector mode PSD mode Table 5 1 Linear Mode BIC Files Mass Range in BIC File Sample Test BIC Optimized at Da Angiotensin_Linear BIC Low mass Calibration and 500 2 000 peptide mix1 Resolution angiotensin ACTH_Linear BIC Peptide mix2 Calibration and 2 000 3 000 Resolution ACTH 18 39 Insulin_Linear BIC Peptide mix2 Resolution 5 000 6 000 insulin Myoglobin_Linear BIC Low mass Resolution 15 000 20 000 protein mix3 myoglobin BSA_Linear BIC BSA Resolution 60 000 70 000 IgG_Linear BIC IgG Resolution 100 000 200 000 Peptide_Sensitivity_Linear BIC Neurotensin Sensitivity 1 000 2 000 in mix Peptide Linear_Negative BIC Low mass Negative ion 500 2 000 peptide mix1 mode 5 4 Applied Biosystems Mass Range specified for acquisition m
467. ospectrometry Workstation User s Guide 3 57 Chapter 3 Preparing Samples It takes 1 or 2 minutes for the sample plate to reach the correct position While the sample plate is moving the Load Eject Status dialog box displays messages about the status of the hardware Voyager DE STR To load sample plates 1 Eject the sample holder as described in Section 3 4 8 Ejecting the Sample Holder 2 Hold the sample plate vertically with the sample surface facing to the right and with the slanted underside of the plate facing toward the back of the instrument 3 Slide the sample plate into the holder from the front until it snaps into place Figure 3 7 The ball bearings on the holder snap into the plate CAUTION If the sample plate does not snap into place it may be inserted into the holder the wrong way and it may jam inside the instrument Remove the plate slide it into the holder with the slanted underside of the plate facing the left and toward the back of the instrument then snap it into place 3 58 Applied Biosystems Loading Sample Plates in the Mass Spectrometer PB100277 Figure 3 7 Loading the Sample Plate in a Voyager DE STR WARNING PHYSICAL INJURY HAZARD Fingers can get caught in the sample holder To avoid injury do not click Load to retract the sample holder when your fingers are near the sample holder 4 From the Sample Plate menu select Load to retract the sampl
468. ot otherwise be reaching the detector For more information see Guide Wire Voltage on page 5 18 Linear mode For Linear mode analysis of peptides in the 1 000 to 2 000 Da range a setting of 0 05 to 0 1 percent is adequate e For Linear mode analysis of ions over 20 000 Da range start with a setting of 0 3 percent and decrease as needed e Settings below 0 02 percent may not give adequate sensitivity 5 56 Applied Biosystems Impact of Changing Instrument Settings Parameters Reflector mode To obtain maximum resolution in Reflector mode for isotopically resolved species set the Guide Wire to 0 e To increase sensitivity in Reflector mode increase the Guide Wire Voltage to e Up to 0 02 for lt 5 000 Da Up to 0 05 for gt 10 000 Da PSD mode In PSD mode use settings between 0 005 and 0 02 percent For more information see Chapter 8 PSD Analysis 5 3 5 Understanding Digitizer Settings This section describes Bin Size e Vertical digitizer settings For more information on default digitizer settings see Mode Digitizer Dialog Box on page 5 24 5 3 5 1 Bin Size The Bin Size nanoseconds parameter is the sampling rate of the digitizer which is equal to the time interval between subsequent data points For information see Effects of adjusting Bin Size on page 5 59 The default Bin Size is 2 nsec Decreasing for To decrease the Bin Size improved 1 Inthe Instrument Settings c
469. ou are familiar with the information in Section 8 4 Acquiring PSD Data with Standard BIC Files in Manual Control Mode WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Angiotensin may cause an allergic skin and respiratory reaction Exposure may cause eye skin and respiratory tract irritation Alpha cyano 4 hydroxycinnamic acid CHCA may cause eye skin and respiratory tract irritation To prepare the practice standard 1 Prepare a 50 pmol ul solution of angiotensin 2 Prepare a cyano 4 hydroxycinnamic acid matrix as described in Section 3 1 2 Preparing Matrix 3 Mix 1 ul of angiotensin with 24 ul of matrix for a final concentration of 2 pmol ul 4 Apply 1 ul of angiotensin matrix to a sample plate Voyager Biospectrometry Workstation User s Guide 8 65 Chapter 8 PSD Analysis 8 6 1 Effects In this section Observing effects of high and low 8 66 laser intensity Observing the of Laser Intensity Adjusting laser intensity affects fragment ion production and signal intensity In this section you will e Observe the effects of setting the laser intensity too high and too low e Determine the laser intensity for your system that yields the best signal for PSD spectra To observe the effects of laser intensity 1 Open the Angio
470. ow diagram 3 66 types of 3 63 3 HPA chemical structure and molecular weight C 10 concentration 3 15 crystals 3 15 3 46 initial velocity setting 5 22 laser intensity relative 5 67 mass spectrum C 4 preparing 3 15 sample concentration 3 15 stability 3 15 when to use 3 3 400 well plate number of cal standards needed 3 39 PLT file 3 67 types of 3 62 3 63 64 well plate number of cal standards needed 3 39 PLT file 3 67 Voyager Biospectrometry Workstation User s Guide 96x2 well plate one calibration standard needed 3 39 PLT file 3 67 position row diagram 3 66 types of 3 64 A a b c ions angiotensin F 6 Absolute counts displaying on right axis 5 70 scaling signal to 4 12 Accelerating Voltage changes compensated for by system 5 17 5 87 description 5 87 effect on calibration 5 17 5 87 effect on resolution 5 50 5 88 effect on signal to noise 5 50 impact on ion acceleration 5 87 in Continuous Extraction mode H 11 in PSD mode 8 44 8 59 maximum configuring 2 38 peptides 5 84 range DE systems 5 17 recommended settings 5 87 Acceptance criteria see Spectrum Acceptance Criteria Accumulate All Spectra data collection mode description 5 45 process that occurs during 6 66 selecting 5 36 Index 1 Index Accumulate All Spectra That Pass Acceptance Criteria data collection mode description 5 46 process that occurs during 6 67 selecting 5 36 Accumulation spectra automatic enabling 6 40 number
471. owing 1 The system sets the laser to one of the following and acquires spectra e If Prescan At First Search Pattern Position Only is enabled Uses the laser setting and search pattern position determined in Prescan mode on page 6 58 and acquires spectra e If Prescan At Each Search Pattern Position is enabled Uses the laser setting and search pattern position determined in Prescan mode on page 6 58 and acquires spectra e If Prescan mode is not enabled Sets the laser halfway between the minimum and maximum specified in the Automatic Control Settings dialog box Starts acquiring data at the first position specified in the search pattern If the signal intensity is e Too high The system decreases the laser setting one laser step as specified in Automatic Control dialog box and acquires a new spectrum e Too low The system increases the laser setting one laser step as specified in Automatic Control dialog box and acquires a new spectrum Voyager Biospectrometry Workstation User s Guide 6 61 Chapter 6 Acquiring Spectra from the Instrument Control Panel NOTE If no minimum or maximum signal intensity criteria is specified the laser is not adjusted The mid range laser setting specified in the Automatic Control dialog box is used e Within range The system saves the data file if Signal to Noise is not enabled or continues with step 2 if Signal to Noise is enabled 2 If Signal t
472. page 1 23 and page 1 35 The detector is not turned on until ions greater in mass than the mass you enter pass the detector Voyager Biospectrometry Workstation User s Guide 5 89 Chapter 5 Optimizing Instrument Settings When to use Optimum setting for starting mass NOTE When Low Mass Gate is enabled the mass range below the Low Mass Gate starting mass is still detected but detector sensitivity below this mass is decreased by 100 to 1 000 fold If strong matrix signal is present below the Low Mass Gate you can use it for calibration if desired Turn on Low Mass Gate when e Analyzing masses greater than 2 000 3 000 Da e Matrix signal is more intense than the sample signal You will need to experiment with the optimum setting for the starting mass threshold for Low Mass Gate As a general guideline e If you are analyzing peptides in the 400 to 2 000 Da range set the Low Mass Gate 350 Da If you are analyzing peptides in the 2 000 to 8 000 Da range set the Low Mass Gate 500 Da e If you are analyzing samples in the 10 000 to 30 000 Da range set the Low Mass Gate 3 000 Da When not to use Do not use Low Mass Gate if 5 90 Applied Biosystems e You are analyzing peaks in the same mass range as matrix peaks e You are using a matrix peak to calibrate NOTE Low mass gate is automatically disabled when you change the instrument mode to PSD Low Mass Gate spike Optimizing Instr
473. pectrum accumulation parameters and spectrum acceptance criteria Manual Control To select Manual Control mode 4 mode 1 Open the Instrument Control Panel 2 Select Instrument Settings from the View menu 3 Click the Manual button on the Instrument Settings control page Laser controls sample positioning and data storage are available for manual adjustment and control For additional information on manual control mode see Section 5 2 1 Instrument Settings Page 4 6 Applied Biosystems Instrument Control Panel Automatic To select Automatic Control mode Control mode 1 Open the Instrument Control Panel 2 Select Instrument Settings from the View menu 3 Click the Automatic button on the Instrument Settings control page 4 Click the Automatic Control button 5 Set laser controls sample positioning and data storage for automatic adjustment and control as described in Section 6 6 2 Setting Instrument Settings for Automatic Control Mode 4 1 3 Accessing the Sequence Control Panel and the Data Explorer Software You can access the Sequence Control Panel and Data Explorer software from the Instrument Control Panel by e Selecting commands from the Applications menu e Clicking toolbar buttons Accessing Data jf you access the Data Explorer software from the Instrument Explorer Control Panel the following happens e The last data file you saved in the Instrument Control Panel is automatically opened in
474. pectrum are disabled Settings remain inactive until you stop the PSD experiment after you acquire all segments of interest e Acquisition of the segment continues until the number of Shots Spectrum specified in Spectrum Acquisition on the Instrument Settings control page is collected or until you select Stop Acquisition from the Acquisition menu e The spectrum is displayed in the Current Trace in the Spectrum window 8 54 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Manual Control Mode Changing settings Accumulating or 6 saving the segment Selecting and 7 acquiring remaining segments During acquisition you can vary laser intensity parameters in the segment list and Shots Spectrum Evaluate the spectrum then do one of the following e Click in the toolbar to accumulate the spectrum You can accumulate as many spectra as needed When the accumulated spectrum is acceptable click on the Accumulated trace then click EJ e Click H in the toolbar to add the segment to the DAT file After you save the segment the Saved check box in the segment list is checked Ifthe current spectrum does not contain significant fragment ion signal and you do not want to accumulate or save the spectrum acquire remaining segments as described below CAUTION Save the current segment if the data is acceptable before starting to acquire the next segment If you do not you will lose the data for
475. pend to an existing optimization file Resetting the Optional External Laser 2 9 Resetting the Optional External Laser When to reset Reset the external laser when you switch from the internal to the external laser when you save or load a BIC file with the external laser specified or run a sequence that contains BIC files with the external laser specified Resetting To reset 1 Click OK in the message dialog box instructing you to reset the external laser 2 Onthe left side of the external laser cabinet Figure 2 28 turn the key located next to the Keyswitch Reset LED from the On position right to the Off position left then to the On position right Stop Start O Off Position AC Power On O r A On Position Laser On O Keyswitch Reset 7 i Key Interlock O Figure 2 28 Left Side of External Laser Cabinet The Laser On LED flashes then remains on The light on the Stop button is illuminated 3 Press the Start button The light on the Stop button goes off and the light on the Start button flashes then goes off The laser is now powered on and ready for acquisition Voyager Biospectrometry Workstation User s Guide 2 71 Chapter 2 Problem with external laser positioning over 2 72 time Applied Biosystems Installing the Voyager Biospectrometry Workstations If the external laser is left on for more than 15 minutes the position at which the la
476. pendix D Log Sheets Contains blank log sheets that you can copy and use to record sample location Appendix E Grid Voltage and Delay Time Settings Contains graphs that illustrate the relationship between these Delayed Extraction tuning parameters xxviii Applied Biosystems How to Use This Guide Chapter Appendix Content Appendix F Reference Standard Information Includes molecular weights and mass to time conversions for standard calibration compounds Also lists theoretical cleavages and observed fragment masses for angiotensin Appendix G Maintenance Log Includes a log sheet for tracking routine maintenance Appendix H Continuous Extraction Mode Includes information for running the system in Continuous Extraction mode for diagnostic purposes Appendix Using the Oscilloscope and Control Stick Describes the front panel controls of the oscilloscope hardware and how to use the Control stick to move sample position start and stop the laser and start and stop acquisition Related The related documents shipped with your system are documentation Voyager Biospectrometry Workstation Getting Started Guide Use this guide to learn the basics of operating the system It provides step by step information for running your first experiment Data Explorer Software User s Guide Refer to this guide for functions in the Data Explorer software that are
477. play the Advanced page parameters Figure 5 6 Mode Digitizer x Instrument Mode Linear Diaitteer Reflector Digitizer Advanced Reflector Settings Mirror to Accelerating Voltage Ratio fi 07 I Timed lon Selector fo Da Figure 5 6 Mode Digitizer Settings Dialog Box with Advanced Tab Displayed Advanced parameters are described in Table 5 8 Voyager Biospectrometry Workstation User s Guide 5 31 Chapter 5 Optimizing Instrument Settings Table 5 8 Advanced Parameters Parameter Description Mirror to Accelerating Voltage Ratio not available in Linear mode Specifies the ratio between the Mirror Voltage and the Accelerating Voltage in Reflector mode to adjust the voltage of the mirror so that it is slightly higher than the Accelerating Voltage A higher voltage is needed at the mirror to reflect ions If the voltage at the mirror is equal to the Accelerating Voltage ions will pass the mirror To reflect intact ions the Mirror to Accelerating Voltage Ratio must be close to or greater than 1 Maximum value is 30 000V Accelerating Voltage NOTE This parameter influences the tuning characteristics and default calibration of the instrument Do not change this setting unless instructed to do so by an Applied Biosystems Technical Representative Changing this value significantly alters the optimum settings of Grid Voltage and Delay Time For information on the voltage applied to the mirror
478. positional tolerance see below approximately 2 540 um around the X Y coordinates is available for analysis of each position Figure 3 12 on page 3 75 Voyager Biospectrometry Workstation User s Guide 3 73 Chapter 3 Preparing Samples Tolerances and When creating search pattern files make sure to non systematic compensate for errors e Positional tolerance related to variability in the position of the plate Positional Tolerance Plate type pm Reusable plates 76 2 Disposable plates 381 NOTE Due to limited surface area and variability in the position of the disposable inserts do not specify more than 384 positions for a disposable plate e Total allowable tolerance includes the positional tolerance plus any non systematic error introduced by sample application Search pattern Note the following radiuS To ensure complete analysis the radius of the search pattern file used must include the radius of the sample spot e To prevent analysis of the wrong sample the radius of the search pattern used must not exceed half of the center to center spacing minus the total allowable tolerance Figure 3 12 3 74 Applied Biosystems Sample Plate Types Center to center distance a HS Daas ra Total ass SN Logs allowable XY AN y s tolerance coordinate SWA l Le rai LION ot Jf N us A x NS A 23 a ak Radius available for analysis Search patter
479. ppendix B 3 Spare Parts for the Sequazyme Kit part number Prepare standards and matrix in 50 50 acetonitrile 0 1 TFA in deionized water For more information see Section 3 1 Preparing Samples WARNING CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves At least 30 minutes before you perform plate optimization turn on high voltage by clicking a in the Instrument Control Panel toolbar Allow the hign voltage power supplies to warm up NOTE By default high voltage turns off after 60 minutes if the instrument is not used You can change the default in Hardware Configuration See High Voltage Configuration on page 2 38 The warm up period allows maximum mass accuracy during plate optimization by reducing variability in accelerating voltages and yields more reproducible ion flight times Running OptiPlate to Optimize Mass Accuracy PLT file To correctly display results in OptiPlate use a PLT file that specifies a maximum of 900 positions with either of the following patterns Figure 2 26 e A grid with a maximum of 30 rows and 30 columns e An offset grid with a maximum 15 rows and
480. ptimization option for a Plate ID and have not optimized all positions on the plate the software interpolates an Extraction Correction for the coordinates that correspond to an unoptimized position using the correction distances from the closest optimized positions If you select the Use Mass Accuracy Optimization option for a Plate ID and no optimization file exists either the plate has not been optimized or the optimization file has been moved or deleted the most recently created optimization file is used regardless of plate type Although using an optimization from another plate does not yield the same degree of mass accuracy as using an optimization specific for a plate it still yields better mass accuracy than using no optimization Depending on your mass accuracy needs you may or may not choose to optimize all sample plates For best mass accuracy use an optimized sample plate select a PlatelD PLT associated with a specific physical plate you have optimized then load the optimized physical plate in the mass spectrometer However you can also do the following items are listed in order of decreasing mass accuracy Use the same type of plate as a plate you have optimized select a PlatelD PLT associated with a plate you have optimized then load a different physical plate of the same type in the mass spectrometer e Use a different type of plate than a plate you have optimized select the PlatelD PLT associated
481. ptimizing Guide Wire Voltage Voyager Biospectrometry Workstation User s Guide 9 21 Chapter 9 Maintenance and Troubleshooting Table 9 4 PSD Troubleshooting Symptom Possible Cause Action Peak selected with Timed ion selector not Call Applied Biosystems Precursor lon Selector not working due to invalid Technical Support appearing or not Flight Length value or appearing at expected Gate Width value mass single stage mirror only Checking the The Windows NT Event Log is a running list of events that Windows NT automatically starts when you run Windows NT An event is Event Log considered any significant occurrence in the system or application that requires the user to be notified You can use Event Viewer to monitor the events that occur in your system You must be logged in with an account that has administrator rights To display Event Viewer 1 Select Administrative Tools from the Program folder on the Windows Start taskbar 2 Click Event Viewer The Event Viewer System Log displays a running log of the events on your system Critical event messages are displayed on your screen Events that do not require immediate attention are logged in the Event Log to provide information without disturbing your work 3 If desired clear the Event Log e Select Save As from the Log menu and save the event log before clearing e Select Clear All Events from the Log menu 4 Sel
482. ptom Possible Cause Action Flat signal on oscilloscope or in spectrum window for sample region matrix peaks seen Accelerating Voltage too low Adjust See Section 5 4 4 2 Setting Accelerating Voltage Sample does not ionize Analyze in negative ion mode continued Before mixing with matrix chemically derivatize sample with amino containing chemical group Use different matrix See Section 3 1 1 Selecting a Matrix Sample not Prepare new sample making well dissolved before sure sample is well dissolved applying to sample then apply to sample plate plate Sample and matrix Prepare new sample and not well mixed before matrix Mix well before applying to sample applying to sample plate plate Sample Prepare sample matrix with a concentration too final sample concentration high or too low appropriate for sample and matrix See Section 3 1 3 Matrix Information Dilute or concentrate sample 5x and run again continued 9 8 Applied Biosystems Troubleshooting Table 9 1 Spectrum Troubleshooting Continued Symptom Possible Cause Action Flat signal on oscilloscope or in spectrum window for sample and matrix region Vertical scaling needs adjustment Adjust See Section 5 3 5 Understanding Digitizer Settings Spectrum window needs adjustment See Section 4 3 Using the Spectrum Window Laser set to 0 Adjust laser by using the s
483. quence continued 7 16 Applied Biosystems Creating a Sequence Table 7 1 Run List Parameters Continued Field Description Data Explorer SET file to use to detect peaks apply monoisotopic peak SET File filtering if specified and calibrate if specified the data file required entry acquired in the row The SET file contains peak detection for Internal and Parameters and the reference masses needed for calibration Internal Update Click the down arrow and select a SET file calibration If you do not specify a SET file for rows that specify External or no calibration the peak detection settings from the default SET file VOYAGERLINEAR SET or VOYAGERREFLECTOR SET are stored in the data file For information see e Section 7 2 Understanding Settings Macros and Calibration e Creating Processing Settings SET Files on page 7 12 Pre Macro Macro to execute before calibration Select a macro by doing either of the following e Click the field and select the macro name from the list All macros and scripts you created in the Data Explorer software are listed e Type the button number to which you assigned the macro For more information see Creating Macros on page 7 8 Hint To display the macro name assigned to the button display the Data Explorer software then place the cursor over a macro button Macro buttons are numbered sequentially from left to right If the Macro
484. quirement B 1 connecting devices to 2 12 2 16 2 20 hard disk size 1 30 1 41 maintenance 9 6 memory 1 30 1 41 minimum configuration 1 30 1 41 A 4 A 7 A 10 monitor connecting Voyager DE STR 2 28 rear panel Voyager DE and Voyager DE PRO 2 10 Index Computer continued Continuous Extraction continued screen savers do not use 1 30 mass accuracy 1 41 troubleshooting H 23 shutting down 2 75 optimizing parameters H 5 technical support for altered resolution H 17 configuration B 1 resolution compared to delayed troubleshooting 9 6 mode 1 13 with Signatec 500 MHz resolution troubleshooting H 21 digitizer 2 11 setting mode 5 25 with Tektronix oscilloscope 2 11 standard Instrument Settings H 4 Concentration see Sample standard instrument settings H 3 concentration troubleshooting H 21 H 23 Conditions environmental A 4 A 7 when to use 1 12 A 10 Control buttons 4 3 4 24 Configuring Control Mode Deflector Gate Width 8 30 description 4 2 4 6 digitizer 2 44 overview 6 2 hardware 2 35 status 4 5 high voltage 2 38 Control pages instrument 2 40 displaying 4 8 instrument type 2 41 docked 4 9 laser 2 42 floating 4 9 Precursor lon Selector 8 30 Instrument Settings 5 9 5 15 sample plate alignment 2 46 layout changing 4 8 Timed lon Selector 2 39 8 30 layout default 4 8 vacuum 2 35 Manual Laser Sample Continuous Extraction Positioning 4 27 see also Instrument Settings overview 4 4 parameters PSD Acquisition settings acceptable
485. r Internal External Calibration Type Internal Update calibration on page 7 18 Continued Voyager Biospectrometry Workstation User s Guide 7 3 Chapter 7 Acquiring Spectra from the Sequence Control Panel File Type Description For information see CAL Contain calibration constants used e Creating Calibration according to specified CAL Files on Calibration Type page 7 11 e External calibration Constants e Calibration File on are applied and saved in a DAT page 7 19 file e Internal calibration CAL optional lf a CAL file is specified and it exists constants are applied to the DAT file before the reference masses in the SET file are matched New calibration constants are generated using the reference masses specified in the SET file and applied to the data Constants are then updated within the DAT file after calibration Internal Update calibration lf a CAL file is specified and it exists constants are applied to the DAT file before the reference masses in the SET file are matched New calibration constants are generated using the reference masses specified in the SET file and applied to the data Constants are then updated within the DAT file after calibration The specified CAL file is updated or created and contains new calibration constants after calibration NOTE If the DAT file contains multiple spectra generated using the Sav
486. r of segments you need to acquire is determined by e Resolution you require e Mass range of interest You see better resolution in the composite spectrum with more segments NOTE If you are interested in selected fragment masses only instead of a complete composite spectrum see Acquiring only select segments on page 8 42 Use the following equation to determine the number of segments to acquire n In m Mp InD where m Fragment ion mass of the lowest mass of interest D Decrement Ratio which determines the size of the segment n Number of segments m Precursor ion mass Acquiring PSD Data with Standard BIC Files in Manual Control Mode Decrement ratio Default Mirror Ratio settings Hint You can use the Windows calculator to determine natural log values To open the calculator select Programs from the Windows Start menu select Accessories then select Calculator If the In function is not displayed when you open the Windows calculator select Scientific from the View menu in the calculator to access advanced functions Hint The Angiotensin_PSD BIC file provided with the software includes 10 segments which is suitable for many applications If the mass you are analyzing differs by more than 300 Da from the mass in the Angiotensin_PSD BIC file 1296 Da you may need to acquire more or fewer than 10 segments to observe the fragment ions of interest The software can automatically c
487. r settings click drag the slider bar on the Manual Laser Intensity Sample Positioning control page For more information see Manually adjusting laser intensity on page 4 28 Adjusting laser intensity depends on the following If you are using Then A BIC file for the same mass Laser intensity may require only minor range and matrix adjustments A BIC file for the same mass range Increase or decrease the laser in 50 to but different matrix 100 step increments or decrements following the strategy described below For more information see Selecting a BIC file on page 5 8 and Laser intensity and matrix on page 5 67 Default Instrument Control Panel This setting is typically too high for many settings The mid range default laser samples and matrix Decrease the laser in 50 setting if a BIC file is not loaded is to 100 step decrements following the strategy approximately 1 800 described below For more information see Laser intensity and matrix on page 5 67 General strategy Asa general strategy for adjusting laser intensity e Start at the default setting for your system approximately 1 800 or the setting in the loaded BIC file Ifthe spectrum you obtain is not acceptable increase or decrease the laser in 50 to 100 step increments or decrements and reacquire For more information on acceptable laser signal see Signal saturation on page 5 69 5 6
488. ragment Mass Designation Designation monoisotopic average monoisotopic average 272 136 272 3 b2 1000 537 1001 2 a8 285 135 285 3 FH 1046 542 1047 2 b8 H 0 326 183 326 4 a3 17 1068 6 1069 2 354 178 354 4 b3 17 1137 596 1138 3 aQ 364 2 364 4 1165 591 1166 3 b9 371 204 371 4 b3 1181 658 1182 3 y9 382 188 382 4 PFH 1183 601 1184 3 b9 H20 400 2 400 5 1296 685 1297 5 MH 416 230 416 5 y3 F 8 Applied Biosystems G Maintenance Log The following page includes a log sheet listing maintenance procedures Copy this page and keep it by your Voyager system Instructions for performing maintenance procedures are listed in Chapter 9 Maintenance and Troubleshooting Voyager Biospectrometry Workstation User s Guide G 1 Maintenance Log for Voyager System Serial Number Record the date and your initials when you perform maintenance procedures Weeky sue up and archive data nw maintenance performed by Applied Biosystems engineer H Continuous Extraction Mode This appendix contains the following sections H 1 Optimizing a Continuous Extraction Standard Instrument BIC Setting H 2 H 2 Obtaining Good Spectra in Continuous Extraction Mode H 8 H 3 Troubleshooting in Continuous Extraction Mode H 19 Voyager Biospectrometry Workstation User s Guide H 1 Appendix H Cont
489. rating Voltage appropriate for the mass range you are analyzing Mass Range Da Accelerating Voltage V lt 1 000 15 000 1 000 to 10 000 20 000 gt 10 000 25 000 If you are analyzing very dilute samples or a sample that ionizes poorly use a setting of 25 000 V Voyager Biospectrometry Workstation User s Guide 5 87 Chapter 5 Optimizing Instrument Settings A lower Accelerating Voltage setting does the following e Provides more data points across a peak for better peak definition when analyzing low mass ions Linear mode only Improves resolution when analyzing compounds below 2 000 Da and the resolution is limited by the digitization rate of the system Reflector mode 5 4 4 3 Setting Guide Wire Voltage 5 88 For more information Applied Biosystems NOTE Guide Wire Voltage is not available on Voyager DE STR models with serial number 4154 and later To optimize sensitivity you can adjust the Guide Wire Voltage e Linear mode Increase in increments of 0 01 percent until you see signal to noise ratio start to decrease e Reflector mode Increase in increments of 0 002 percent Settings above 0 05 percent do not typically improve signal to noise ratio NOTE In Reflector mode Guide Wire Voltage also affects mass resolution To fine tune signal to noise ratio and resolution at the same time adjust the Guide Wire Voltage in increments smaller than 0 01 pe
490. ration For more information see the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating Setting PSD 1 Open the Angiotensin_PSD BIC file provided with the Acquisition software This is a PSD mode BIC file parameters gt in the Calibration section of the Instrument Settings control page select e The matrix you are using External File then select the PRECURSOR_UNKNOWN CAL file you created for the unknown in Generating an external calibration for the unknown precursor ion on page 8 15 3 Inthe PSD Acquisition Settings control page e Type the accurate Precursor mass e Inthe Calibration section select External File then select the he ANGIO PSD CAL file you created in Generating a PSD calibration on page 8 13 4 Select Save Instrument Settings As from the File menu then save the instrument setting file with a new name 8 16 Applied Biosystems PSD Quick Start Acquiring PSD 1 Inthe Manual Laser Intensity Sample Positioning segments control page select the same sample position from which you acquired the precursor spectrum 2 From the View menu select Data Storage Set parameters as needed See Setting Data Storage parameters on page 6 14 for information Hint Include a_PSD suffix when you name PSD data files to help you distinguish them from non PSD data files For example if you type in Experiment1_PSD as the file name the complete data file name will be
491. rcent For more information see e Section 5 4 3 3 Optimizing Guide Wire Voltage e Section 5 3 4 Understanding Guide Wire Voltage Optimizing Instrument Settings Parameters 5 4 4 4 Setting Shots Spectrum NOTE This parameter is dimmed if the system is set to Single Shot mode For information see page 2 44 Overview A higher number of Shots Spectrum can improve signal to noise ratio and also improve the dynamic range of the acquisition However acquiring a higher number of Shots Spectrum increases acquisition time When random noise is present in a spectrum the improvement in signal to noise ratio is approximately proportional to the square root of the number of Shots Spectrum taken Manually The maximum number of Shots Spectrum is determined by accumulating to the digitizer on your system see Shots Spectrum on override limit Page 5 19 You can acquire a greater number of Shots Spectrum than the maximum number of Shots Spectrum noted above by manually accumulating spectra For more information see Section 6 2 2 Manually Accumulating Spectra from Multiple Acquisitions 5 4 4 5 Setting Low Mass Gate Description Low Mass Gate suppresses matrix peaks that can interfere with ion detection which can saturate the detector as laser intensity increases This saturation effect is less pronounced on systems with Linear High Current Detectors For serial numbers of systems that include High Current Detectors see
492. rds 3 24 Applied Biosystems e Many samples adhere strongly to plastic tubes and pipet tips You can minimize sample loss by preparing samples in 30 percent acetonitrile with 5 to 10 percent TFA e Do not dilute sample with phosphate buffered saline PBS or other buffer solutions A high salt concentration can interfere with sample ionization and may cause increased sodium and potassium adduct peaks Prepare samples in water If samples contain organic solvents they will dissolve the matrix applied to the sample plate If you require mass accuracy greater than the accuracy provided by external calibration for example gt 0 05 on a Voyager DE system use an internal standard See Appendix A Specifications for the mass accuracy on other systems Add two standards of known molecular weight to the sample Standards should e Closely bracket the molecular weight of your unknown e Be easily distinguished from the unknown As a starting point use an internal standard concentration in the same range as your sample concentration Because an internal standard may affect the intensity of the sample signal it is a good idea to prepare sample with several different internal standard concentrations Preparing Samples 3 1 5 Sample Cleanup Cleaning Use the following techniques to clean samples techniques Washing e Drop dialysis floating membrane e Cation exchange e ZipTips When to clean Sample cleanup is needed if sampl
493. re creating PLT 3 77 search pattern file for 3 73 selecting 3 56 3 59 SymBiot plates creating for 3 84 x y coordinates determining 3 83 PLT files creating editing 3 83 new 3 76 non grid positions 3 83 using Create PLT File 3 79 using Notepad 3 83 Plumbing CID 8 31 Index 28 Applied Biosystems Polarity setting displayed in status bar 4 5 setting Positive or Negative 5 25 Switch Delay in configuration 2 38 Polymers matrix for 3 3 methods for sample loading 3 21 nonpolar synthetic matrix for C 10 polar synthetic matrix for C 7 C 10 sample plate to use 3 62 Porcine Trypsin molecular weight F 2 Position number including in data file name 7 16 7 46 Positional tolerance of sample plates 3 74 Positive ion mode setting 5 25 Switch Delay Time 2 38 Post source decay see PSD Potassium adduct ion effect on masses 9 17 from buffer 3 24 Potential field gradient definition 5 51 increasing 5 51 maximum allowed 5 53 Power requirements Voyager DE and Voyager DE PRO 2 3 Voyager DE STR 2 7 Powering up mass spectrometer 2 73 system components 2 73 Precursor lon Selector configuring 2 39 8 30 disabling to troubleshoot 8 29 8 45 effect of 8 69 8 76 enabling 8 45 8 60 function 8 20 optimizing 8 29 Precursor lon Selector continued resolution 8 29 A 5 A 9 screening out prompt fragments 8 69 see also Timed lon Selector setting tracks Timed lon Selector 8 44 width too small 8 29 Precursor mass determining in Refle
494. re 1 5 Time of Flight Analysis The time required for ions to reach the detector at the opposite end of the flight tube drift time is measured The number of ions reaching the detector at any given time is also measured and is referred to as ion intensity abundance or signal intensity Voyager Biospectrometry Workstation User s Guide 1 9 Chapter 1 Introducing the Voyager Biospectrometry Workstations Drift time is proportional to the square root of the mass as defined by the following equation m 2 2KE z where drift time drift distance mass kinetic energy number of charges on ion NX30 aS Approximate ion mass is determined using the equation above A calibration procedure using a reference standard of known mass can be used to establish a more accurate relationship between flight time and the mass to charge ratio of the ion Advantages of Advantages of MALDI TOF technology include MALDI TOF Can analyze a theoretically unlimited mass range greater than 300 000 daltons Da Mass range is limited by ionization ability not analyzer physics e Can obtain complete mass spectrum from a single ionization event This is also referred to as multiplexing or parallel versus serial detection e Is compatible with buffers normally used in biological assays reducing the need for sample cleanup e Can analyze mixtures and different classes of biopolymers including peptides oligonu
495. rease the laser intensity and acquire a new spectrum If the signal decreases at the higher Delay Time setting increase the laser and acquire a new spectrum Hint If you are analyzing a mixture and the resolution of the peak of interest is not acceptable observe the resolution of a higher mass peak and a lower mass peak If resolution is better on the higher mass peak decrease the Delay Time by 100 nsec for Linear mode or 50 nsec for Reflector mode If resolution is better on the lower mass peak increase the Delay Time by 100 nsec for Linear mode or 50 nsec for Reflector mode Collect a spectrum and observe Leave the Grid Voltage setting unchanged 5 78 Applied Biosystems 3 Optimizing Instrument Settings Parameters If the resolution improves by at least 20 percent 10 percent continue increasing the Delay Time in 100 nsec for Linear mode or 50 nsec for Reflector mode increments Table 5 12 through Table 5 14 list valid Delay Time settings for different systems and mass ranges If the resolution does not improve decrease the Delay Time by 100 nsec for Linear mode or 50 nsec for Reflector mode acquire a new spectrum and observe Table 5 12 Voyager DE Voyager DE PRO and Voyager DE STR Linear Mode Delay Time and Grid Voltage Values Linear Mode Mass Range Da Delay Time Grid nsec Voltage 500 2 000 50 150 90 95 2 000 10 000 50 400 90 95 10 000 20 000 200 500
496. region used to accelerate ions Includes e Sample plate and sample stage An area supplied with voltage 0 to 25 000 V for acceleration of ions into the flight tube e Variable voltage grid A grid supplied with additional voltage to fine tune ion acceleration e Ground grid Ground surface for formation of potential gradient e Collision cell Provided with CID Collision Induced Dissociation option for enhanced fragmentation in PSD analysis e Grounded aperture Entrance to flight tube For more information on the ion source and voltages see Section 5 4 4 2 Setting Accelerating Voltage and Section 5 4 3 5 Optimizing Grid Voltage 1 34 Applied Biosystems Parts of the Voyager DE STR System Video camera A camera that displays a real time sample image 100 times magnification on the video monitor Vacuum system A pumping system and a sealed enclosure that creates and maintains a high vacuum environment for unobstructed ion drift For more information see Section 1 6 3 Vacuum System Flight tube and ion focusing lens A field free region no additional accelerating forces are present in which ions drift at a velocity inversely proportional to the square root of their masses The fixed voltage 50 percent of the Accelerating Voltage applied to the ion focusing lens refocuses ions on the detector NOTE Voyager DE STR models with serial number 4153 and earlier include a beam guide wire instead of a
497. registered trademarks and Voyager Biospectrometry Data Explorer and Sequazyme are trademarks of PE Corporation or its subsidiaries in the U S and certain other countries Microsoft MS and Windows NT are registered trademarks of Microsoft Corporation Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated IBM is a registered trademark of International Business Machines LeCroy is a registered trademark of LeCroy Corporation ZipTip and Milli Q are registered trademarks of Millipore Corporation Pentium is a registered trademark of Intel Corporation Biacore is a registered trademark of Biacore AB LIQUI NOX is a trademark of Alconox Inc PhastGel is a trademark of Pharmacia Teflon is a registered trademark of E I Du Pont de Nemours and Co Trans Blot is a registered trademark of Bio Rad Laboratories Acqiris is a registered trademark of Acairis SA Tektronix is a registered trademark of Tektronix Inc Voyager products and their use are covered by one or more of the following US Patents 5 288 644 5 643 798 5 453 247 5 625 184 5 627 369 5 760 393 5 885 775 5 827 659 5 821 063 Additional US and foreign patents are pending Part Number 4317707 Rev A 08 2000 Printed on recycled paper Table of Contents Table of Contents Safety and Compliance Information xV How to Use This Guide xxix Chapter 1 Introducing the Voyager Biospectrometry 1 1
498. rence from other compounds Specificity of ion selection is determined by the resolution of the Precursor lon Selector also called Timed lon Selector on your system See Appendix A Specifications for more information PSD fragment ions Focusing fragment ions with Mirror Ratio setting Overview of PSD Analysis At higher laser intensities some molecular ions decompose into PSD fragment ions in the flight tube after they leave the ion source the post source decay process Before fragmentation the intact molecular ion travels with a kinetic energy of KE 1 2 mv where KE kinetic energy accelerating voltage m mass v velocity After fragmentation the fragment ions continue travelling with the same velocity as the original ion but with reduced kinetic energy Fragment ions travel with the original precursor ion until they reach the reflector where they separate from the original ion and behave as though they received less initial acceleration Therefore PSD fragment ions are not correctly focused and appear at a mass higher than the expected mass After fragmentation the fragment ion travels with a kinetic energy of m KE tragment ion KE original ion RUE original ion where KE kinetic energy accelerating voltage m mass Consider an ion M H fragmenting during flight into two fragments A and B of lower mass than the original ion Both of the following reactions occur MHt
499. reset to the 0 to 4 000 range for the external laser Laser Rate NOTE This parameter is available only on systems with the appropriate hardware For more information contact Applied Biosystems Specifies the laser firing rate e Default The default firing rate 3 Hz e Optimized Fastest possible firing rate for e Digitizer used on the system e Mass Range described on page 5 19 e Bin Size setting described on page 5 29 Acqiris digitizers allow a laser firing rate of 20 Hz and are independent of Mass Range and Bin Size settings To operate at the fastest laser firing rate when using other digitizers increase the Bin Size or decrease the Mass Range These parameters affect the Number of Data Points Digitized which in turn affects the laser firing rate If you switch to faster optimized laser firing rates you may need to increase or decrease the laser intensity Laser firing rate does not affect resolution For optimum mass accuracy use the same laser setting optimized or default for calibrants and unknowns 5 26 Applied Biosystems continued Instrument Settings Parameter Descriptions Table 5 6 Instrument Mode Parameters Continued Parameter Description Laser Rate NOTE Laser firing rate does not have a linear relationship to Bin Size For example if an acquisition of 100 Shots Spectrum takes 5 seconds with a Bin Size of 2 nsec an acquisition of 100 Shots Spectrum may not
500. ria is selected or if the laser can no longer be adjusted the system begins a new acquisition from the next search pattern position depending on the Spectrum Accumulation conditions and Spectrum Acceptance Criteria Acceptance criteria are defined in Setting spectrum acceptance and laser adjustment criteria on page 6 42 e If collection mode is set to Save First Passing and an acceptable spectrum is collected the system saves the data file and the run is complete e If acceptance criteria are not met or collection mode is set to Save All Save All Passing or Save Best the acquisition sequence is repeated at the next position specified in the random search pattern or the search pattern file 7 Ifthe system moves across an entire sample position and does not find an acceptable signal it e Logs the error in the Automatic Control tab in the Output window of the Instrument Control Panel Does not create a data file 6 64 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel 6 6 7 Process that Occurs when Accumulating Spectra from Multiple Search Pattern Positions NOTE These processes occur when you acquire using a BIC file that has Use Automated Sample Positioning enabled You can obtain a single spectrum from multiple positions within a single sample position by specifying a search pattern To do so enable the Automatic Control mode in Instrument Settings control page and
501. right and top to bottom across the plate With units in microns To create PLT files that specify staggered positions for example the 384 well plate shown in Figure 3 9 on page 3 66 or positions that are not evenly spaced or to edit an existing PLT file see Creating or editing a PLT file using Notepad editor on page 3 83 Using Create PLT To create a PLT file using Create PLT File File 1 Perform the procedure in Preparing the sample plate on page 3 76 2 Use the Manual Laser Sample Positioning control page see Figure 3 14 on page 3 78 or the control stick to determine the center Absolute X and Y coordinates for the first and last positions on the plate for which you are creating the PLT file see Figure 3 13 on page 3 77 3 Inthe Instrument Control Panel select Create PLT File from the Tools menu to display the Create PLT File dialog box Figure 3 15 Voyager Biospectrometry Workstation User s Guide 3 79 Chapter 3 Preparing Samples Create PLT File x Number of columns 10 Number of rows LUE Position width 2540 microns Position height 2540 microns Position depth mm r Position Shape Position Numbering Ellipse Numeric Rectangle Alphanumeric r First Position upper left Absolute 15875 microns Absolute Y 473075 microns Last Position lower igh j Absolute x 473075 microns Absolute Y 15875 microns m Plate File PLT pe Note
502. ring Samples 3 1 5 4 ZipTips When to use What you need Use this technique for peptides proteins and oligonucleotides when you know the sample contains salt buffer or glycerol contaminants This technique is faster easier and more effective than dialysis for removing contaminants NOTE This technique introduces organic solvent into the sample which is not compatible with thin layer sample application If you will apply sample using the thin layer application technique remove the organic solvent from the sample before loading sample on the plate For more information see Section 3 2 4 Loading Samples Thin Layer Application Millipore ZipTips can be used for a wide range of applications and are compatible with digestion procedures You can expect 50 to 70 percent recovery using the following procedure NOTE ZipTips can also be used for sample concentration and fractionation of complex mixtures To clean samples with ZipTips you need e Micro adsorptive C8 pipette ZipTips e Acetonitrile ACN e TFA WARNING CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Voyager Biospectrometry Workstation User s Guide 3 31 Chap
503. ritation Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Matrix concentration 50 mg ml Additive concentration 50 mg ml diammonium citrate in deionized water Final sample concentration 1 10 pmol ul Solvents 50 percent acetonitrile and deionized water NOTE HPLC grade water may vary in salt concentration Do not use for oligonucleotide analysis Preparation Follow the procedure in Preparing matrix on page 3 5 and combine 8 1 3 HPA diammonium citrate Air dry sample plate after loading sample and matrix Crystals Needle like crystals inside a ring see Figure 3 2 on page 3 46 Stability Prepare weekly Voyager Biospectrometry Workstation User s Guide 3 15 Chapter 3 Preparing Samples DHB Use DHB for e Neutral carbohydrates e Small molecules DHB for neutral carbohydrates WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Table 3 6 DHB Matrix Information for Neutral Carbohydrates Matrix concentration 10 mg ml Final sample concentration 10 pmol ul Solvents Deionized water Preparation Follow the procedure in Preparing matrix on page 3 5 D
504. riteria Evaluation Mass Range From To 0 Da 2000 Da r Signal to Noise Baseline Mass Range From To Cancel Figure 6 16 Spectrum Acceptance Criteria Dialog Box 2 Set Spectrum acceptance and Laser adjustment criteria parameters as described in Table 6 3 Table 6 3 Acquiring in Automatic Mode from the Instrument Control Panel Spectrum Acceptance Criteria Parameters Parameter Description Acceptance Criteria Minimum Signal Intensity Used for spectrum acceptance and laser adjustment Sets the minimum signal intensity accepted for the most abundant peak local base peak within the mass range of interest Also used to determine laser adjustment When the system adjusts the laser it checks that the signal intensity is above the minimum specified If signal intensity is not above the minimum the system increases the laser intensity If signal intensity is not above the minimum when the laser is at the highest setting specified in Automatic Control the system moves to the next search pattern position Maximum Signal Intensity Used for spectrum acceptance and laser adjustment Sets the maximum signal intensity for the most abundant peak local base peak within the mass range of interest Also used to determine laser adjustment When the system adjusts the laser it checks that the signal intensity is below the maximum specified If signal intensity is not below the m
505. rol mode parameters Acquiring data Laser Intensity Adjustment Criteria Spectrum Accumulation Criteria accumulating spectra 6 65 accumulation conditions 6 40 Acquisition mode 6 61 Automatic Control tab 4 5 6 45 before you begin 6 36 checking disk space 6 58 enabling 6 38 evaluating data 6 42 laser intensity adjustment criteria 6 42 laser intensity setting 6 39 laser not aligned with sample position 2 48 9 23 number of spectra to acquire 6 40 Index 4 Applied Biosystems Automatic Control mode continued optimizing BIC for Sequence run 7 7 optimizing BIC in Manual Control mode before using 6 37 overview 4 6 6 3 Prescan mode description 6 58 process that occurs during acquisition 6 58 PSD analysis 8 57 resolution filtering 6 44 resolution filtering peak height used 6 44 sample plate aligning 2 46 6 36 sample plate required 3 41 sample position selecting 6 41 saving conditions 6 40 search pattern SP file 6 46 signal to noise filtering 6 43 spectrum acceptance criteria 6 42 Automatic Control mode parameters accessing 5 16 5 33 Automated Laser Intensity Adjustment 5 34 Automated Sample Positioning 5 37 Laser Intensity Adjustment Criteria see Laser Intensity Adjustment Criteria Minimum and Maximum Laser Intensity 5 34 Number of spectra to acquire 5 35 Prescan 5 34 random search pattern 5 37 Search Pattern file 5 38 Spectrum Acceptance Criteria see Spectrum Acceptance Criteria S
506. ropanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache and so on Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To wash 1 Apply sample and matrix to the sample plate and allow to dry 2 Add 1 to 3 ul of solvent to the dry sample matrix 3 Wait 10 seconds then remove the liquid NOTE Try not to touch the sample plate with the pipet tip If you do touch the spot do not analyze the area you touched Preparing Samples Repeat step 2 and step 3 one more time Allow the sample plate to dry before analyzing If one wash does not improve results you can wash again 3 1 5 2 Drop Dialysis Floating Membrane When to use What you need Procedure Use this technique on polar compounds when you know contaminants are of low molecular weight This technique works well for DNA and polar proteins such as glycoproteins For drop dialysis you need a membrane To perform drop dialysis 1 With a pore size of 0 025 um or smaller That does not adhere to your sample Fill a small container for example a pipet tip box with about an inch of deionized water Place the container on a stable surface Use forceps to place the membrane in th
507. rs unless instructed to do so by an Applied Biosystems Technical Representative Altering these settings may cause your Voyager Biospectrometry Workstation to function improperly 4 Click OK to exit 2 6 4 Instrument Configuration 2 40 Applied Biosystems To check the instrument configuration 1 In the Instrument Control Panel select Hardware Configuration from the Instrument menu Click the Instrument tab to display the Instrument page Figure 2 18 Hardware Configuration Hardware Configuration xi Vacuum Control Stick Laser Timed lon Selector High Voltage Instrument Digitizer m Type Voyager DE PRO Delayed Extraction R Installed Information Laboratory Name FE Biosystems Instrument Name Mai TOF Serial Number fi 000 Figure 2 18 Instrument Configuration 3 Check the following fields as needed _ Instrument type Read only Displays your instrument type e Delayed Extraction Read only Reflects whether your system has delayed extraction hardware installed e Laboratory Name You can edit this field to display your laboratory name Names listed in this field are included in DAT files and on printouts Instrument Name You can edit this field to display your instrument name Names listed in this field are included in DAT files and on printouts e Serial Number Displays the serial number for the connected instrument 4 Click OK to exit
508. rts of the Voyager DE and Voyager DE PRO Systems Two vacuum pumps create the vacuum environment e Fore pump Creates a vacuum in the sample loading chamber creates a lower than atmospheric pressure condition before the turbo pump starts and provides backing pressure to the turbo pump Turbo pump Creates a high vacuum condition in the main source chamber Vacuum is maintained in the chambers by valves that isolate the chambers The Voyager DE Biospectrometry Workstation includes two vacuum gauges e BA1 Bayard Alpert Gauge Monitors pressure in the main source chamber e TC2 Monitors pressure in the sample chamber Readings from the vacuum gauges are displayed in the System Status Control page in the Instrument Control Panel See Section 2 11 Checking System Status and Pressures Voyager Biospectrometry Workstation User s Guide 1 27 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 5 3 2 Voyager DE PRO Vacuum System Vacuum The Voyager DE PRO Biospectrometry Workstation includes chambers three vacuum chambers Figure 1 16 Main source chamber e Sample loading chamber Mirror chamber E Mirror E chamber Turbo high vacuum pump 2 LI Foreline valve 2 Foreline valve 1 1 Main Turbo source chamber pump 1 m high vacuum Sample loading chamber low vacuum Figure 1 16 Voyager DE PRO Biospectrometry Works
509. rum ActiveDocument SpecSetup DeisotopeBaseFormula C6H5NO processing ActiveDocument Spec View DeisotopeSpectrum here ActiveDocument SpecView PrintTrace Next J End Sub Figure 7 4 Example Macro to Deisotope and Print a Data File Containing Multiple Voyager Spectra 7 3 3 Creating Calibration CAL Files Create calibration CAL files in the Data Explorer software if you plan to specify calibration in the Sequence Control Panel e External calibration CAL file required Internal calibration CAL file optional e Internal Update calibration CAL file name required but CAL file does not have to exist If the specified CAL file exists constants are applied before the reference masses in the SET file are matched If the specified CAL file does not exist no constants are applied New calibration constants are generated using the reference masses specified in the SET file and applied to the data file Constants are updated within the DAT file The specified CAL file is updated or created and contains new calibration constants after calibration For information see the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating Voyager Biospectrometry Workstation User s Guide 7 11 Chapter 7 Acquiring Spectra from the Sequence Control Panel 7 3 4 Creating Processing Settings SET Files 7 12 Overview Processing settings only For more information Applied Biosystems Crea
510. rum resolution 8 25 number to acquire 8 25 8 40 optimum resolution observed near Max Stitch Mass 8 23 8 42 8 49 reacquiring 8 56 saving 8 55 segment list defaults 8 47 selecting for acquisition 8 54 8 63 size and Decrement Ratio correlation 8 41 size collecting different 8 42 size default 8 42 Pulse width laser 1 22 1 34 Pump see Fore pump see Turbo pump Pumping down time required to reach pressure after venting 2 77 Purifying sample see Sample cleanup Q Quality of data 6 6 R RA number B 5 Random search pattern 5 37 Range display see Display Range Read only instrument setting files provided 5 3 setting attributes 5 13 Index 32 Applied Biosystems Rear panel computer Voyager DE and Voyager DE PRO 2 10 mass spectrometer Voyager DE STR 2 23 Reflector benefits of single stage 1 24 1 36 description 1 24 1 36 detector description 1 24 1 36 function 1 24 1 36 Reflector mode BIC files 5 5 cannot see ions in H 20 improving resolution 5 88 path length Voyager DE PRO 1 4 path length Voyager DE STR 1 6 setting 5 25 Related documents xxix Removing traces 4 15 Repetition firing rate laser 5 26 Resolution mass and laser threshold in Continuous Extraction mode H 11 and signal to noise ratio H 8 automatic spectrum evaluation 6 44 calculating during acquisition 6 30 checking in Continuous Extraction mode H 17 comparison between delayed and continuous modes 1 13 filtering during acquisition 6 44
511. rument Settings BIC Files Provided If a BIC file for the mass range you are analyzing does not exist open a standard BIC file with the closest higher mass Hint To optimize a wide mass range select a BIC file with the mass of the highest component in the range Save the BIC file under a new name Standard BIC files are read only and cannot be saved Loading Modifying and Saving Instrument Settings Modifying for To modify the instrument settings file for Manual Control Manual Control mode mode 1 Ifthe Instrument Settings control page Figure 5 2 is not displayed select Instrument Settings from the View menu Instrument Settinas x m Instrument Mode Reflector Positive Mode Digitizer m Control Mode Manual Automatic Automate Bontral m Voltages Accelerating 20000 Y Grid s x Guide Wire fo 005 x Delay Time 180 nsecs m Spectrum Acquisition Shots Spectrum 20 Mass Range Da jo to 5000 M Low Mass Gate Da 200 r Calibration Matrix 2 Cyano 4 hydroxycinnamic acid 7 Default C Extemal File al Figure 5 2 Instrument Settings Control Page 2 Click Mode Digitizer to select settings For parameter descriptions see Linear Reflector Digitizer parameters on page 5 28 Voyager Biospectrometry Workstation User s Guide 5 9 Chapter 5 Optimizing Instrument Settings 5 10 Optimizing Modifying for Aut
512. ry down quickly under vacuum for even response If you allow to air dry you will see uneven response during analysis Crystals Milky amorphous appearance for promoting cationization see Figure 3 3 on page 3 46 Difficult to see crystals when vacuum dried Stability Prepare weekly 3 16 Applied Biosystems DHB for small molecules Table 3 7 DHB Matrix Information for Small Molecules Preparing Samples WARNING CHEMICAL HAZARD Please read the MSDS before handling any chemical mentioned below and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Methanol is a flammable liquid and vapor Exposure may cause eye skin and respiratory tract irritation and central nervous system depression and blindness Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness and headache and so on Matrix concentration 10 mg ml Final sample concentration Highly sample dependent Ideally a minimum of 10 200 pmol l 10 20 ng l With lower concentrations sample signal may be difficult to distinguish from matrix signal Solvents Any solvent in which molecules are soluble deionized water to 100 methanol or acetone Preparation Follow the procedure in Preparing matr
513. ry tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache and so on Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin Exposure may cause central nervous system depression and liver damage Table 3 3 Sinapinic Acid Matrix Information for Thin Layer Application Matrix concentration 20 mg ml Final sample concentration Up to 0 1 pmol ul Materials and solvents e Pure nitrocellulose for example Bio Rad Laboratories Trans Blot 162 0146 e Acetone e Isopropanol continued Voyager Biospectrometry Workstation User s Guide 3 11 Chapter 3 Preparing Samples Table 3 3 Sinapinic Acid Matrix Information for Thin Layer Application Continued Preparation 1 Dissolve nitrocellulose to a concentration of 20 mg ml in acetone by vortexing for 15 minutes 2 Add isopropanol at a ratio of 1 1 3 Add a cyano 4 hydroxycinnamic acid to a final concentration of 20 mg ml Air dry sample plate after loading matrix Dry with gentle air flow after adding sample NOTE If the dry matrix is a mustard yellow color instead of bright yellow it may contain impurities To recrystallize purify dissolve the a cyano matrix in warm ethanol Filter and add about two volumes of deionized w
514. s For a description of parameters contained on the control pages see Section 5 2 Instrument Settings Parameter Descriptions and Chapter 8 PSD Analysis 4 4 Applied Biosystems Instrument Control Panel Spectrum window The Spectrum window provides a display of data The data Status bar displayed depends on your digitizer option e Signatec LeCroy or Acqiris digitizers Displays a live real time spectrum trace as data is acquired Trace changes from Live to Current when acquisition ends Tektronix oscilloscope No trace displayed during acquisition Displays a Current spectrum trace when acquisition is complete When acauisition is complete peaks can be detected and labeled For more information see Section 4 3 Using the Spectrum Window The status bar Figure 4 2 at the bottom of the Instrument Control Panel displays status for Instrument State High Voltage Source BA1 and Mirror BA2 Chamber Pressure Acquisition status Control Mode set in the loaded BIC Instrument Mode and lon Polarity set in the loaded BIC the Active Position selected for analysis and the current laser intensity SeN High Vokage OFF SANSE ME Acquistion OFF a Output window Control Mode MANUAL Instrument Mode LIN POS Active Wel 31 Laser1800 Figure 4 2 Status Bar The Output window includes two tabs at the bottom that display information Data Storage Displays the file name of the
515. s larger Bin Size settings typically yield a lower Number of Data Points Digitized The Bin Size settings available on your system depend on the sampling rate of your digitizer Default is 2 nsec For more information see Section 5 3 5 Understanding Digitizer Settings Number of Data Displays the number of data points that the digitizer will Points Digitized sample based on Bin Size setting and Mass Range which has a relative impact on laser firing rate A higher number of data points results in a lower laser firing rate Number of Data Points Digitized is dependent on Bin Size Setting larger Bin Size settings typically yield a lower Number of Data Points Digitized NOTE On LeCroy digitizers a larger Bin Size setting can yield a larger Number of Data Points Digitized caused by hardware control restrictions However the actual number of data points saved in the data file will be smaller in proportion to the larger Bin Size setting as described in Effects of adjusting Bin Size on page 5 59 For more information see Section 5 3 5 Understanding Digitizer Settings NOTE The relationship between Number of Data Digitized and the laser firing rate is not linear The actual number of data points saved in the data file may be less than the Number of Data Points Digitized depends on your digitizer continued Voyager Biospectrometry Workstation User s Guide 5 29 Chapter 5 Optimizing Instrume
516. s Centroid is described in the Data Explorer Software User s Guide 6 6 Applied Biosystems 6 1 3 Calibrating the Mass Scale This section describes e Types of calibration When to calibrate e Calibration equations e Default calibration e Generated calibration e Acquiring calibration standards Before You Begin Types of The Voyager software includes a default calibration routine that provides adequate mass accuracy for many applications calibration If you require optimum mass accuracy you can generate a calibration based on the observed time of flight of the known masses of calibration standards The Calibration function in the Voyager system allows you to generate four types of calibration Default calibration Provides typically 0 1 accuracy in Linear mode and typically 0 01 or better accuracy in Reflector mode External calibration Provides 0 05 accuracy in Linear mode and 0 01 or better accuracy in Reflector mode To perform external calibration you create a calibration CAL file in the Data Explorer software using standards of known mass You then specify the file in the Instrument Control Panel when acquiring data or apply the file to existing data in the Data Explorer software Internal calibration Provides accuracy of 0 02 or better in Linear mode and 0 002 20 ppm in Reflector mode See Appendix A Specifications for the internal calibration mass accuracy specification for your
517. s Use double deionized water such as Milli Q grade 18 mQ which is appropriate for most applications WARNING CHEMICAL HAZARD Refer to the Material Safety Data Sheet MSDS provided by the chemical manufacturer before handling solvents or matrixes Preparing matrix To prepare matrix 1 Label a 1 5 ml microcentrifuge tube with the name of the matrix the final concentration and the date prepared 2 Before weighing out the matrix zero the balance with the labeled tube 3 Weigh out the matrix into the tube See Matrix Information on page 3 6 to determine the proper concentration for your matrix NOTE It is not necessary to weigh out the exact amount of matrix You can record the weight of the matrix and adjust the final concentration accordingly NOTE Use a fresh tip each time you pipette a different substance 4 Add a volume of appropriate solvent to achieve the needed concentration for your matrix See Matrix Information on page 3 6 The solvent should be miscible with the sample Voyager Biospectrometry Workstation User s Guide 3 5 Chapter 3 Preparing Samples 5 Cap the tube and vortex thoroughly for approximately 1 minute or until dissolved You can shake the tube by hand if you do not have a vortex mixer 6 Microcentrifuge the tube for 30 seconds at 2 000 to 5 000 rpm Alternatively allow the solution to settle for about 10 minutes You may see a precip
518. s and externally calibrate the samples using the closest calibration standard The Mass Accuracy Optimization option compensates for differences in calibration across a sample plate and increases the throughput of sample analysis by e Reducing the number of standard positions needed to obtain the optimum mass accuracy For more information see Section 3 2 2 Locating Standards for Optimum Mass Accuracy e Allowing larger distances between samples and standards which makes more positions available for sample analysis Before using Mass Accuracy Optimization optimize sample plates using the OptiPlate software For more information see Section 2 8 Running OptiPlate to Optimize Mass Accuracy To use the Mass Accuracy Optimization option select the Use Mass Accuracy Optimizations option when you load a sample plate in the Instrument Control Panel described in Section 3 4 4 Loading Sample Plates The software automatically retrieves the optimization information created by the OptiPlate software for the Plate ID associated with the plate To compensate for differences in calibration across a sample plate the software applies the Extraction Correction calculated by the OptiPlate software for each position on the sample plate as the data is acquired If positions have not been optimized If no optimization found Optimization strategy Loading Sample Plates in the Mass Spectrometer If you select the Use Mass Accuracy O
519. s range and optimize other parameters as needed See Chapter 5 Optimizing Instrument Settings In the Calibration section of the Instrument Settings control page select the matrix you are using See Calibration on page 5 20 From the View menu select Data Storage Set parameters as needed See Setting Data Storage parameters on page 6 14 In the Manual Laser Intensity Sample Positioning Control page select the sample to acquire See Selecting sample position and laser intensity on page 6 13 To start acquiring select Start Acquisition from the Acquisition menu or click Acquire the spectrum using a laser intensity that does not generate fragments Save the data file by clicking E Acquiring PSD Data with Standard BIC Files in Manual Control Mode Generating an external calibration for the precursor ion The precursor spectrum is displayed in the Current trace in the Spectrum window and the DAT file is saved in the directory specified To obtain maximum mass accuracy for the precursor ion generate an external calibration file that you will use when you perform the PSD acquisition This external calibration is used to determine the t value precursor ion flight time needed for the PSD calibration equation described on page 8 28 The t value is determined using the standard calibration equation t in the standard equation described on page 6 9 NOTE The calibration you specify on the
520. s the parts of the Instrument Control Panel how to manipulate traces in the Spectrum window how to control the workstation from the software and how the Instrument Control Panel works with the Sequence Control Panel Voyager Biospectrometry Workstation User s Guide xxvii How to Use This Guide Chapter Appendix Content Chapter 5 Optimizing Instrument Settings Describes instrument settings BIC files and how to optimize them Chapter 6 Acquiring Spectra from the Instrument Control Panel Describes how to acquire mass spectra from single samples using the Instrument Control Panel in Manual Control mode and Automatic Control mode Chapter 7 Acquiring Spectra from the Sequence Control Panel Describes how to acquire mass spectra from multiple samples using the Sequence Control Panel Chapter 8 PSD Analysis Describes using PSD analysis software and exploring the impact of system settings on the quality of data obtained Chapter 9 Maintenance and Troubleshooting Lists routine maintenance procedures performed by Applied Biosystems Contains troubleshooting information and error codes Appendix A Specifications Includes system specifications Appendix B Warranty Service Information Contains warranty service return and spare parts information Appendix C Matrixes Lists chemical structures and preparation information for common matrixes Ap
521. s whether or not spots overlap e Spot Diameter Defines the size of the spots Default value is 50 microns 30 steps e Do Not Overlap Spots When enabled prevents spots from overlapping If the Number of Spots specified of the size specified by the Laser Spot Diameter cannot fit in the generation area when this option is enabled an error message is displayed when you generate the pattern and only the spots that fit in the generation area without overlapping are created 3 Click OK The search pattern spots are displayed in the sample position area 4 Use the drawing tools to adjust and refine the search pattern if necessary For details see Drawing a search pattern on page 6 56 Voyager Biospectrometry Workstation User s Guide 6 55 Chapter 6 Acquiring Spectra from the Instrument Control Panel Drawing a search Use the drawing tools in the sample position area to add pattern insert edit or delete a spot When you select a drawing tool it remains selected and active until you select another tool or another menu item To Click Menu Cursor Then then select changes to Add a spot Add spot Click on the sample position at the location where you want to add a spot If you click and hold when you add you can adjust the position of the spot before it is added Insert a spot Insert spot Move the cursor to the location within t the existing chain of spots where you
522. s yield acceptable results in Manual Control mode before setting to Automatic Control mode 2 Inthe Instrument Settings control page select Automatic Control mode Voyager Biospectrometry Workstation User s Guide 6 37 Chapter 6 Acquiring Spectra from the Instrument Control Panel 3 Click Automatic Control to display the Automatic Control dialog box Figure 6 15 NOTE The Automatic Control button is dimmed if Automatic Control mode is not selected Automatic Control Figure 6 15 Automatic Control Dialog Box 6 38 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Laser 4 Select Use Automated Laser Intensity Adjustment NOTE To manually control the laser intensity when acquiring in Automatic Control mode deselect Use Automated Laser Intensity Adjustment For more information see Section 4 5 2 Adjusting Laser Intensity and Selecting Sample Position 5 If you enable Use Automated Laser Intensity Adjustment specify the Minimum and Maximum Laser Intensity and the Step Size to use For more information see Laser Intensity Adjustment on page 5 34 6 If you enable Use Automated Laser Intensity Adjustment and Use Automated Sample Positioning described in step 10 on page 6 41 select Use PreScan if desired then select an option e First Search Pattern Position Only Each Search Pattern Position NOTE Prescan options are not avail
523. same extension as reflector or linear CAL files If you select a reflector or linear CAL file when in PSD mode an error message is generated Filling in the 9 Fillin the segment list as described in Section 8 4 4 segment list Filling in the Segment List and Saving the BIC File Voyager Biospectrometry Workstation User s Guide 8 61 Chapter 8 PSD Analysis 8 5 2 Setting Laser Increment and Saving the BIC File Setting Laser Set the Laser Increment for each segment The Laser Increment value is added to the starting laser intensity set in the Manual Laser Sample Positioning control page Increment For example Initial Laser Laser Laser Intensity Segment sete oe Intensity Increment for Acquisition 1 800 1 20 1 820 2 40 1 840 3 100 1 900 NOTE You typically need a higher laser intensity to optimize signal intensity for segments with lower Mirror Ratio settings Saving the BIC To save the instrument settings BIC file select Save Instrument Settings As from the File menu then save the BIC file with a new name 8 62 file Applied Biosystems Acquiring PSD Data with Standard BIC Files in Automatic Control Mode 8 5 3 Acquiring PSD Segments in Automatic Control Mode Acquiring PSD To acquire PSD segments in Automatic mode segments 1 inthe Manual Laser Intensity Sample Positioning control page select the same sample position from which you acquired the
524. scribed in Drawing a search pattern on page 6 56 and Setting x y coordinates on page 6 57 Units display only Displays the units for the SP file Default is microns New SP files you create with the Search Pattern Editor default to micron units Existing SP files you edit retain their original units microns or steps Well Width Displays the width and height dimensions of the sample Well Height position retrieved from the loaded PLT file Default units are display only steps if you display this dialog box when no SP file is open If you display this dialog box when an SP file is open Well Width and Well Height values are displayed in the units that correspond to the open SP file continued 6 54 Applied Biosystems Acquiring in Automatic Mode from the Instrument Control Panel Table 6 4 Search Pattern Generator Parameters Continued Parameter Description Pattern Select a generation method e Random Generates randomly placed spots within the generation area e Spiral Generates spots within the generation area that spiral out from the Center X Y coordinates specified Pattern Type Select a distribution method e Uniform Evenly distributes spots in the generation area e Center Bias Clusters spots around the center of the generation area e Edge Bias Clusters spots around the edge of the generation area continued Spot Settings Determine
525. ser strikes the sample may drift If this occurs press the Stop button on the left side of the external laser cabinet to reset the laser Then press the Start button to reset the laser CAUTION If you do not press the Start button after pressing the Stop button no power is supplied to the laser The software will allow you to start acquisition but the laser will not fire If the laser is left on for long periods of time there may be a problem with laser positioning on the sample If this occurs press the Stop button then the Start button to reset the laser Startup and Shutdown 2 10 Startup and Shutdown In this section This section describes e Powering up Initializing e Reinitializing e Powering down system components e Powering down the mass spectrometer Powering up To power up the Voyager Workstation 1 Turn on the main power switch The power switch is located e On the right side panel of the mass spectrometer cabinet on Voyager DE and Voyager DE PRO systems e On the back panel of the mass spectrometer cabinet on the Voyager DE STR system The vacuum pumping system begins running After two minutes the turbo pump starts You will hear a whine as the vacuum system spins up to speed 2 Turn on remaining system devices in this order e Video monitor e External digitizer if your system includes one e Oscilloscope if your system includes one e Computer e Printer 3 If your system
526. sheets you can copy and use to log samples before loading Voyager Biospectrometry Workstation User s Guide D 1 Voyager Biospectrometry Sample Log Plate Date 1 6 Voyager Biospectrometry Sample Log Plate Date Page of Path and File Name Samp Matrix Sample Linear Reflector ls 2 20 21 22 23 24 25 Voyager Biospectrometry Sample Log Plate Date Page of Path and File Name Samp Matrix Sample Linear Reflector 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Voyager Biospectrometry Sample Log Plate Date Page of Path and File Name Samp Matrix Sample Linear Reflector 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Voyager Biospectrometry Sample Log
527. signal is much higher than the sample signal NOTE Low mass gate is automatically disabled when you change the instrument mode to PSD See Section 5 4 4 5 Setting Low Mass Gate for more information Calibration Matrix Displays the matrix used for the analysis Available matrixes in this field are a cyano 4 hydroxycinnamic acid e Sinapinic acid e 2 5 Dihydroxybenzoic acid e 3 Hydroxypicolinic acid If you do not specify a matrix a default value of 300 m sec is used Corrects for the initial velocity contributed by the matrix for a more accurate calibration Correction factors for each matrix are listed in Matrix influence on page 5 22 If your matrix is not listed you can add other matrixes to this list by editing the Matrix reference file For information see Modifying the matrix reference file on page 5 23 continued 5 20 Applied Biosystems Instrument Settings Parameter Descriptions Table 5 4 Instrument Settings Parameters Continued Parameter Description Calibration continued Default Enables default calibration For more information see Default calibration on page 6 9 External file Specifies calibration using a specified external CAL file Click H to select a CAL file previously generated in the Data Explorer software For more information see the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating 1
528. sition H 21 me one position alignment a misalignment The center of the sample Laser spot J position is not aligned with Sample position H respect to the laser spot You adjust misaligned sample positions to ideal Figure 2 23 Sample Position Misalignment alignment Camera misalignment The laser spot is not centered on the video Laser spot monitor The camera requires adjustment Do not perform the sample Fi 2 24 C Misali plate alignment procedure nmen igure amera Misalignme PEE E Technical Support 2 48 Applied Biosystems Aligning the Sample Plate What you need To align the sample plate you need the following materials e Sample plate with a cyano 4 hydroxycinnamic acid CHCA matrix spotted in four corner sample positions as described in Table 2 1 For more information see Section 3 5 5 Adjusting the Laser Position for a Custom PLT File WARNING CHEMICAL HAZARD Alpha cyano 4 hydroxycinnamic acid CHCA matrix may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves NOTE If your monitor screen is marked with the position at which the laser strikes the sample plate and you are aligning plates with wells or etched sample positions you do not need to spot the sample plate with matrix before aligning You can
529. sity in the same increments as the fine laser control buttons Selecting the active sample position in Plate view Displaying coordinates of active position Controlling the Workstation The active sample position is the sample position from which data is acquired Select the active sample position from the Plate view by doing any of the following e Type a position name or number in the Active Position field Select a number from the drop down list in the Active Position field e Click on a sample position ToolTip displays the position number e Use the control stick to move to a sample position For more information on using the control stick see Section 1 3 Using the Control Stick NOTE When you use the control stick to select a sample position if the software determines that you have gone beyond the boundary of a position it will automatically change to the closest Active Position Position boundaries are determined calculated from the Sample Plate Template PLT file For more information see Effect of plate type on area available for analysis on page 3 73 The location X Y coordinates of the active position are displayed at the bottom of the Manual Laser Sample Position control page e Relative Displays the X Y coordinates microns relative to the center of the Active Position e Absolute Displays the X Y coordinates microns relative to the home position position in the lower l
530. spectrometry mass spectrometer are shown in Figure 1 18 Variable voltage Laser grid attenuator Reflector Reflector detector electrostatic Sample a e mirror plate SETI TT TT Main source chamber l SLT T TTT i Q Flight Video tube camera Ground grid Linear Timed lon detector Aperture grounded Selector Collision cell optional gt lon path in reflector mode Ae P gt Laser path Figure 1 18 Voyager DE STR Mass Spectrometer NOTE Voyager DE STR models with serial number 4153 and earlier include a Beam Guide Wire instead of an lon Focusing lens Voyager Biospectrometry Workstation User s Guide 1 33 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 Parts of the mass The Voyager DE STR mass spectrometer includes spectrometer Laser attenuator and prism A nitrogen laser that operates at 337 nm and ionizes sample It produces 3 nanosecond duration pulses Laser rate can be set to Default 3 Hz or Optimized see page 5 26 Maximum possible rate is 20 Hz actual rate depends on the digitizer installed and the digitizer Bin Size setting NOTE Optimized laser rate is available only on Voyager DE STR systems with serial number 4161 and later The laser attenuator varies the intensity of the laser beam reaching the sample The prism deflects the laser beam into the ion source e lon Source A high voltage
531. spectrum on page C 3 MW 154 03 Da MW 168 Da Mixture MW 322 03 Da COOH OH HO PB100253 COOH CH 0 PB100775 Voyager Biospectrometry Workstation User s Guide C 9 Appendix C Matrixes Table C 1 Matrix Information Matrix Applications Color Matrix Solution Concentration Characteristic Matrix lons monoisotopic 2 4 hydroxy Applications e 1 3 mg ml in 243 077 phenylazo benzoic Proteins 90 50 water e 265 059 acid HABA Lipopoly acetonitrile or in spectrum on Polar ad acetonitrile page C 4 nonpolar methanol MW 242 07 Da synthetic 10 mg ml in polymers ethanol or coon Color of crystals Rares r N solution Le Orange 3 hydroxypicolinic Applications Make 9 1 dilution of 96 045 acid 3 HPA Oligonucleotides Matrix diammonium e 140 035 citrate e 279 062 see mass Color of crystals spectrum on solution Matrix 50 mg ml in 235 072 page C 4 Light b 50 50 water 234 064 eer acetonitrile 233 056 MW 139 03 Da Diammonium E COOH citrate 50 mg ml in S Tee eee 7 water nS Za PB100257 C 10 Applied Biosystems Table C 1 Matrix Information Matrixes Matrix Solution Characteristic Matrix Applications Color i Matrix lons Concentration monoisotopic Dithranol Applications 10 mg ml in e 225 055 see mass Nonpolar tetrahydrofuran 226 063 spectrum on synthetic Silver trifluoroacetate e 227
532. station User s Guide 3 35 Chapter 3 Preparing Samples Types of sample Three types of 100 position reusable sample plates are plates available Polished blank sample plates with or without sample numbers Liquid is held in place by surface tension of sample matrix mixture Advantage of this type of plate is that you can observe crystallization pattern and the actual sample spot is visible e Laser etched sample plates Liquid is held in place by laser etched indentation in plate Advantage of this type of plate is that the sample position on the plate is well defined Use laser etched sample plates when running in Automatic Control mode Welled sample plates Liquid is held in place by indentation in plate Advantage of this type of plate is that the sample position on the plate is well defined Use welled sample plates e When running in Automatic Control mode e To contain larger sample matrix volumes that will spread beyond the sample position boundaries due to volatile solvent content In addition to 100 position reusable sample plates the Voyager software supports other reusable and disposable plates For more information see e Section 3 5 Sample Plate Types e Section 3 5 1 Sample Plate Types and Applications 3 36 Applied Biosystems Loading Samples on Sample Plates Handling sample To prevent contamination of your analysis plates Start with a clean sample plate See Section 3 3 Cleaning Sample Pla
533. stem sample plates with various formats available Beam guide technology for high mass sensitivity Video camera and monitor for sample viewing e Low mass gate matrix suppression e Intuitive Microsoft Windows NT based software Features unique to the Voyager DE PRO Biospectrometry Workstation include e High performance reflector e Timed ion selector e Post source decay PSD analysis capability Linear or reflector mode operation e Collision induced dissociation CID option 1 4 Applied Biosystems Voyager DE STR System Overview 1 2 Voyager DE STR System Overview Voyager DE STR The Voyager DE STR Biospectrometry Workstation Figure 1 3 is a floor standing MALDI TOF matrix assisted laser desorption ionization time of flight mass spectrometer that includes a reflector analyzer PB100427 Figure 1 3 Voyager DE STR Biospectrometry Workstation Voyager Biospectrometry Workstation User s Guide 1 5 Chapter 1 Introducing the Voyager Biospectrometry Workstations Biospectrometry Biospectrometry is the application of mass spectrometry in the 1 field of the life sciences This field uses fast chromatographic techniques enzymatic chemistries and surface chemistries and combines them with mass spectrometry and advanced software to better enable biomol
534. strument Control Panel See Section 2 11 Checking System Status and Pressures for more information 1 6 4 Front Panel Indicators The front panel of the Voyager DE STR system is shown in Figure 1 20 TURBO PUMPS SYSTEM SOURCE O O HIGH VOLTAGE O Locic O ENABLED START UP NORMAL REFLECTOR O O O INTERLOCK O ENABLE O PB100267 Figure 1 20 Voyager DE STR Front Panel Indicators Front panel indicators are described in Table 1 1 Voyager Biospectrometry Workstation User s Guide 1 39 Chapter 1 Introducing the Voyager Biospectrometry Workstations Table 1 1 Front Panel Indicators Indicator Indication When Lit Laser Laser N Red Laser power is on Enabled Red Laser is firing Turbo Pumps Startup Yellow One or both turbo pumps are initializing Normal Green Both turbo pumps are operating at normal speed Fault Red One or both turbo pumps are off Should not be lit during normal operation System High Voltage High voltage is on Interlock An interlock error door open or panel off Automatically disables laser and high voltage Logic Internal control board in mass spectrometer has been powered up Enabled Computer is controlling mass spectrometer 1 40 Applied Biosystems 1 6 5 Computer Components Hardware The Voyager DE STR Biospectrometry Workstation includes the following IBM compatible computer hardwar
535. strument Settings Parameter Descriptions 5 14 5 2 1 Instrument Settings Page ccceceeeeeeeeeeeeeeeaaeaaeeteeeeeeeeeeees 5 15 5 2 2 Mode Digitizer Dialog BOX ccccceeeeeeeeaneeeeeeeaaaaeeeeeeeaaaeeeeees 5 24 5 2 3 Automatic Control Dialog BOX ccceceeeeeeeeeeeeeeaeaeeeneeneeenees 5 33 5 2 4 Description of Spectrum Accumulation Options e 5 39 Impact of Changing Instrument Settings Parameters 5 49 5 3 1 Summary of Parameters cccceeeeeeeee cee eeeeeeeeeeeeeeeeeeeeeeeeaeaaes 5 49 5 3 2 Understanding Grid Voltage ecceceeeeeeee eee eneeeeeteeeteeeeeeeeeees 5 51 5 3 3 Understanding Delay Time ccc eeeee eee eeeeeeeeaaeeeeeeeeeaaaeeeeees 5 54 5 3 4 Understanding Guide Wire Voltage sceeeseeeeeeeeeeeeeees 5 56 5 3 5 Understanding Digitizer Settings c c sseeeeeeeeeeeeeeeeeeeeees 5 57 5 3 5 1 BIN SIZE ton Late Madani eas Ante tase 5 57 5 3 5 2 Vertical Digitizer Settings 5 60 Optimizing Instrument Settings Parameters 5 64 5 4 1 Optimization Strategy 5 65 5 4 2 Determining the Laser Setting cccceeccsceeeeeeeeeeeeeeeeeeeeeeees 5 67 Voyager Biospectrometry Workstation User s Guide vii Table of Contents 5 5 5 4 3 Optimizing Resolution 20 0 ee eee ee cee
536. symboles suivants peuvent tre affich s sur le syst me Dans ce document ces symboles peuvent aussi appara tre c t des avertissements auxquels ils s associent Le tableau suivant donne la signification de tous les symboles lectriques qui figurent sur les appareils En pr sence de l un de ces symboles il est imp ratif de se conformer aux consignes de s curit appropri es Position MARCHE de l interrupteur d alimentation principal Position ARR T de l interrupteur d alimentation principal Positions MARCHE ARRET de l interrupteur d alimentation principal bouton poussoir Borne pouvant tre reli e la mise la terre d un autre appareil Ce n est pas une borne de mise la terre prot g e Borne de mise la terre de protection devant tre reli e la terre avant d effectuer tout autre raccordement lectrique a l appareil Borne recevant ou fournissant une tension ou un courant de type alternatif Borne pouvant recevoir ou fournir une tension ou un courant de types alternatif et continu ja B O0O Ce symbole appara t c t des valeurs des fusibles requis par le syst me Voyager Biospectrometry Workstation User s Guide xvii Safety and Compliance Information AVERTISSEMENT Indique la pr sence d une haute tension et avertit l utilisateur de proc der avec pr caution AVERTISSEMENT Avertit l utilisateur de la n cessi
537. systems How to Use This Guide How to Use This Guide Purpose of this guide Audience Structure of this guide The Applied Biosystems Voyager Biospectrometry Workstation User s Guide describes the procedures for installing using maintaining and troubleshooting Voyager Biospectrometry workstations This guide is intended for novice and experienced Voyager workstation users who are analyzing biomolecules The Applied Biosystems Voyager Biospectrometry Workstation User s Guide is organized in chapters and appendixes Each chapter page is marked with a tab anda header to help you find information The table below describes the material covered in each chapter Chapter Appendix Content Chapter 1 Introducing the Voyager Biospectrometry Workstations Describes the parts of the system and software and gives background information on MALDI TOF and Delayed Extraction technology Chapter 2 Installing the Voyager Biospectrometry Workstations Provides procedures for installing the system attaching components such as the video monitor installing the software starting up and shutting down Chapter 3 Preparing Samples Describes how to prepare matrix and sample how to load sample on sample plates and how to load plates in the mass spectrometer Also describes sample plate types and plate PLT files Chapter 4 Voyager Instrument Control Panel Basics Describe
538. systems Using the Oscilloscope and Control Stick CAUTION Check to see if acquisition has already stopped automatically before pressing the control stick button If acquisition has stopped the Instrument Control Panel status bar is blank it displays an Acquiring Data message during acquisition If acquisition has stopped and you press a control stick button you will begin a new acquisition and overwrite the current spectrum NOTE To move the sample under the laser beam deflect the control stick in the appropriate direction Longer deflection will result in faster movement of the sample plate Glossary a b and c ions Generic description of potential ions that are formed by fragmentation of a parent peptide protein a b and c ions are fragments that retain the charge atthe amino end n terminus of the molecule See also x y z ions an bn cn p co NHC Rn Rn 1 ASCIl american Standard Code for Information Interchange A file format that contains codes that constitute the 128 character ASCII set and allows exchange of data between information processing systems communication systems and associated equipment Accelerating voltage Potential difference between the ion source and ground used to accelerate ions Actual acceleration in a TOF instrument usually occurs in the first 1 to 2 cm of the flight tube lons then drift the remaining flight tube distance Arcing Electrical disch
539. t de consulter le manuel pour obtenir davantage d informations et de proc der avec pr caution gt gt Symboles non Le tableau suivant donne la signification des symboles lectriques d alertes de s curit non lectriques qui figurent sur les appareils AVERTISSEMENT Danger associ la pr sence d un appareil de chauffage Proc der avec pr caution pour viter de se br ler au contact de pi ces ou d l ments chauds Indique que l appareil renferme un laser b xviii Applied Biosystems Before operating this instrument Material Safety Data Sheets MSDSs Safety and Compliance Information Ensure that anyone involved with the operation of the instrument is instructed in both general safety practices for laboratories and specific safety practices for the instrument Make sure you have read and understood all related Material Safety Data Sheets Some of the chemicals that may be used with your system are listed as hazardous by their manufacturer When hazards exist they are prominently displayed on the labels of all chemicals In addition MSDSs supplied by the chemical manufacturer provide information about e Physical characteristics e Safety precautions e Health hazards e First aid e Spill clean up e Disposal procedures WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet provided by the manufacturer and observ
540. t 6 16 9 21 mass spectrometer 9 23 annotating 4 16 changing colors to black before printing 4 18 color 4 22 no matrix peaks 9 9 Current 4 14 no sample peaks 9 7 displaying as vertical bars 4 22 peak shape 9 11 9 16 H 20 do not print 4 19 4 22 poor crystallization on sample expanding 4 13 plate 3 25 labels spectrum 6 29 PSD mode 9 21 line type 4 22 resolution 5 76 9 10 H 21 linking 4 13 sample plate crystallization 9 9 Live 4 14 saturated signal 9 9 maximum number 4 14 sensitivity 3 25 9 10 Not Used 4 14 Sequence Control Panel 9 21 Shots Spectrum 9 20 signal to noise 9 13 9 14 9 15 spectrum 9 7 Spectrum window is not updated with every laser shot 9 21 tail on spectrum 3 25 overview 4 13 previewing 4 18 printing 4 18 removing 4 15 scaling mode setting 4 11 4 12 traces do not print 4 18 types of 4 14 Trypsinogen molecular weight F 3 white does not print 4 18 Turbo pump Voyager DE zooming 4 13 function 1 27 Trihydroxy acetophenone see THAP vacuum gauge 1 27 Trimers troubleshooting 9 15 Turbo pump Voyager DE PRO function 1 29 Troubleshooting ae onde 2 active position 9 20 CID 8 36 Turbo pump Voyager DE STR computer 9 6 function 1 37 continuous mode spectrum H 20 vacuum gauge 1 39 dimers trimers in spectrum 9 15 error codes vacuum gauge panel 9 27 Instrument Control Panel 9 19 laser 9 20 Index 42 Applied Biosystems U Uniform search pattern 5 37 Unzooming 4 13 Upda
541. t Exit from the File menu A message is displayed Click Yes The Sequence Control software closes If you started the Instrument Control Panel by double clicking the Sequence Control Panel icon the Instrument Control software closes 2 If you started the Instrument Control Panel by double clicking the Instrument Control Panel icon select Exit from the File menu in the Instrument Control Panel window The Instrument Control software closes Hardware Configuration 2 6 Hardware Configuration CAUTION Do not alter the Hardware Configuration unless instructed to do so by an Applied Biosystems Technical Representative Altering these settings may cause your Voyager Biospectrometry Workstation to function improperly In this section This section describes e Vacuum configuration e High voltage configuration e Timed Precursor ion selector configuration Instrument configuration e Laser configuration e Digitizer configuration 2 6 1 Vacuum Configuration To check the vacuum configuration 1 Inthe Instrument Control Panel select Hardware Configuration from the Instrument menu 2 Click the Vacuum tab to display the Vacuum page Figure 2 15 on page 2 36 Voyager Biospectrometry Workstation User s Guide 2 35 Chapter 2 Installing the Voyager Biospectrometry Workstations Hardware Configuration xj High Voltage Instrument Digitizer Vacuum Laser Timed lon Selector m Source Chamber B
542. t detector available on The valid range for Guide Wire Voltage is 0 000 to 0 300 Voyager DE STR of the Accelerating Voltage models with serial Linear mode Use 0 05 to 0 3 as suggested by the number 4154 and later standard instrument settings and increase the Grid Voltage with increasing mass e Reflector mode Use 0 00 to 0 050 as suggested by the standard methods e In PSD mode Use settings lt 0 02 Higher settings may compromise the selectivity of the Precursor lon Selector See Section 5 3 4 Understanding Guide Wire Voltage and Section 5 4 3 3 Optimizing Guide Wire Voltage for more information Delay Time nsec Works in conjunction with the Delayed Extraction parameter Time in nanoseconds after the laser ionizes the sample at which full Accelerating Voltage is applied creating the potential gradient that accelerates ions Recommended range is 0 to 3 000 nsec Optimize in conjunction with Grid Voltage described on page 5 17 See Section 5 3 3 Understanding Delay Time and Section 5 4 3 4 Optimizing Delay Time for information continued 5 18 Applied Biosystems Instrument Settings Parameter Descriptions Table 5 4 Instrument Settings Parameters Continued Parameter Description Shots Spectrum Determines the number of laser shots that each spectrum will contain For more information see Section 5 4 4 4 Setting Shots Spectrum The maximum number of Shots per Spec
543. t from the Acquisition menu The PSD data file is closed You cannot view the PSD data file in the Data Explorer software until you stop the experiment CAUTION If you stop an experiment without saving any segments no DAT file is created Voyager Biospectrometry Workstation User s Guide 8 11 Chapter 8 PSD Analysis Determining if PSD 1 Click in the Instrument Control Panel toolbar to calibration is open the PSD data file in the Data Explorer The needed software stitches together portions of the fragment spectra and displays a composite spectrum see Figure 8 6 on page 8 26 for an explanation of how the software generates the composite spectrum Your angiotensin composite spectrum should be similar to the spectrum shown in Figure 8 3 Stitched PSD BP 1183 7 57835 100 5 8E 4 1181 6 90 805 705 60 2 269 16 amp 50 354 20 255 14 405 784 41 304 513 28 110 08 20 10 619 36 04 LL lit eM 0 0 260 520 780 1040 1300 Mass m z Figure 8 3 Angiotensin Spectrum 2 Examine the masses in the stitched spectrum and compare them to the expected masses listed in Table 8 3 8 12 Applied Biosystems PSD Quick Start Table 8 3 Expected Masses in Angiotensin Spectrum Expected Mass lon Type 110 08 His 255 16 b2 17 269 16 y2 354 20 b3 17 513 08 y4 619 36 a5 784 41 b6 1 181 6 y9 If masses are not within 0
544. t the LSA1000 LeCroy digitizer board to the mass spectrometer Connection on Connection on right side panel of mass Connection on rear digitizer see panel of computer Cable Figure 2 5 Spectrometer see see Figure 2 4 g Figure 2 2 g 10 100 Base T None Integrated network Cross over connection not network add in network card cable 100 Base T supplied TRIG1 TRIG None Trig BNC CH1 CH 1 None Ch 1 BNC CH2 CH 2 None Ch 2 BNC Voyager DE PRO only 2 16 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations 2 3 5 Connecting the Acgiris Digitizers This section describes how to connect the Acqiris 500 MHz and 2 GHz digitizer boards to the Voyager DE and Voyager DE PRO mass spectrometers If you have an oscilloscope or an external digitizer on your system disregard this section Refer to the following table when you connect the Acairis 500 MHz digitizer board to the mass spectrometer Connection on right side panel of mass spectrometer see Connection on rear panel of computer Cable see Figure 2 4 Figure 2 2 TRIG EXTERNAL Trig BNC on mass spectrometer to EXTERNAL BNC on board CH 1 INPUT Ch1 BNC on mass spectrometer to 90V BNC Spark Gap to 3d BNC Attenuator to INPUT BNC on board Ground wire to computer chassis ground screw Voyager Biospectrometry Workstation User s Guide 2 17 Chapter 2 Installing
545. t the rows to fill then select Fill Down from the Edit menu The first entry selected is copied to all selected rows below Filling down the You can set the Sample Position field to fill down in two ways sample position Copy the first sample position to all selected rows below by click dragging to select the rows to fill then selecting Fill Down from the Edit menu Increment sample positions in all selected rows below based on the type of sample plate loaded in the Instrument Control Panel as described below Incrementing _ To increment sample positions when filling down sample positions 4 inthe Instrument Control Panel select Select Sample Plate from the Sample Plate menu then select the Sample Plate you will use for the sequence For more information see Section 3 4 4 Loading Sample Plates 2 Inthe Sequence Control Panel select Preferences from the View menu In the Preference dialog box e Select Fill Down Sample Position Sequentially Based on PLT File e Type the number of positions contained on the loaded sample plate in the Default Number of Rows in the Grid field e Click OK For more information see Section 7 8 2 Setting Sequence Control Panel Preferences Voyager Biospectrometry Workstation User s Guide 7 21 Chapter 7 Acquiring Spectra from the Sequence Control Panel Importing and exporting the run list File requirements 7 22 for importing Applied Biosystems 3
546. tation Vacuum Chambers 1 28 Applied Biosystems Parts of the Voyager DE and Voyager DE PRO Systems Vacuum pumps Three vacuum pumps create the vacuum environment e Fore pump Creates a vacuum in the sample loading chamber creates a lower than atmospheric pressure condition before the turbo pumps start and provides backing pressure to the turbo pumps e Turbo pump 1 Creates a high vacuum condition in the main source chamber e Turbo pump 2 Creates a high vacuum condition in the mirror chamber Vacuum is maintained in the main source chamber and sample loading chamber by valves that isolate the chambers Vacuum is maintained in the mirror chamber by a differential pumping baffle Vacuum gauges The Voyage DE PRO Biospectrometry Workstation includes three vacuum gauges e BA1 Bayard Alpert Gauge Monitors pressure in the main source chamber e BA2 Bayard Alpert Gauge Monitors pressure in the mirror chamber e TC2 Monitors pressure in the sample loading chamber Readings from the vacuum gauges are displayed in the System Status Control page in the Instrument Control Panel See Section 2 11 Checking System Status and Pressures for more information Voyager Biospectrometry Workstation User s Guide 1 29 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 5 4 Computer Components Hardware The Voyager DE and Voyager DE PRO Biospectrometry Workstations include the following IBM co
547. tations manufactured in 1995 or earlier Gold 64 well disposable for use with disposable sample V503476 plate holder part number V700314 Disposable sample plate holder for use with gold 64 well V700314 disposable plate part number V503476 Biacore Chip holds 4 biochips V700697 Voyager Biospectrometry Workstation User s Guide B 7 Appendix B Warranty Service Information Description Part Number Membrane Gels V700698 Hydrophobic plastic surface flat 400 position V700699 96 x 2 position flat hydrophobic plastic surface plate V700813 B 8 Applied Biosystems C Matrixes This appendix provides information for commonly used matrixes The information includes see Table C 1 on page C 6 e Matrix mass spectra e Chemical structure e Applications e Description of physical appearance e Suggested solution concentration e Characteristic matrix ions Refer to Figure C 1 through Figure C 9 for characteristic peaks patterns and masses For additional matrix information refer to the bibliography WARNING CHEMICAL HAZARD Refer to the Material Safety Data Sheet MSDS provided by the chemical manufacturer before handling solvents or matrixes Voyager Biospectrometry Workstation User s Guide C 1 Appendix C Matrixes Spec 1 BP 207 0 30435 igi 207 0 3 0E 4 90 225 0 Intensity 0 Goo 180 260 340 420
548. te Readjusting the 5 86 laser after optimization Applied Biosystems Figure 5 25 Optimizing Signal to Noise Ratio After you have optimized Accelerating Voltage Guide Wire Voltage and Shots Spectrum for optimum signal to noise you can further fine tune by adjusting the laser intensity For information on adjusting the laser intensity see Adjusting laser intensity on page 5 68 Optimizing Instrument Settings Parameters 5 4 4 2 Setting Accelerating Voltage Overview Accelerating Voltage defines the energy of ions as they travel Recommended setting in the flight tube and reach the detector Efficiency of detection particularly for high mass ions increases with higher ion energy Therefore the maximum Accelerating Voltage typically yields optimum performance when analyzing masses above 10 000 Da However a lower Accelerating Voltage can increase flight times and can improve resolution in spectra NOTE The calibration of the mass scale changes significantly when you change the Accelerating Voltage Default calibration adjusts for these changes However you will observe more accurate calibration if you use an external calibration CAL file generated with the same Accelerating Voltage See the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating for more information Higher masses decrease detector sensitivity To overcome this effect set the Accele
549. te Types Applications and Benefits Continued Sample Plate Type Applications Benefits Special Sample Plates Stainless steel polished e Allows customized sample positioning and blank preparation using an automated sample preparation device Disposable gold coated Can derivatize the surface of the sample plate for 100 position protein or enzyme immobilization e Long term storage of samples e Eliminates cross contamination Biacore Chip e Direct analysis of affinity isolated species e Confirmation of binding constant determined by surface plasmon resonance Membrane Gels The base surface of the plate is recessed to accommodate the depth of gels or membranes and can be used with PVDF or nitrocellulose membranes e PAGE gels for example PhastGel Teflon surface e Increases sensitivity because hydrophobic walls 384 position or concentrate the sample into a small area 400 position e Accurate sample positioning for high throughput Voyager Biospectrometry Workstation User s Guide 3 63 Chapter 3 Preparing Samples Table 3 11 Sample Plate Types Applications and Benefits Continued Sample Plate Type Applications Benefits 96 x 2 192 position flat hydrophobic plastic surface plate Increases sensitivity because hydrophobic walls concentrate the sample into a small area Useful for close external calibration due to the proximity o
550. te calibration description 7 11 how to program every X minutes 3 39 overview 7 5 selecting 7 18 Username obtaining from system administrator 2 74 Users guides online 2 31 V Vacuum configuring 2 35 pressure ranges 2 76 pressure status 2 77 4 5 startup 2 73 time required to reach operating pressure 2 77 Vacuum gauge panel Voyager DE and Voyager DE PRO buttons 9 26 error codes 1 1 9 27 location 9 25 pressure ranges 9 26 Vacuum gauge panel Voyager DE STR buttons 9 26 error codes 1 1 9 27 location 9 25 pressure ranges 9 26 Vacuum gauges CID disruption caused by unpurged lines 8 34 pressure ranges 8 34 Vacuum system Voyager DE Index see also Vacuum gauge panel chambers 1 26 diagram 1 26 function 1 25 gauges 1 27 pumps 1 27 Vacuum system Voyager DE PRO see also Vacuum gauge panel chambers 1 26 1 28 diagram 1 28 function 1 25 gauges 1 29 Vacuum system Voyager DE STR see also Vacuum gauge panel chambers 1 37 diagram 1 38 function 1 37 gauges 1 39 Valves vacuum 1 26 1 28 1 38 Velocity focusing description 1 15 in PSD mode 8 74 Velocity initial see Initial velocity Vertical bars displaying traces as 4 22 traces do not print 4 19 4 22 Vertical Offset digitizer impact of changing 5 62 setting 5 30 suggested setting 5 62 Vertical position oscilloscope l 5 Vertical Scale digitizer impact of changing 5 60 PSD 8 48 setting 5 30 setting on oscilloscope I 5 suggested
551. te processing settings SET files in the Data Explorer software if you plan to specify Internal or Internal Update calibration or if you want peak detection settings other than default settings stored with the data file The SET file can also apply monoisotopic peak filtering NOTE If you do not specify a Pre Macro that performs peak deisotoping or you do not set monoisotopic peak filtering set the Mass Tolerance for Reference Matching in Autocalibration settings narrow enough to ensure that the monoisotopic peak is correctly identified before calibration When you create the SET file save processing settings only Graphic settings are not applied in the Sequence Control Panel even if they are present in the SET file For more information on SET files see the Data Explorer Software User s Guide e Section 1 4 2 Customizing Processing and Graphic Settings SET e Section 3 2 3 Setting Peak Detection Parameters e Section 3 3 4 Sorting Filtering and Printing the Peak List e Section 5 4 2 Importing and Specifying Automatic Calibration Settings Creating a Sequence 7 4 Creating a Sequence This section includes e Setting General Sequence parameters e Creating a run list Parts ofa A sequence includes sequence General sequence parameters Apply to all entries in the run list described in Section 7 4 1 Setting General Sequence Parameters Run list parameters Apply to individual entries
552. tems continued Instrument Settings Parameter Descriptions Table 5 4 Instrument Settings Parameters Continued Parameter Description Voltages Accelerating Voltage Voltage applied to the first stage ion source Valid range is 0 to 25 000 V For information on settings for different mass ranges see Section 5 4 4 2 Setting Accelerating Voltage NOTE The calibration of the mass scale changes significantly when you change the Accelerating Voltage Default calibration adjusts for these changes However you will observe more accurate calibration if you use an external calibration CAL file generated with the same Accelerating Voltage See the Data Explorer Software User s Guide Section 5 3 2 Manually Calibrating for more information Grid Voltage Voltage applied to the grid above the sample plate Valid range is determined by the Accelerating Voltage Optimize in conjunction with Delay Time described on page 5 18 See Section 5 3 2 Understanding Grid Voltage and Section 5 4 3 5 Optimizing Grid Voltage for more information continued Voyager Biospectrometry Workstation User s Guide 5 17 Chapter 5 Optimizing Instrument Settings Table 5 4 Instrument Settings Parameters Continued Parameter Description Guide Wire Voltage Voltage applied to the beam guide wire Overcomes the NOTE Guide Wire dispersion effect from the source and refocuses ions on the Voltage is no
553. tensin_PSD BIC file provided with the software 2 Inthe PSD segment list select the first row with a Mirror Ratio setting of 1 0 3 Acquire a spectrum 4 Observe the 1 180 to 1 190 Da mass region for the fragment ion cluster typically seen for angiotensin Figure 8 14 Intensity 3 4E 4 Figure 8 14 Angiotensin I Fragment lons Precursor lon Selector On Applied Biosystems Exploring PSD Mode 5 Note the behavior of the signal intensity for the first few spectra and subsequent continued samplings at a given laser power NOTE Sample preparations that contain high salt contamination or other impurities often yield increased signal intensity after an initial period of low intensity as the top layer of sample is consumed You typically see signal intensity reach maximum and decrease more quickly than in non PSD mode as sample is consumed You may need to move around on the sample spot to maintain signal intensity 6 Continue increasing the laser intensity and observing the signal Figure 8 15 shows the effect of higher laser intensity on the angiotensin spectrum Current Spec 1183 4 6 2E 4 1184 5 Intensity 180 3 1150 7 1164 1178 Mass m z Figure 8 15 Angiotensin I Fragment lons at High Laser Intensity Voyager Biospectrometry Workstation User s Guide 8 67 Chapter 8 PSD Analysis Hint As you initially experiment a
554. tential gradient is applied and ions are accelerated 1 12 Applied Biosystems Voyager DE Delayed Extraction Technology In Continuous Extraction mode e Accelerating voltage is continuously applied and the potential gradient exists when sample is ionized e lons are immediately accelerated Figure 1 7 and Figure 1 8 show the improved resolution obtained in Delayed Extraction mode 1236 67 1297 6 sean Peaks are no longer eos resolved gt L 1288 1290 1292 1234 1236 1298 1300 1902 1904 1306 1256 1230 1232 1234 1236 1238 1500 1302 1304 1306 Maze ma Maze mis Delayed Extraction Continuous Extraction Figure 1 7 Comparison of Angiotensin Resolution with Delayed and Continuous Extraction Linear Mode 2466 2 2466 2 Peaks are no longer resolved 2466 18 24652 2467 18 2458 2460 2462 2464 2466 2468 2410 2de adm 24582460 2462 2464 2466 2465 2410 dt dt Mace ms Mass mis Delayed Extraction Continuous Extraction Figure 1 8 Comparison of ACTH Clip 18 39 Resolution with Delayed and Continuous Extraction Reflector Mode Voyager Biospectrometry Workstation User s Guide 1 13 Chapter 1 Introducing the Voyager Biospectrometry Workstations Benefits of Delayed Extraction of ions overcomes many of the adverse Delayed effects of Continuous Extraction Extraction Benefits of Adverse Effects of Delayed Extraction Continuous
555. tep Size laser 5 34 Stop Conditions 5 38 Automatic Control tab description 4 5 displayed during acquisition 6 45 maximum number of lines displayed 4 6 Axes changing scale on right axis 5 70 customizing 4 21 right changing scale 5 70 right turning on and off 4 21 scaling turning off right axis 4 11 B BAT Voyager DE description 1 27 E09 error 9 27 maximum operating pressure 2 36 pressure displaying 2 77 4 5 pressure range 9 26 wait time 2 37 BA Voyager DE PRO description 1 29 E09 error 9 27 maximum operating pressure 2 36 pressure displaying 2 77 4 5 pressure range 9 26 wait time 2 37 BA1 Voyager DE STR description 1 39 E09 error 9 27 maximum operating pressure 2 36 pressure displaying 2 77 4 5 pressure range 9 26 wait time 2 37 BA2 Voyager DE description 1 29 pressure displaying 2 78 4 5 pressure range 9 26 Index BA2 Voyager DE PRO description 1 29 E09 error 9 27 pressure displaying 2 78 4 5 pressure range 9 26 BA2 Voyager DE STR description 1 39 E09 error 9 27 pressure displaying 2 78 4 5 pressure range 9 26 Back panel see Rear panel Backing up 9 6 Bandwidth see Input Bandwidth Base peak intensity scaling to 4 11 4 12 Basics Instrument Control Panel 4 2 Sequence Control Panel 4 32 Batch acquisition 6 3 7 2 Beam guide wire function 1 23 5 18 5 56 replaced by lon focusing lens on STR models with serial number 4154 and later 1 33 1 35 voltage 5 18 Bench space requiremen
556. ter 2 Evaluating results Saving Deleting data files 2 70 Applied Biosystems Installing the Voyager Biospectrometry Workstations NOTE If the reference position is located in the center of the plate most corrections tend to be negative values Do not expect to see all colors in the color legend represented in position results Examine colors to ensure e No positions display a black edge indicating a problem with optimization e The reference position corresponds to the color legend at position 0 00 e Colors of positions change evenly and gradually with increasing distance from the reference position When acauisition of all positions is complete and you have examined results click Save The optimization file is assigned the same name as the Plate ID given an OPT_ prefix and an XML extension and stored in the same directory as the PLT file For example if you run OptiPlate for a Lab1 Plate ID an optimization file called OPT_Lab1 XML is created Do not delete or move the XML files These files are automatically accessed by the software when you select the Optimize Mass Accuracy option when loading a sample plate After performing plate optimization and saving the optimization file you can delete the data files created during this procedure OptiPlate results are associated with the XML file that was created described above not with data files Data files are no longer needed even if you ap
557. ter 3 Preparing Samples Procedure To clean samples with ZipTips 1 Wash a C 8 ZipTip in the following order with e 10 ul of ACN e 10 ul of 50 50 ACN 0 1 TFA e 10 ul of 0 1 TFA e Repeat wash of 10 ul of 0 1 TFA To wash draw a few microliters of a wash solution up into the ZipTip and expel to waste 2 Draw a few microliters of the sample up and down in the ZipTip several times 3 Discard the liquid The sample is bound to the C4g surface in the ZipTip 4 Wash the ZipTip again with 10 ul of 0 1 TFA by drawing up into the ZipTip and expel to waste 5 Repeat step 4 two times 6 Elute the sample with 2 to 3 ul of organic solvent or matrix containing approximately 50 percent ACN by drawing solvent or matrix solvent up and down in the ZipTip several times NOTE A 50 percent organic concentration should give 50 to 70 percent sample recovery You can vary the organic concentration to optimize recovery 7 Elute samples directly on the plate as described in Section 3 2 3 Loading Samples Dried Droplet Application or into a microcentrifuge tube for storage 3 32 Applied Biosystems Preparing Samples 3 1 6 Mixing Sample and Matrix Dried Droplet Application When to use Premixing sample and matrix Use dried droplet application if you are analyzing samples with a concentration gt 0 01 pmol ul If you are analyzing samples with a concentration lt 0 01 pmol ul use the thin layer application t
558. ter 8 PSD Analysis Benefits The benefits provided by CID include e Fragmentation of ions that does not occur under normal PSD conditions e Side chain fragmentation that may allow you to distinguish between Leucine and Isoleucine isomers e Greater number of immonium ions generated for peptide analysis Figure 8 10 and Figure 8 11 are sample spectra from low and mid mass ranges that illustrate the impact of CID gas 32004 CID off 410 24000 A o be te kog e 8 7 T 1 mt Lise 16000 CID on enhanced peaks A labeled 7 bh te 8000 o W 0 T T T T T T T 55 75 95 115 135 155 175 195 Figure 8 10 Low Masses Impact of CID Angiotensin 1 In the top trace CID off typical fragments are seen and labeled In the bottom trace CID on fragments not seen without CID or fragments that are significantly enhanced with CID are labeled 8 32 Applied Biosystems Enhancing Fragmentation with CID 400004 CID off 320004 1017 y10 y11 17 y13 b7 18 240004 2 T tr we booed oat nes Ainaa PO ha ERNE NAL BAIAS A Mel adenan an han Anaan Aan a CID on enhanced peaks labeled 16000 650 850 4050 1250 i 1450 Mass mz Figure 8 11 Mid Masses Impact of CID Glu 1 Fibrinopeptide In the top trace CID off typical fragments are seen and labeled In the bottom trace CID on w fragments not seen without CID are labeled
559. teria holds the data in memory and averages current data with previous data If it fails the system displays an error message in the Output window describing which spectrum failed and which acceptance criteria specified have failed The data is discarded 7 Ifthe data meets the Acceptance Criteria stays on the same search pattern position If the data fails the Acceptance Criteria moves to the next search pattern position Voyager Biospectrometry Workstation User s Guide 6 67 6 68 Applied Biosystems Chapter 6 Acquiring Spectra from the Instrument Control Panel Repeats step 2 through step 7 in subsequent search pattern positions until any of the following is true Number of spectra to acquire that you select in the Automatic Control dialog box is reached e All search pattern positions have been scanned e Stop conditions are met Saves the averaged spectrum to disk in the directory designated in the Data Storage control page described in Setting Data Storage parameters on page 6 14 Chapter 7 Acquiring Spectra 7 from the Sequence Control Panel This chapter contains the following sections 7 1 OVEFVIGW nm nain ie tree 7 2 7 2 Understanding Settings Macros and Calibration ice rites mimi tn 7 3 7 3 Before Creating a Sequence 7 7 7 4 Creating a SEQUENCE n seeren 7 13 7 5 Preparing to Run a Sequence 7 24 7 6 Running a Sequence
560. teria Save Conditions which save individual multiple spectra in one data file as described below the recommended maximum Number to Acquire is 500 Acquiring more than 500 individual spectra in one file generates a very large data file that can be slow to open and process Spectrum The following Spectrum Accumulation conditions create Accumulation one data file that contains multiple spectra e Save all spectra e Save all spectra that pass acceptance criteria If you select either of these conditions the recommended maximum Number of spectra to acquire is 500 Acquiring more than 500 individual spectra in one file generates a very large data file that can be slow to open and process You can display the Chromatogram window in the Data Explorer software for DAT files containing multiple spectra For more information see Section 5 2 4 Description of Spectrum Accumulation Options continued Voyager Biospectrometry Workstation User s Guide 5 35 Chapter 5 Optimizing Instrument Settings Table 5 10 Automatic Control Parameters Spectrum Accumulation Continued Parameter Description Spectrum Accumulation continued The following Spectrum Accumulation conditions create one data file that contains one spectrum e Save the first spectrum to pass acceptance criteria e Save the best spectrum e Accumulate all e Accumulate all passing For more information see Section 5 2 4 Descripti
561. ternal PSD calibration below which is used to determine the values fora B and y needed for the PSD calibration equation NOTE If default calibration yields acceptable mass accuracy for your application an external calibration is not required In the PSD Acquisition Settings control page type the Precursor mass NOTE Precursor mass is used for PSD calibration Type in an accurate value with appropriate precision for example type 1296 68 not 1297 Select Precursor lon Selector if it is not already selected NOTE Leave the Precursor lon Selector enabled when performing PSD analysis The only time you may want to disable the Precursor lon Selector is to determine if it is working Voyager Biospectrometry Workstation User s Guide 8 45 Chapter 8 PSD Analysis 8 46 Applied Biosystems To set PSD calibration select the default calibration or select a previously generated external PSD CAL file For more information see the Data Explorer User s Guide Chapter 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files NOTE The calibration you specify on the PSD Acquisition Settings control page is used to determine the values fora B andy needed for the PSD calibration equation described on page 8 28 The calibration you specified on the Instrument Settings control page in step 4 is used to determine the value for t precursor ion flight time needed for
562. tes for more information e Handle the sample plate by the edges e Use powder free gloves if you wear gloves Guidelines for To ensure good crystallization f good e Mix sample and matrix before applying If you are mixing crystallization sample and matrix directly on the sample plate apply sample before matrix to prevent matrix from drying Ifyou are using a welled plate fill the entire well when loading the sample plate if possible especially if acquiring in Automatic control mode Surface tension and sample availability may determine whether you fill the well completely Do not touch the surface of the sample well with the pipette tip may cause uneven crystallization Recommended Use finely tapered pipette tips to dispense sample matrix pipette tips solution on the sample plate Tips with blunt ends do not easily dispense the small volumes used for sample loading Voyager Biospectrometry Workstation User s Guide 3 37 Chapter 3 Preparing Samples 3 2 2 Locating Standards for Optimum Mass Accuracy 3 38 Without Mass Accuracy Optimization With Mass Accuracy Optimization Applied Biosystems In a typical analysis run that requires optimum mass accuracy you include samples interspersed with many calibration standards and externally calibrate the samples using the closest calibration standard The number and location of standards you need depends on whether or not you use the Mass Accuracy Op
563. the first passing spectrum is found One DAT file containing one spectrum is created If no spectra meet criteria no DAT file is created NOTE If you set the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified number of spectra to acquire or the number of search pattern positions in the SP file Voyager Biospectrometry Workstation User s Guide 5 43 Chapter 5 Optimizing Instrument Settings Save the best In Save the Best Spectrum mode Figure 5 11 spectrum Automatic Sample Positioning is required e Acquisition is performed on each search pattern position until the specified number of spectra to acquire is reached NOTE In Save the Best Spectrum mode acquisition moves to the next search pattern position for every acquisition even if acceptance criteria pass e All Acceptance Criteria are evaluated e If the first spectrum passes all acceptance criteria it is displayed in the Spectrum window as an accumulated trace and considered the current best spectrum e When a subsequent spectrum that passes all acceptance criteria is acquired its Signal to Noise result is compared to the Signal to Noise result of the current best spectrum If the result is higher the new spectrum replaces the current best spectrum in the accumulated trace If the result is lower the new spe
564. the PSD calibration equation NOTE If default PSD calibration yields acceptable mass accuracy for your application an external calibration is not required NOTE PSD CAL files are named with the same extension as reflector or linear CAL files If you select a reflector or linear CAL file when in PSD mode an error message is generated Acquiring PSD Data with Standard BIC Files in Manual Control Mode 8 4 4 Filling in the Segment List and Saving the BIC File This section describes e Filling in the list e Typing or selecting new values Using the Fill Down command e Adding or deleting rows e Saving the BIC file Filling in the list To fill in the segment list If you are using 1 If you are using the standard instrument settings BIC Angiotensin_PSD file provided with the software Angiotensin_PSD BIC BIC the segment list contains 10 segments with the Mirror Ratio settings listed in Default Mirror Ratio settings on page 8 41 If the precursor ion mass you are analyzing differs by more than 300 Da from the mass in the Angiotensin_PSD BIC 1296 Da you may need to acquire more or fewer than 10 segments to observe the fragment ions of interest You can add or delete segments or change the Mirror Ratio for a segment as needed If you are creating If you are creating a new BIC file click f to add the anew BIC file required number of rows for the number or segments to acquire For
565. the Voyager Biospectrometry Workstations Refer to the following table when you connect the Acqiris 2 GHz digitizer board to the mass spectrometer Connection on right side panel of mass spectrometer see Figure 2 2 Connection on rear panel of computer see Figure 2 4 Cable TRIG EXTERNAL Trig BNC on mass spectrometer to EXTERNAL BNC on board CH 1 INPUTA Ch1 BNC on mass spectrometer to 90V BNC Spark Gap to 3d BNC Attenuator to INPUTA BNC on board Ground wire to computer chassis ground screw CH 2 DE PRO only INPUTB Ch2 BNC on mass spectrometer to 90V BNC Spark Gap to 3d BNC Attenuator to INPUTB BNC on board Ground wire to computer chassis ground screw 2 18 Applied Biosystems Connecting Voyager DE and Voyager DE PRO Workstations 2 3 6 Connecting the Tektronix Oscilloscope If you have an internal digitizer board or external digitizer in your computer disregard this section Figure 2 6 shows the front panel of the oscilloscope To TRIG on side panel of mass spectrometer PB100770 on side panel on side panel of mass of mass spectrometer spectrometer DE PRO systems only Figure 2 6 Oscilloscope Connections Voyager Biospectrometry Workstation User s Guide 2 19 Chapter 2 Installing the Voyager Biospectrometry Workstations Refer to the following table when you connect the oscilloscope to the mass spectrometer and t
566. the current segment Repeat step 3 through step 6 to collect remaining segments You can collect segments in any order and do not have to acquire all segments in the list You can also acquire multiple segments with the same Mirror Ratio NOTE Segments are listed in the Data Explorer software in the order in which they are acquired If segments with duplicate Mirror Ratios are contained in the file the software uses the last acquired segment when it generates the composite spectrum Voyager Biospectrometry Workstation User s Guide 8 55 Chapter 8 PSD Analysis Reacquring a 8 To reacquire a segment Segment Select the segment to reacquire Click 2 2 A new row is added to the end of the segment list and contains the settings from the selected segment e Set parameters as needed e Repeat step 3 through step 6 Stopping the 9 When you have acquired all necessary segments select experiment Stop Experiment from the Acquisition menu The PSD data file is closed CAUTION If you stop an experiment without saving any segments no DAT file is created 8 56 Applied Biosystems Acquiring PSD Data with Standard BIC Files in Automatic Control Mode 8 5 Acquiring PSD Data with Standard BIC Files in Automatic Control Mode Before you begin Steps to acquire During PSD analysis in Automatic Control mode e The laser is automatically adjusted for PSD segments with different Mirror Ratios
567. the sample position Importing and exporting the run list Sample order in The type of calibration you perform may require a specific the run list sample order in the run list Internal or external calibration List samples in any order e Close external calibration internal update List samples and standards as described in Section 7 7 3 Performing Close External Calibration 7 14 Applied Biosystems Creating a Sequence Creating a run list To create a new run list 1 Select New Sequence from the File menu A new sequence run list grid opens Figure 7 6 Sequence Satay SemercelieNare fods Ormak Far Stata prenas Elegued Tase I G w Cameri Erny indes a Parsarweg Ertas a Run list Figure 7 6 Sequence Run List Hint You can show and hide columns by selecting a column then selecting Show Hide from the View menu 2 Click the scroll bar at the bottom of the grid to display columns in the grid that are not in view 3 Click a cell to activate it then enter Run List parameters as described in Table 7 1 You can also import parameters into the run list See page 7 22 Voyager Biospectrometry Workstation User s Guide 7 15 Chapter 7 Acquiring Spectra from the Sequence Control Panel Table 7 1 Run List Parameters Field Description Sample Position required entry Position from which to acquire data Data File required entry Base name of the
568. timization option when you analyze samples described in Section 3 4 2 Using the Mass Accuracy Optimization Option To obtain optimum mass accuracy without using the Mass Accuracy Optimization option you must e Spot standards in every other position relative to samples e Locate the standards as close as possible to samples as on the 96 x 2 sample plate Do not use the outer wells on the sample plate when using flat or polished blank plates If you use the Mass Accuracy Optimization option the number and location of standards you need to obtain optimum mass accuracy depends on Whether or not you use an optimized sample plate The type of sample plate you use Loading Samples on Sample Plates With an optimized To obtain optimum mass accuracy using the Mass Accuracy plate Optimization option and a plate you have optimized with OptiPlate you can Spot one or a few standards on the plate The number of standards required depends on the plate you use and your mass accuracy needs Plate Number of Standards Needed 96 x2 1 center 64 100 384 400 plates 5 with positions located near center and 4 the physical edge of the plate corners Locate standards anywhere on the plate for example in a center position on a 96 x 2 plate and in four corner positions on other plates that have positions located near the physical edge of the plate When you analyze set the run to update the calibratio
569. tion 0 02 Reflector mode External Calibration 0 008 Internal Calibration 0 001 Mass Accuracy of Fragment lons 1 Da Resolution of PSD Precursor Timed lon Selection 80 Table A 8 Voyager DE STR Mass Spectrometer Specifications Condition Specification Mass range 2300 000 Da Upper limit set by suitably ionized biomolecule Reflector Single stage with optimized optics for PSD Analysis Flight tube horizontal Linear mode 2 0 m e Reflector mode 3 0 m lon source Two stage lon source voltages Tunable e Accelerating Voltage Up to 25 000 V Grid Voltage Range determined by Accelerating Voltage Laser Nitrogen 337 nm 3 ns pulse 20 Hz maximum firing rate Actual laser firing rate dependant on digitizer See Section A 4 Digitizer Specifications Digitizer 2 GHz digitization for enhanced resolution See Section A 4 Digitizer Specifications Voyager Biospectrometry Workstation User s Guide A 9 Appendix A Specifications Table A 8 Voyager DE STR Mass Spectrometer Specifications Continued Condition Specification Vacuum system Dual differential turoomolecular pumping for ultra high vacuum e Multi vacuum gauge capability for independent monitoring of source and analyzer regions lon detection Positive and negative Sample analysis Automated single plate sample loading system sample plates of v
570. tion see Section 4 5 1 Using Toolbar Buttons and Instrument Menu Commands Voyager Biospectrometry Workstation User s Guide 4 3 Chapter 4 Voyager Instrument Control Panel Basics Control pages The Instrument Control Panel contains five control pages e Instrument Settings Controls settings for instrument mode voltages spectrum acquisition and calibration For more information see Chapter 5 Optimizing Instrument Settings e Data Storage Controls data storage information such as file location and file name For more information see Setting Data Storage parameters on page 6 14 e System Status Contains information regarding the status of the instrument For more information see Section 2 11 Checking System Status and Pressures e Manual Laser Sample Positioning Allows you to manually control the laser intensity and sample plate position For more information see Section 4 5 2 Adjusting Laser Intensity and Selecting Sample Position e PSD Acquisition Settings Allows you to control PSD acquisition For more information see Chapter 8 PSD Analysis NOTE The PSD Acquisition control page is displayed only if you are in PSD mode on a Voyager DE PRO or Voyager DE STR system You can access a control page by selecting it from the View menu or clicking the corresponding toolbar button You can display any combination of control pages For information see Section 4 2 Using the Control Page
571. to acquire multiple samples using different instrument settings BIC files e Data Explorer processing software Allows post processing analysis of mass spectral data 1 7 1 Control Software Instrument and Sequence Control Panels Instrument The Voyager Instrument Control Panel allows you to directly Control Panel control the Voyager mass spectrometer to acquire mass spectra one at atime The Instrument Control Panel provides the following features Mass calibration mass resolution calculator and signal to noise ratio calculator functions e Direct control of instrument hardware including high voltages load eject and acquisition start stop e Real time viewing and manipulation of mass calibrated default or external calibration and peak labeled spectral traces as data is acquired with Acqiris LeCroy and Signatec digitizers 1 42 Applied Biosystems Software Overview e Ability to zoom in on up to four different areas of a trace e Ability to acquire single samples in Manual or Automatic Control mode e Manual accumulation of mass spectra from multiple acquisitions into a single data file The Instrument Control Panel Figure 1 21 is displayed when you start the Voyager Control Panel software The Instrument Control software is described in Chapter 4 Voyager Instrument Control Panel Basics Voyager Instrument Control Panel SPEC Default Instrument Settings L File Edit View Instrument Acq
572. toolbar is not displayed in the Data Explorer software select Toolbar from the View menu select Macros then click Close continued Voyager Biospectrometry Workstation User s Guide 7 17 Chapter 7 Acquiring Spectra from the Sequence Control Panel Table 7 1 Run List Parameters Continued Field Description Internal External Calibration type to use for the row Click the down arrow and Calibration select one of the following e Blank No calibration is applied during processing Acquisition calibration calibration specified in the BIC file used to acquire is maintained e External Applies the constants in the specified CAL file to the data file acquired in the row e Internal lf a CAL file is specified and it exists constants are applied to the DAT file before the reference masses in the SET file are matched New calibration constants are generated using the reference masses specified in the SET file and applied to the data Constants are then updated within the DAT file after calibration For more information see Section 7 7 4 Internal Standard Calibration Considerations e Internal Update lIf a CAL file is specified and it exists constants are applied to the DAT file before the reference masses in the SET file are matched New calibration constants are generated using the reference masses specified in the SET file and applied to the data Constants are then updated within the D
573. tor mode 4 25 PSD mode 8 55 single shot mode 2 45 Scaling see also Display Range digitizer signal 5 30 to Absolute Value 4 12 to Base Peak 4 11 to Display window 4 11 to minimum and maximum counts 4 12 Screen savers do not use 1 30 1 41 Search Pattern Editor automatically generating spots 6 52 drawing a pattern 6 56 Search Pattern Generator 6 52 setting x y coordinates 6 57 using 6 50 Search pattern file see also Search Pattern Editor see also Search pattern random converting version 4 to version 5 5 92 DEFAULT SP 6 48 definition 6 46 description 6 47 difference between version 4 and version 5 5 92 example for internal calibration 7 41 for custom plates 3 72 for PLT files 3 73 internal calibration creating for separate spots 7 38 7 40 location 6 46 maximum number of positions 6 47 radius to ensure analysis of correct sample 3 74 specifying 5 38 SPIRAL SP 6 49 units of measure 6 48 Index 36 Applied Biosystems Search pattern random 5 37 Segments PSD see PSD segments Select button on oscilloscope I 5 Sensitivity see also Intensity signal see also Signal to noise impact of changing instrument setting parameters 5 49 improving 5 30 5 56 Linear mode 5 25 range Voyager DE 1 4 range Voyager DE PRO 1 4 range Voyager DE STR 1 6 troubleshooting 9 10 Vertical Scale parameter effect on 5 30 SEQ files 7 20 Sequence see also Sequence Control Panel acquiring 7 25 before creating
574. traces do not print change the line width or color Voyager Biospectrometry Workstation User s Guide 4 19 Chapter 4 Voyager Instrument Control Panel Basics 4 20 Dedicating a printer to landscape orientation Print Setup Applied Biosystems To dedicate the printer to landscape orientation set the orientation from the Windows desktop 1 2 3 4 5 Click Start then select Settings Click Printers Select the printer name in the list displayed Click on File and select Document Defaults In the Page Setup Tab select Landscape orientation NOTE This printer setting will affect all applications that use the printer not just the Voyager software NOTE If you cannot select Landscape orientation you do not have access permission See your administrator Print Setup allows you to select a printer and set other printer options For more information on Print Setup and connecting printers to your computer refer to the documentation provided with your computer Customizing the Instrument Control Panel 4 4 Customizing the Instrument Control Panel Undocking toolbars Customizing the toolbar Customizing the display The toolbar at the top of the Instrument Control Panel is divided into sections A section is preceded by a double vertical bar You can undock each section of the toolbar and move it anywhere within the Instrument Control Panel by click dragging the doub
575. traction Technology seernes aaa nes ant tn 1 11 1 5 Parts of the Voyager DE and Voyager DE PRO Systems 1 17 1 6 Parts of the Voyager DE STR System 1 31 1 7 Software Overview 1 42 Voyager Biospectrometry Workstation User s Guide 1 1 Chapter 1 Introducing the Voyager Biospectrometry Workstations 1 1 Voyager DE and Voyager DE PRO System Overview The Voyager DE and Voyager DE PRO Biospectrometry Workstations are designed for use by mass spectrometrists biochemists molecular biologists and life scientists Voyager DE The Applied Biosystems Voyager DE Biospectrometry Workstation Figure 1 1 is a benchtop MALDI TOF matrix assisted laser desorption ionization time of flight mass spectrometer Voyager Delayed Extraction technology provides improved resolution and mass accuracy ae El Rs amp PB100465 Figure 1 1 Voyager DE Biospectrometry Workstation 1 2 Applied Biosystems Voyager DE M and Voyager DE PRO System Overview Voyager DE PRO The Voyager DE PRO Biospectrometry Workstation Figure 1 2 is a benchtop MALDI TOF matrix assisted laser desorption time of flight mass spectrometer that includes a reflector analyzer Voyager De
576. traction Mode 6 21 6 3 1 What Is a Good Spectrum ccecceeeeeeeeeeeeeeeeeeeeeeeaaeeanaaaes 6 21 6 3 2 Laser Intensit aed cea ieee 6 24 6 3 3 Parameters Affecting Resolution and Signal to Noise Ratio ccccccceeeeeeeeeeeeeeeeeeeeeseeaeaaaeneeeeeeenees 6 25 Making Accurate Mass Measurement 6 26 Applied Biosystems 6 5 6 6 Table of Contents Evaluating Data in the Instrument Control Panel 6 28 6 5 1 Detecting Integrating and Labeling Peaks 6 28 6 5 2 Calculating Mass Resolution cceceeeeeeeeeeeeeeeeeeeeeeeeeeeeaeaaes 6 30 6 5 3 Calculating Signal to Noise Ratio cccceeeeeeeeeeeeeeeeeeeeeaees 6 33 Acquiring in Automatic Mode from the Instrument Control Panel 6 35 6 6 1 Before Acquiring in Automatic Control Mode 6 36 6 6 2 Setting Instrument Settings for Automatic Control Mode 6 37 6 6 3 Automatically Acquiring Evaluating and Saving Spectra 6 45 6 6 4 Search Patterns iii ieiieeerreesnse 6 46 6 6 5 Creating and Editing SP Files Using the Search Pattern Editor 00 0 ce cecceec cece een eeaeeneesuesneeeaeanees 6 50 6 6 6 Process that Occurs During Acquisition in Automatic Mode 6 58 6 6 7 Process that Occurs when Accumulating Spectra from Multiple Search Pattern Positions cccccceceeeeeeeeeeeeeeeeeees 6 65 6 6 7
577. trix Sample Time of Flight TOF Applied Biosystems Figure 1 4 Matrix Assisted Laser Desorption lonization Time of flight mass spectrometry works on the principle that if ions are accelerated with the same potential from a fixed point and at a fixed initial time and are allowed to drift the ions will separate according to their mass to charge ratios Lighter ions drift more quickly to the detector Heavier ions drift more slowly Figure 1 5 lons generated by MALDI exhibit a broad energy spread after acceleration in a stationary electric field By forming ions in a field free region and then applying a high voltage pulse after a predetermined time delay to accelerate the ions this energy spread can be minimized See Section 1 4 Voyager DETM Delayed Extraction Technology for more information MALDI TOF MS Technology Overview For acquiring TOF spectra time measurement depends on extraction mode Delayed Extraction mode Measurement of the ion signal starts when the extraction pulse is applied The time at which the extraction pulse is applied is user settable See Section 1 4 Voyager DE Delayed Extraction Technology for more information e Continuous Extraction mode The extraction field is continuously applied Measurement of the ion signal starts when the laser pulses Heavier Lighter Lighter Heavier inne lanes ions ions we Se ass Intensity oe Ce Flight Path Time Figu
578. trum as it is acquisition acquired Mass range for the segment is set to Mass Equivalent to Start Precursor mass 4 which is equal to Precursor flight time 2 End Mirror to Accelerating Voltage Ratio x Precursor mass which is equal to Mirror to Accelerating Voltage Ratio x Precursor flight time All instrument settings are disabled Settings remain inactive until you stop the PSD experiment or all segments are acquired e Acquisition of the segment continues until the number of Shots Spectrum specified in Spectrum Acquisition on the Instrument Settings control page is collected or until you select Stop Acquisition from the Acquisition menu e The segment spectrum is displayed in the Current Trace in the Spectrum window and saved to the data file After all segments are acquired the software automatically stops the experiment then closes and saves the PSD data file 8 64 Applied Biosystems Exploring PSD Mode 8 6 Exploring PSD Mode Preparing the practice standard To be successful in PSD analysis you need to understand how ions behave in PSD mode and how to optimize acquisition conditions Before running samples spend some time practicing with standards In this section you will observe the effects of e Laser intensity on fragment ion production and signal intensity e Precursor lon Selector on prompt fragments e Grid Voltage on resolution This section assumes that y
579. trum for each digitizer are as follows e Signatec 10 000 e LeCroy 1 000 e Acqiris 10 000 e Tektronix 10 000 NOTE The actual number of times the laser fires may be greater than the number of Shots Spectrum specified due to the limited averaging speed of the digitizer For example if 250 000 data points are recorded every other laser shot is skipped by the digitizer In any case the specified number of Shots Spectrum will be stored in the data file NOTE This parameter is dimmed if the system is set to Single Shot mode For information see page 2 44 Spectrum Acquisition Mass Range Da Determines the mass range collected Valid ranges are e Start Mass 0 to End Mass e End Mass Start Mass to End Mass Mass Range can affect optimized laser rate described on page 5 26 continued Voyager Biospectrometry Workstation User s Guide 5 19 Chapter 5 Optimizing Instrument Settings Table 5 4 Instrument Settings Parameters Continued Parameter Description Spectrum Acquisition continued Low Mass Gate Da Turns on the detector voltage after the ions of the Mass specified have passed the detector lon masses below this entry are not considered during analysis Suppresses matrix peaks that can interfere with ion detection and saturate the detector as laser intensity increases Turn on Low Mass Gate when e Analyzing masses greater than 2 000 to 3 000 Da e Matrix
580. truments avoid full scale signals 64 000 counts which may be saturated To avoid the saturation problem accumulate spectra with appropriate evaluation criteria See Setting spectrum acceptance and laser adjustment criteria on page 6 42 for more information e Signal intensity of reference and analyte peaks above 1 000 counts Signal intensity above 1 000 counts with a Shots Spectrum setting of 50 or greater ensures adequate peak statistics needed for accurate assignment of peak position 6 26 Applied Biosystems During mass calibration and peak detection Making Accurate Mass Measurements e Signal intensity of reference and analyte peaks is in the same range Signal intensity of reference and analyte peaks should be of similar magnitude If they are not in the same range you may see weak analyte peaks with poor signal to noise ratio while the internal standard is adequate or saturated internal standard peaks with adequate analyte peaks __No contaminants present Unresolved contaminants can affect peak shape Two internal reference masses that bracket the mass range of interest Best results are obtained when masses above and below the mass of interest are correctly identified NOTE Although not required using more than two reference masses can help to minimize the effects of an asymmetrical reference peak Internal reference masses are within the same range as analyte masses Best results are
581. ts peripherals 2 3 Voyager DE and Voyager DE PRO 2 2 Voyager DE STR 2 7 A 10 Biacore Chip sample plate 3 63 BIC files see also Instrument Settings files BIC Linear mode 5 4 list of 5 3 location 5 3 opening 5 7 opening from DAT file 5 7 parameters saved in 5 2 Voyager Biospectrometry Workstation User s Guide Index 5 I N D E X Index BIC files continued provided with system 2 30 5 3 PSD mode 5 6 Reflector mode 5 5 saving after acquisition to store laser intensity 6 19 Sequence Control Panel requirements 7 8 7 27 Sequence Control Panel selecting 7 16 Bin size digitizer default 5 57 description 5 29 5 57 impact on data resolution 5 50 5 59 impact on file size 5 59 Biospectrometry definition 1 3 1 6 BLANK PLT 3 77 Bovine Trypsin molecular weight F 2 Bradykinin mass to time conversion F 4 molecular weight F 2 BSA BIC file 5 4 buffers 9 14 H 21 mass to time conversion F 5 molecular weight F 3 C C18 sample cleanup 3 31 Cables required 2 12 CAL file creating 6 7 PSD selecting 8 46 8 60 selecting 6 13 CAL file Sequence Control Panel creating 7 11 description 7 4 selecting 7 19 Index 6 Applied Biosystems Calibration accurate mass 6 26 automatic see Calibration automatic correction factors to improve mass accuracy 2 53 default see Calibration default deisotoping to improve mass accuracy 7 10 equation 6 9 external see Calibration external high voltage warmup
582. ts Defines the units of the sample positions that are displayed in the optional Manual Laser Sample Positioning control page see Figure 4 9 on page 4 31 If you do not include the WellUnits parameter the software uses the default Allowable values e 1 Steps default e 2 Microns Automatically set to microns if you create the PLT file using the Create PLT File function described in Creating a PLT file using Create PLT File on page 3 79 WellShape Defines the shape of the sample positions that are displayed in the optional Manual Laser Sample Positioning control page If you do not include the WellShape parameter the software uses the default Allowable values e 1 Elliptical default e 2 Rectangular Corresponds to Position Shape parameter in the Create PLT File dialog box see Table 3 14 Create PLT File Parameters on page 3 81 WellWidth Defines the width of the sample positions that are displayed in the optional Manual Laser Sample Positioning control page If you do not include the WellWidth parameter the software uses the default based on the value selected for WellUnits Default values e Default Step value 800 e Default Micron value 2 540 Corresponds to Position Width parameter in the Create PLT File dialog box see Table 3 14 Create PLT File Parameters on page 3 81 Automatically set to microns if you create the PLT file using Create PLT File contin
583. ttern position is acquired if the Use Automated Sample Positioning option is disabled acquisition is performed on the positions you manually select instead of search pattern positions All search pattern positions may not be analyzed e Acceptance criteria are not used e One DAT file containing multiple spectra is created r Spectrum Accumulation Acquire E spectra under conditions Save all spectra gt 1 spectrum from each position Each spectrum has 50 shots as specified in the instrument settings Spectrum saved in DAT file has 50 shots 50 shots 1 spectral Number of DAT files 1 Figure 5 8 Save All Spectra Mode Automatic Control Dialog Box Voyager Biospectrometry Workstation User s Guide 5 39 Chapter 5 Optimizing Instrument Settings Examples If Random search pattern is selected and Number to Acquire is 5 five positions are analyzed and one DAT file containing five spectra is created If an SP file with 20 positions is selected and Number to Acquire is 5 five positions are analyzed and one DAT file containing five spectra is created If an SP file with 20 positions is selected and Number to Acquire is 25 20 positions are analyzed and one DAT file containing 20 spectra is created Save all spectra In Save All Spectra That Pass Acceptance Criteria mode that pass Figure 5 9 acceptance e Each spectrum that meets the specified acceptance criteria criter
584. ued Voyager Biospectrometry Workstation User s Guide 3 69 Chapter 3 Preparing Samples Table 3 13 PLT File Parameters Continued Parameter Description WellHeight optional Defines the height of the sample positions that are displayed in the Manual Laser Sample Positioning control page If you do not include the WellHeight parameter the software uses the default based on the value selected for WellUnits Default values e Step value 800 e Micron value 2 540 Corresponds to Position Height parameter in the Create PLT File dialog box see Table 3 14 Create PLT File Parameters on page 3 81 Automatically set to microns if you create the PLT file using Create PLT File WellDepth optional Depth mm of indentation of a non flat plate for example a welled plate from the surface of the plate to the bottom of the indentation If not present the system assumes a depth of 0 equal to a flat plate Corresponds to Position Depth mm parameter in the Create PLT File dialog box see Table 3 14 Create PLT File Parameters on page 3 81 X and Y The coordinates of the position center in reference to the lower left corner of the sample plate To determine the coordinates to enter in the PLT file display the Sample View see Figure 4 9 on page 4 31 and record the Absolute X Absolute Y logical coordinates pairs for each specified position See Logical Coordinates in Plate
585. uence Control Panel at the left or right of the desktop and the Instrument Control Panel in the opposite location Voyager Biospectrometry Workstation User s Guide 4 35 Chapter 4 Voyager Instrument Control Panel Basics 4 36 Applied Biosystems 5 Optimizing Instrument Settings This chapter contains the following sections 5 1 Loading Modifying and Saving Instrument Settings 5 2 5 2 Instrument Settings Parameter Descriptions 5 14 5 2 1 Instrument Settings Page eeeeeceeeeeeeeteeeeeeeeeeeees 5 15 5 2 2 Mode Digitizer Dialog Box 5 24 5 2 3 Automatic Control Dialog BOX sses 5 33 5 2 4 Description of Spectrum Accumulation Options 5 39 5 3 Impact of Changing Instrument Settings Parameters 5 49 5 3 1 Summary of Parameters cccceeeeeeeeeeeaeeeneeneeeees 5 49 5 3 2 Understanding Grid Voltage s e 5 51 5 3 3 Understanding Delay Time 5 54 5 3 4 Understanding Guide Wire Voltage 5 56 5 3 5 Understanding Digitizer Settings cee 5 57 5 4 Optimizing Instrument Settings Parameters 5 64 5 4 1 Optimization Strategy ccceeeeeeeeeeeeeeeeeeeeseeeneeeees 5 65 5 4 2 Determining the Laser Setting ceeeceeeeeeeeeeees 5 67 5 4 3 Optimizing Resolution eececeeeeeeeeee eee eeeeeeneeeeeeees 5 71 5
586. uence Status control page general status of run e Instrument Control Panel Checking the The Acquisition Status field in the run list displays the status of Acquisition each spectrum as it is acquired Possible states are Status field Acquiring Acquiring a sample e Processing Applying macros or calibrating the data file Acq Error Error occurred during acquisition Proc Error Error occurred during processing e Success Sample processing successful NOTE The Acquisition Status field is updated each time a spectrum is acquired and processed for a line in the sequence When acquisition and processing is complete for the line the status reflects the status for the last spectrum acquired and processed It does not reflect the overall status of all acquisitions performed by the line Voyager Biospectrometry Workstation User s Guide 7 29 Chapter 7 Acquiring Spectra from the Sequence Control Panel Checking the Select Status Panel from the View menu to check the status Sequence Status of a sequence The Sequence Status panel Figure 7 7 is panel displayed Sequence Status Sequence File Name Autos eql Overall Run Status OFF Elapsed Time CS Current Entry Index CS Remaining Entries CS Figure 7 7 Sequence Status Panel Sequence status parameters include Field Description Sequence File Name Displays the name of the SEQ file currently running Overall
587. uired see page 5 37 e Search pattern SP file The software analyzes positions that correspond to specific x y coordinates defined in the SP file For more information see Search pattern SP files below You specify the search pattern option in the Automatic Control Settings of the Instrument Settings control page See Section 5 2 3 Automatic Control Dialog Box for more information NOTE Before using search pattern files created in version 4 software convert to version 5 format See Section 5 5 Converting Version 4 Methods and Search Pattern Files You can create customized search pattern files or use the DEFAULT SP or SPIRAL SP files provided for 100 position plates For details see Creating and Editing SP Files Using the Search Pattern Editor on page 6 50 Acquiring in Automatic Mode from the Instrument Control Panel A search pattern file is an ASCII text file that contains a list of relative X Y position pairs with respect to the center of the current sample position measured in microns that represent points in the sample position On a 100 position plate a sample position is 2 540 um in diameter with the origin 0 0 at the center of the position The centers of the sample positions are 5 080 um apart Figure 6 17 illustrates the location of different coordinates The system sequentially reads each entry in the search pattern file as it analyzes a sample The first entry in the file is
588. uisition SamplePlate Display Tools Applications e fu lelweles alm le m Data Storage Filename I Autosequence Filenames Sample Description Comment Manual Laser Intensity 18 44 Active Pos 91 X 100 well plate plt Current Directoy E WoyagenData m id Lu Linear Positive Mode Digiizer Manual Sample Positioning SE 1B dE ooo 000 see eee o o Le Le o o Le CODEC Kay Ce Lhe Ke oy ter Instrument Mode p Control Mod_e Manual C Automatic Automatic Control r Voltages Accelerating 20000 v Grid 34 0 0 110 0 Guide Wire 0 05 0 000 0 300 Delay Time 100 nsec Spectrum Acquisition Shots Spectrum 50 Mass Range Da 500 to 5000 F Low Mass Gate Da 500 Intensity Calibration _______________ Matrix 3 Cyano 4 hydroxycinnamic acid Default C Extemal File 0 1200 1600 2000 Mass m z TESTE TRIN Resutt A Automated Control Data Storage 7 Aeee High Voltage OFF Somes ased Mron 25200500 Acquisition OFF Control Mode MANUAL instrument Mode LIN POS Figure 1 21 Voyager Instrument Control Panel Voyager Biospectrometry Workstation User s Guide 1 43 Chapter 1 Introducing the Voyager Biospectrometry Workstations Sequence Control The Sequence Control Panel works with the Instrument 1 Panel Control Panel to allow you to acquire multiple samples using differ
589. uisition in Automatic Mode Starts firing the laser Acquires the specified number of Shots Spectrum at the first search pattern position Stops firing the laser Holds the data in memory and averages current data with previous data Moves to the next search pattern position Repeats step 2 through step 6 in subsequent search pattern positions until any of the following is true Number of spectra to acquire that you select in the Automatic Control settings is reached e All search pattern positions have been scanned e Stop conditions are met Saves the averaged spectrum to disk in the directory designated in the Data Storage control page described on page 6 14 Acquiring in Automatic Mode from the Instrument Control Panel 6 6 7 2 Process that Occurs when Accumulating Passing Spectra When accumulating only the spectra that meet the Acceptance Criteria Accumulation mode is determined by the Spectrum Accumulation conditions described in Section 5 2 3 Automatic Control Dialog Box the system does the following during acquisition 1 Sets the laser intensity as described in Section 6 6 6 Process that Occurs During Acquisition in Automatic Mode Starts firing the laser Acquires the number of Shots Spectrum at the first search pattern position Stops firing the laser Evaluates the data against the Acceptance Criteria in Section 5 2 3 Automatic Control Dialog Box 6 Ifthe data meets the Acceptance Cri
590. ultiple samples using different conditions Toolbar Run list Sequence Status Yee Dre miter Deij tte uf Sagares Sisa CLE a Oadet TETE tisset Ten i Cut tony res f 1 Figure 4 10 Sequence Control Panel The Sequence Control Panel includes e Toolbar Contains buttons that control the software and the instrument For a description of a toolbar button place the cursor on it A brief description of the button ToolTip is displayed below the button e Sequence Status Displays information about the currently running entry in the sequence e Run list Allows you to define sample position data file name instrument settings file Data Explorer SET file processing macros calibration type and CAL file used sample description comments for each sample acquisition status and a run checklist column to track completed samples 4 32 Applied Biosystems How the Instrument and Sequence Control Panels Interact When you start the Sequence Control Panel the Instrument Control Panel is automatically started and tiled horizontally at the bottom of the screen You can hide the Instrument Control Panel by deselecting Instrument Control Panel from the View menu For more information see Section 4 7 How the Instrument and Sequence Control Panels Interact and Chapter 7 Acquiring Spectra from the Sequence Control Panel 4 7 How the Instrument and Sequence Control Panels Interact This section inclu
591. um Forthe first spectrum you acquire the laser setting saved in the BIC file selected is applied If the laser intensity in the BIC is not optimized for the mass range you may not see a signal Make subsequent laser adjustments as described in Adjusting laser intensity on page 5 68 e To locate an acceptable signal click drag the scroll bars on the Manual Laser Sample Position control page to aim the laser at different parts of the sample position Samples often contain hot spots areas of high ion intensity and cold spots areas of low ion intensity When you locate a hot spot stop acquiring and restart e When acquiring actual data do not move the sample position You observe the best resolution and mass accuracy when you collect ions produced from one location NOTE When analyzing high masses or samples of low concentration or when using thin film matrices for example in polymer analysis or in PSD mode you may run out of sample if you acquire from the same area in the sample position You may need to move around on the sample position when acquiring to build up adequate signal to noise ratio Before adjusting laser intensity for subsequent spectra stop acquisition The laser setting recorded with the data file is the last laser setting used NOTE It is good practice to use one laser setting to acquire a spectrum but you may need to adjust the laser intensity to maintain adequat
592. umber of data points collected Voyager Biospectrometry Workstation User s Guide 5 59 Chapter 5 Optimizing Instrument Settings 5 3 5 2 Vertical Digitizer Settings This section describes e Vertical settings e Vertical Scale e Vertical Offset Input Bandwidth Vertical settings The digitizer settings include three vertical parameters that affect the signal acquired e Vertical Scale Adjusts the dynamic range of the digitizer scale to accommodate the signal you are acquiring For more information see e Vertical Scale on page 5 30 e Vertical Scale on page 5 60 e Vertical Offset Applies a Y Offset to the signal For more information see e Vertical Offset on page 5 30 e Vertical Offset on page 5 62 e Input Bandwidth not available with Signatec digitizers Reduces higher frequency noise For more information see e Input Bandwidth not available with Signatec digitizers on page 5 30 e Input Bandwidth not available with Signatec digitizers on page 5 63 Vertical Scale A correct Vertical Scale setting ensures optimum vertical dynamic range for the acquired data Figure 5 18 If the Vertical Scale is set incorrectly the following occurs Too high Decreases the dynamic range of the data Too low Truncates the tops of peaks decreases observed mass resolution and distorts the relative distribution of ion abundances in multicomponent mixtures 5
593. ument Sellinas l File Edt View Instrument Acquistion SamplePlate Display Tools Applications la gt Toolbar jokue ele s kle iee messe Date Storage REZ Dia Vaso A Cente xx Unes Postive Mode Digiizer Filename Control Mode Data Storage Somes A Here Control page pme ie cam poy Instrument Grid E 00 1100 1 2 GudeWie 0o05 0000 0300 Settings Manual Laser Intensity p C 50 Delay Tine MT neee h ontrol page avi fa Spectrum Acquisition o oon Manual Sample Positioning Shote Spectum 50 ActivePos i z Pa Mass Range Da 500 te 5000 P 100 well plate plt Fo Manual Laser Spee oe Sess F Lou Mass Gate Da iad ol FE oooo0o000000 j Calibration Sample Position boooooo0oo00o0o0 TI E looo0000000 2 Defaut Control page loooo000000 C Biteral Fle loo 00000000 a mi loo 00000000 loo 00000000 loo 00000000 g ao ado 0 160 2000 booooooooo Mass m Output window O i AC Autre Contol_ Data Storage 7 Status bar RON High Votage OFF SOREA SN SE Accuston OFF Corro ode MANUAL Instrument Mode LIN POS Spectrum window Figure 4 1 Instrument Control Panel Toolbar The toolbar contains buttons that control the software and the instrument For a description of a toolbar button place the cursor on it A brief description of the button Tooltip is displayed below the button For more informa
594. ument Settings Parameters When Low Mass Gate is turned on a characteristic spike may occur in the spectrum Figure 5 26 This spike is typically only seen in Reflector mode NOTE Systems with high current detectors Voyager DE systems with serial number 1128 and later Voyager DE PRO systems with serial number 6007 and later Voyager DE STR systems with serial numbers 4112 4113 and 4116 and later do not typically exhibit this spike Older systems with a dual channel plate detector in Linear mode may exhibit a 50 to 1 000 mV spike Intensity Spec 1 AS MC BP 1018 0 64531 Low Mass Gate spike 3772 607 5 962 1671 727 2973 632 3481 4303918 423 2800 3700 Mass m z Eliminating Low Mass Gate spike when acquiring Eliminating Low Mass Gate spike after acquiring Figure 5 26 Low Mass Gate Spike The spike occurs at a time that corresponds to just below the Mass for the Low Mass Gate entered in the BIC file For example if the Mass is set to 400 Da you would see the spike at approximately the time that corresponds to 370 Da You can identify the Low Mass Gate spike by its sharp lift off its narrow width and the noise as it returns to baseline You can eliminate the Low Mass Gate spike from the data by setting the Mass for acquisition slightly higher than Low Mass Gate Mass You can eliminate the Low Mass Gate spike after you acquire by using the Truncate Spectrum function in the Dat
595. umns are displayed Hidden columns that require an entry are automatically displayed if you select a row to run and it does not contain the necessary entry If you import information into the run list entries are imported into hidden columns To arrange the Sequence Control Panel and Instrument Control Panel on the desktop select a command from the Desktop menu e Tile Horizontal Places the Sequence Control Panel at the top or bottom of the desktop and the Instrument Control Panel in the opposite location Tile Vertical Places the Sequence Control Panel at the left or right of the desktop and the Instrument Control Panel in the opposite location Customizing the Sequence Control Panel 7 8 2 Setting Sequence Control Panel Preferences To set preferences 1 Select Preferences from the File menu to display the Preferences dialog box Figure 7 12 Auto Size IV Append Sample Position r to Data Filename Fill Down Sample Position r Sequentially Based On PLT File Enable Low Memory Dialog IV Default Number of Ps Rows In The Grid fa OK Cancel Figure 7 12 Preferences Dialog Box 2 Specify preferences as needed Table 7 2 Sequence Control Panel Preferences Preference Description Autosize Automatically enabled when you are in Workbook mode The run list is automatically resized and displayed appropriately in tabbed frames when you resize the window When Workbook mode is disabled Autosi
596. us 2 77 turning high voltage on off 4 25 warmup before calibration 2 56 6 4 7 24 Horizontal position oscilloscope l 5 Horizontal scale oscilloscope l 5 Hot spots in signal intensity 6 5 How to use this guide xxvii HPA see 3 HPA HPLC grade waiter use of 3 5 3 13 3 15 Humidity operating A 4 A 7 A 10 HV Tune Ratio replaced by new parameter 5 95 Hydroxypicolinic acid see 3 HPA l IAA chemical structure and molecular weight C 12 concentration 3 19 crystals 3 19 mass spectrum C 6 preparing 3 19 sample concentration 3 19 when to use 3 3 Idle Power 2 39 IgG BIC file 5 4 Immonium ions common 8 23 in PSD mode 8 25 Importing into Sequence run list 7 22 Indoleacrylic acid see IAA Initial Velocity correction factors for each matrix 5 20 definition 5 22 overcoming effects of 1 15 selecting for matrix in Instrument Settings 6 13 values for matrices 5 22 Initializing hardware 2 74 Input Bandwidth digitizer impact of changing 5 63 impact on resolution and signal to noise 5 50 PSD 8 48 setting 5 30 suggested settings 5 63 Installation cables 2 12 computer Voyager DE and Voyager DE PRO 2 10 2 16 2 20 computer with Signatec 500 MHz digitizer 2 11 Index Installation continued computer with Tektronix oscilloscope 2 11 digitizer Acqiris Voyager DE and Voyager DE PRO 2 17 digitizer Acqiris Voyager DE STR 2 26 digitizer Lecroy LSA 2 15 digitizer Signatec 2 13 initial 2 2 mass spectrometer Voyager DE
597. utes calibration update may be required less frequently or not at all depending on your mass accuracy needs To set the run to update the calibration list the standard position in the sequence run list every fifth line or so the precise line on which to list the standard depends on the time required for each line in the sequence to run and specify Internal Update calibration For more information see Section 7 Acquiring Spectra from the Sequence Control Panel 3 40 Applied Biosystems Loading Samples on Sample Plates 3 2 3 Loading Samples Dried Droplet Application NOTE If you are loading a plate for acquisition or automated sample positioning in Automatic Control mode use a laser etched or welled sample plate to provide a reference point for sample application When to use Use this technique for most applications when sample concentration is gt 0 1 pmol ul Before loading Note the following samples Some organic solvents such as methanol acetone and THF spread very easily on metal surfaces If the sample contains these solvents try to load smaller volumes 0 5 ul or less e Try to place the sample in the center of the sample position If the sample is not in the center the laser position shown on the Sample View see Figure 4 9 on page 4 31 may not be centered on the sample WARNING CHEMICAL HAZARD Methanol is a flammable liquid and vapor Exposure may cause eye skin and resp
598. valid parameter to red e Displays an error message indicating that invalid parameters have been found and allows you to run rows that do not contain errors 7 26 Applied Biosystems Running a Sequence Iferroris fan error is displayed check the following and correct as displayed needed Sample positions correspond to the type of PLT file loaded in the Instrument Control Panel All directories specified for data files exist All specified BIC SET and CAL files exist NOTE If a CAL file specified for an External calibration line does not exist the software checks preceding rows in the sequence to see if an Internal Update calibration row will create the CAL file For more information see Types of files and calibration on page 7 3 All BIC files specify Automatic Control mode All BIC files specify Linear or Reflector Operation mode PSD mode is not allowed All BIC files specify the same laser type internal or external You cannot switch between internal and external lasers when running a sequence If you specify an internal laser BIC file then an external laser BIC file the second BIC file is flagged as invalid when you try to run the sequence Voyager Biospectrometry Workstation User s Guide 7 27 Chapter 7 Acquiring Spectra from the Sequence Control Panel 7 6 3 During and After Acquiring a Sequence Pausing and To pause a sequence click m resuming a The sequence pauses after t
599. werful Tool for The Mass and Sequence Analysis Of Natural And Modified Oligonucleotides Nucleic Acids Research 1993 21 3191 3196 Smirnov l P M T Roskey P Juhasz E J Takach S A Martin and L A Haff Sequencing Oligonucleotides by Exonuclease Digestion and Delayed Extraction Matrix Assisted Laser Desorption lonization Time of Flight Mass Spectrometry Anal Biochem 1996 238 19 25 Tang K N I Taranenko S L Allman C H Chen L Y Chang and K B Jacobson Picolinic Acid as a Matrix for Laser Mass Spectrometry of Nucleic Acids and Proteins Rapid Commun Mass Spectrom 1994 8 673 677 Wu K J A Steding and C H Becker Matrix assisted Laser Desorption Time of flight Mass Spectrometry of Oligonucleotides Using 3 Hydroxypicolinic Acid as an Ultraviolet sensitive Matrix Rapid Commun Mass Spectrom 1993 7 142 146 In Gel Digestion UCSF In Gel Digest Procedure 2 2 00 http donatello ucsf edu ingel html Rosenfeld et al Anal Biochem 1992 203 173 179 Hellman et al Anal Biochem 1995 224 451 455 Lipooligosaccharides Gibson B W J J Engstrom C M John W Hines and A M Falick Characterization of Bacterial Lipooligosaccharides by Delayed Extraction Matrix Assisted Laser Desorption lonization Time of Flight Mass Spectrometry J Am Soc Mass Spectrom 1997 8 645 658 Genespectrometry B j B L j 0 G R A P H Y Haff L P Juhasz S Martin
600. y a fixed setting use the same values for Min and Max NOTE To automate laser adjustment you must specify adjustment criteria as described in Setting spectrum acceptance and laser adjustment criteria on page 6 42 If you do not specify adjustment criteria the laser is not adjusted The mid range setting between the minimum and maximum is used Step Size Sets the size of the increments the system uses when automatically adjusting the laser intensity Maximum step size cannot exceed the difference between the minimum and maximum laser intensity settings Use Prescan Enables or disables Prescan mode Prescan mode determines the laser setting within the specified range needed to meet the Signal Intensity criteria There are two Prescan options e First Search Pattern Position Only e Each Search Pattern Position For more information see Prescan mode on page 6 58 5 34 Applied Biosystems Instrument Settings Parameter Descriptions Table 5 10 Automatic Control Parameters Spectrum Accumulation Parameter Description Spectrum Accumulation Number to Acquire Specifies the number of spectra to save or accumulate This field is dependent on the selected Spectrum Accumulation option described on page 5 39 If you select Save First Spectrum to Pass Acceptance Criteria the number to acquire is restricted to 1 If you select Save All Spectra or Save All Spectra that Pass Acceptance Cri
601. y times the system must acquire until it obtains five spectra that pass acceptance criteria One DAT file containing five spectra is created NOTE If you set the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified number of spectra to acquire or the number of search pattern positions in the SP file Voyager Biospectrometry Workstation User s Guide 5 41 Chapter 5 Optimizing Instrument Settings If an SP file with 7 positions is selected and the Number to Acquire is 5 the number of positions analyzed is determined by whether or not acceptance criteria fail e If all fail seven positions are analyzed total number of positions in the SP file and no DAT file is created e If the first position fails but all remaining pass the first two positions are analyzed and one DAT file containing five spectra is created e If the first and second positions fail but all remaining pass the first three positions are analyzed one DAT file containing five spectra is created and so on e If acceptance criteria never fail only the first position is analyzed and one DAT file containing five spectra is created NOTE If you set the Stop After X Consecutive Failing Acquisitions stop condition acquisition stops when the specified number of failing acquisitions is reached regardless of the specified num
602. yager software uses to calculate the mass for fragment ions is shown below a R mg P B ty tp V t 1 aa mp where m Fragment ion mass a B y Calibration constants Ri Mirror Ratio ty Fragment ion flight time b Precursor ion flight time at R 1 calculated using the standard calibration equation described on page 6 9 Mp Precursor mass entered in Instrument Settings see page 8 45 Default PSD jf you select default PSD calibration the Voyager software calibration uses the calibration equation above and substitutes the following values for calibration constants e o Calculates from instrument geometry Band y Uses zero 0 8 28 Applied Biosystems Overview of PSD Analysis 8 2 6 Optimizing the Precursor lon Selector The Precursor lon Selector called Timed lon Selector in Reflector mode allows you to analyze the ion of interest by deflecting ions until the time that corresponds to the mass of the ion of interest At the time that corresponds to the mass of the ion of interest the Precursor lon Selector voltage is turned off and the ion of interest passes to the reflector After the ion passes through voltage is turned on again in the ion selector NOTE Leave the Precursor lon Selector enabled when performing PSD analysis The only time you may want to disable the Precursor lon Selector is to determine if it is working The width of the mass window in which the Precursor l
603. you can see ions in Linear mode it indicates that voltages or laser power need adjusting e If you cannot see ions in Linear mode refer to Section 9 2 Troubleshooting Refer to Table H 8 if you are having spectrum trouble Table H 8 Spectrum Troubleshooting Symptom Possible Cause Action Very wide peaks Refer to Poor resolution Refer to Poor resolution in Continuous Extraction in Continuous Extraction mode symptom on mode action on page H 21 page H 21 H 20 Applied Biosystems Troubleshooting in Continuous Extraction Mode Table H 8 Spectrum Troubleshooting Continued Symptom Possible Cause Action Poor resolution in Continuous Extraction mode continued on next page Laser intensity too high Adjust laser by using the slider controls on the Manual Laser Control page Accelerating Voltage incorrect Adjust Guide Wire Voltage too high Adjust See Section 5 3 4 Understanding Guide Wire Voltage Current calibration not correct for sample off by a factor of 2 or 3 Use correct calibration for sample Sample concentration too high Prepare sample matrix with a final sample concentration appropriate for sample and matrix See Matrix Information on page 3 6 Excess matrix in sample preparation Increase sample matrix ratio Too much salt or buffer in sample Clean up sample See Section 3 1 5 Samp
604. yze 4 To start acquiring select Start Acquisition from the Acquisition menu or click During During acquisition acquisition e Laser is adjusted sample position is adjusted and Spectrum Acceptance Criteria are applied to each search pattern position in a spectrum For more information see Spectrum Accumulation on page 5 35 and Section 6 6 6 Process that Occurs During Acquisition in Automatic Mode e Each spectrum is saved or accumulated as determined by the conditions selected in Spectrum Accumulation in the Automatic Control dialog box For more information see Section 5 2 4 Description of Spectrum Accumulation Options e Information about the acquisition is displayed in the Automated Control tab of the Output window Voyager Biospectrometry Workstation User s Guide 6 45 Chapter 6 6 6 4 Search Search patterns 6 46 Search pattern SP files Applied Biosystems Acquiring Spectra from the Instrument Control Panel Patterns This section describes e Search patterns e Search pattern SP files e Units in search pattern files e Default search pattern file e Spiral search pattern file e Search pattern for custom plates A search pattern determines the path that the laser follows when scanning the selected Active Position using Automatic Control Mode You have two options for search patterns Random tThe software randomly determines search pattern positions as data is acq
605. zation 7 Click OK The sample plate is loaded and aligned as needed For more information see How the system aligns a plate on page 2 47 It takes 1 or 2 minutes for the sample plate to reach the correct position While the sample plate is moving the Load Eject Status dialog box displays messages about the status of the hardware 3 60 Applied Biosystems Sample Plate Types 3 5 Sample Plate Types Overview The Voyager Instrument Control software supports a variety of reusable and disposable plates Each type of physical plate has a corresponding plate PLT file that defines the location and dimensions of the positions on the plate Standard PLT files for all standard plates are provided with the software You can e Customize the standard plate types by copying and editing their PLT files See Section 3 5 3 Guidelines for Defining Custom Plate Types These additional plate types are custom options Contact Applied Biosystems for more information e Define your own plate types of unlimited positions by editing existing or creating new PLT files For information see Section 3 5 2 Editable Configuration Plate PLT Types Provided with the System and Section 3 5 4 Creating and Editing PLT Files In this section This section describes e Sample plate types and applications e Editable configuration plate types provided with the system e Guidelines for defining custom plate types e Creating and edit
606. ze is not available You can manually resize and move the run list window to any dimensions continued Voyager Biospectrometry Workstation User s Guide 7 45 Chapter 7 Acquiring Spectra from the Sequence Control Panel Table 7 2 Sequence Control Panel Preferences Continued Preference Description Append Sample Position to Data File Name Includes position number in the data file name suffix For example if you specify a base name of SAMPLE the final data file name is SAMPLE_A1_0001 DAT for the data file acquired from position A1 Fill Down Sample Position Based on PLT File When you use the fill down function increments sample positions in all selected rows below based on the type of sample plate loaded in the Instrument Control Panel See Incrementing sample positions on page 7 21 Enable Low Memory Dialog Verifies before each acquisition step that at least 150 MB of disk space are available on the destination drive Default number of rows Determines the number of rows displayed when you create a new sequence You can override the default and add or delete rows as needed 7 46 Applied Biosystems Click OK 8 PSD Analysis This chapter contains the following sections 8 1 PSD Quick Start 8 2 8 2 Overview of PSD Analysis 8 20 8 3 Enhancing Fragmentation with CID 8 31 8 4 Acquiring PSD Data with Standard BIC Files

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