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HP Data Explorer 4 Series User's Manual

1. Figure 5 7 Automatic Calibration Settings Dialog Box 4 Click Select New File then select a calibration reference file For information on creating a reference file see Section 5 3 3 Creating or Modifying a Calibration Reference File REF 5 30 Applied Biosystems Automatic Calibration 5 To add upto 10 reference masses to the Masses to Match list do either of the following e Click Add All to add the first 10 reference masses from the reference file e Click Add Reference To List to individually select reference masses to add NOTE If the current list already contains 10 reference masses you must delete a mass before you click Add References to List If you click Add Reference to List the Select or Create Reference Peak Information dialog box Figure 5 8 is displayed and lists all masses in the reference file If you click Add All this dialog box is not displayed it shoud is Erm ate litriem e Peat lesen ESI Chiaie oa eer a pDr Eqpkeer Fioge A gee ret Jrs fe iow Cancel fees immm E epoke hope Mave jm Dots tintin ac E bap Him Figure 5 8 Select or Create Reference Peak Information Dialog Box Data Explorer Software User s Guide 5 31 Chapter 5 Examining Spectrum Data 6 Specify reference masses to add by doing either of the following Click a mass then click OK e Type new reference mass information in the Name Theoretical m z Charge
2. Data Explorer Software User s Guide 1 7 Chapter 1 Data Explorer Basics Additional files Additional file types you may see on your system are types described below Table 1 2 Additional File Types Category File Type File Content Data PKT Text file containing a chromatogram or a spectrum peak list that you can save from the Output window See Output window on page 1 15 TXT Data file exported to an ASCII text file See Section 1 6 3 Converting to and Exporting ASCII Data Data SPC Data file format for files acquired before Version 3 0 of Mariner the Mariner Instrument Control Panel only NOTE Voyager data files in SPC format have a file structure different from Mariner data files in SPC format and are not supported in the Data Explorer software MS Data file format for files acquired before Version 5 0 of Voyager the Voyager Instrument Control Panel only MSA and PSD data file format for composite and fragment files MSF acquired before Version 5 0 of the Voyager Instrument Voyager Control Panel only NOTE The Data Explorer software cannot generate composite spectra from MSF fragment files MSB Baseline corrected data file format for composite and Voyager fragment files acquired before Version 5 0 of the only Voyager Instrument Control Panel Procedure TUN AutoTune method See the Mariner Workstation User s Mariner Guide only 1 8 Applied Biosy
3. Mass difference from adjacent peaks Applies the labels to peaks that have the specified mass difference relative to the adjacent labeled peak of lower m z Select Match Charge State spectra only if you want the charge state of a peak evaluated before applying the user label When this function is enabled a peak must occur within the specified Mass Tolerance described below and have the specified charge state before the user label is applied Hint The Match Charge State function allows you to screen out peaks that are within the specified Mass Tolerance but are not the charged species you are interested in Peak Labeling 6 To manually enter label settings click The User Label Entry dialog box is displayed Figure 3 24 User Label Entry xj Label Parameters Label Peak Mass m z Mass Tolerance m z Charge fe fi 0 OK Cancel Figure 3 24 User Label Entry Dialog Box Spectrum 7 Set the following parameters Spectrum Parameter Description Specifies Label Text of the label to display Peak Mass if Label Type selected Mass of the peak to which the label applies is Mass Mass Difference if Label Type Difference in mass that must exist between selected is Difference peaks to apply user labels Mass Tolerance Mass tolerance that the peak must occur within to apply user labels NOTE If a peak meets the criteria for more than one user label multiple user l
4. NOTE If the original spectrum is displayed in vertical bars instead of lines select Graphic Options from the Display menu click the trace tab of interest then select Lines for the Line Type in the Plot Setup section of the dialog box For more information see Section 1 5 Setting Graphic Options Mass Deconvolution Mariner Data Only 5 6 Mass Deconvolution Mariner Data Only NOTE Mass deconvolution is not supported for Mariner DAD data NOTE The Mass Deconvolution software is an option in the Data Explorer software The Multiple Charge command on the Process menu is dimmed if you have not purchased the option Overview You can use the Data Explorer Mass Deconvolution features to generate a theoretical zero charge spectrum that represents the molecular mass of a protein e Mass Deconvolution Use when you have a spectrum with clearly resolved multiply charged peaks You specify the m z values for the peaks to include This function requires at least two adjacent peaks within the same charge envelope e Convert to Zero Charge Spectrum Use when you have a spectrum with overlapping charge envelopes or a noisy baseline You specify a mass range for the peaks to include and a mass range for the molecular mass Mass To use the Mass Deconvolution function Deconvolution 4 Click the Spectrum window 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after process
5. Data Explorer Software User s Guide 6 9 Chapter 6 Using Tools and Applications Figure 6 6 Periodic Table 3 Click an element to select it and to display the Isotope dialog box Figure 6 7 6 10 Applied Biosystems Using the Elemental Composition Calculator Isotope Mass Abund Minimum Maximum 0 500 12C 12 00000 0 99 0 0 13C 13 00335 0 01 0 0 14C 14 00324 0 00 0 0 Figure 6 7 Isotope Dialog Box NOTE Ignore the column of check boxes to the left of the Isotope column if it is displayed Change the Minimum and Maximum number of occurrences for the first isotope of the element as needed NOTE The software ignores changes you make to the individual isotope minimum and maximum values Click OK two times to return to the Element Limits dialog box Repeat step 2 through step 5 to add other elements as needed then click OK to return to the Elemental Composition Calculator Data Explorer Software User s Guide 6 11 Chapter 6 Using Tools and Applications Setting limits for To set limits for amino acid DNA RNA or carbohydrate result other result types 6 12 Applied Biosystems types 1 Click the limits button displayed for the selected result type in the Elemental Composition Calculator dialog box see Figure 6 1 on page 6 3 The Limits dialog box for the selected Result Type is displayed Figure 6 8 shows the Amino Acid Limits dialog box Figure 6 8 Amino A
6. Symptom Possible Cause Action Spectrum peaks labeled with incorrect charge state when charge state labels are selected continued Charge state determination parameters are set such that peaks are determined to have no charge Adjust parameters See e Peak Processing parameters spectrum data only on page 3 26 e Section 3 2 5 Charge State Determination and Examples Noise between isotope peaks is detected Increase Base Peak Intensity or Max Peak Area to eliminate noise See Basic Settings spectrum data only on page 3 22 Apply noise filter See Section 5 7 Noise Filtering Smoothing Monoisotopic peak not labeled correctly Peak detection charge state parameters are not set correctly the software is identifying the tallest peak in a cluster as the monoisotopic peak Adjust parameters to correctly identify the monoisotopic peak See Peak Processing parameters spectrum data only on page 3 26 Data Explorer Software User s Guide 9 21 Chapter 9 Troubleshooting 9 22 Applied Biosystems A Warranty 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 extra charges for supporting non standard computer configurations or components that have not been supplied o
7. 2 From the Process menu select Add Subtract Spectra 3 Click then right click drag over the peak in the extracted ion chromatogram The spectrum range is displayed in the Spectra To Be Added list in the Add and Subtract Spectra dialog box Figure 7 6 4 Inthe Add and Subtract Spectra dialog box click anywhere in the Spectra To Be Subtracted list 5 Inthe TIC right click over a region of baseline that does not contain any peaks NOTE When selecting spectra to subtract select from the TIC The baseline in the extracted ion chromatograms is for a selected mass and does not represent all components which may be present Mariner Data Examples The spectrum range is displayed in the Spectra To Be Subtracted list in the Add and Subtract Spectra dialog box Figure 7 6 m List Spectra To Be Added alk 775 793 m List Spectra To Be Subtracted 794 806 m Add Sub Mode Average C Accumulate OK Cancel Figure 7 6 Subtracting Spectra 6 Click OK The subtracted spectrum is displayed Figure 7 7 on page 7 10 Data Explorer Software User s Guide 7 9 Chapter 7 Data Explorer Examples 7 10 Spec TSSOP oo DPE 1g 41s Creating extracted ion chromatogram Figure 7 7 Subtracted Spectrum The peak at 391 is still present which indicates one of the following conditions e You did not subtract sufficient baseline e The peak is a coeluting c
8. Number of data points 41 Figure 3 1 Example Detection Ranges Calculated by Software Based on Number 3 4 Applied Biosystems of Data Points Per Peak Overview Overlapping peak To accommodate spectral peaks that occur on the boundary of detection ranges two peak detection ranges the software creates detection ranges that overlap Figure 3 2 Peak A Peak B Peak C Peak D p Range 3 wa a Range 2 Range 4 Range 1 at Figure 3 2 Overlapping Detection Ranges If a peak occurs in an overlapping range for example Peak B in Figure 3 2 the software detects the peak using the settings from the detection range within which the entire peak occurs Range 2 in Figure 3 2 If a peak occurs completely within two ranges for example Peak D in Figure 3 2 the software uses the settings from the higher m z detection region Range 4 in Figure 3 2 PSD peak For PSD data the mass time correlation is different from that detection for of standard TOF spectra Therefore resolution dependent Voyager data settings are not supported for PSD data Data Explorer Software User s Guide 3 5 Chapter 3 Peak Detection and Labeling 3 2 Peak Detection This section includes Strategy for Mariner peak detection Strategy for Voyager peak detection Setting peak detection parameters Peak detection parameter descriptions Charge state determination and examples 3 2
9. Reacquire spectrum Save DAT file Error message displayed when opening PSD data file You are currently acquiring the PSD DAT file and have not stopped the experiment Stop the Experiment See the Voyager Biospectrometry Workstation User s Guide Parts of other software windows are displayed on top of the Data Explorer window or toolbar buttons or status indicators are not displayed Multiple application windows overlap Close applications not in use then minimize and maximize the Data Explorer window to refresh the display Failed to create empty document message displayed when you open a data file Chromatogram and Spectrum windows are too small Resize the Output window click drag border so that at least a small part of the Chromatogram or Spectrum window is displayed Graphic Option settings are not applied to all traces Did not select Use same settings for all graphs before entering settings Select Use same settings for all graphs then click Apply Data Explorer Software User s Guide 9 3 Chapter 9 Troubleshooting Table 9 1 General Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action M z range in data files converted to centroid does not match m z range in original data file M z range in a data file that is converted from profile to centroid is determined by the peak detection range set
10. e Enable Allow Overlapping Labels select Peak Label from the Peaks menu If overlapping labels are not enabled some peaks may not be labeled NOTE Before using the MS Fit MS Tag toolbox additional data preparation is recommended See Section Using MS Fit MS Tag Toolbox Data Explorer Software User s Guide C 3 Appendix C Data Explorer Toolbox Visual Basic Macros C 3 Accessing the Macros C 4 Applied Biosystems To access the macros 1 Open the Data Explorer software Open a data file 2 3 Prepare the data as described in the previous section 4 From the Tools menu select Macros 5 In the Macros dialog box select modToolBoxPalette Toolbox_Palette then click Run The Toolbox Palette dialog box is displayed Taolbos Falete Pephds Fragmentation Tobos Pakam Aruban Tonka BS FEJ MS Tag Toolbox a Figure 3 8 Toolbox Palette Dialog Box Using the Ladder Sequencing Toolbox NOTE If the modToolBoxPalette Toolbox_Palette is not listed you must import the macro into the Data Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 Hint You can assign the modToo lBoxPalette Toolbox_Palette macro to a macro button in the Data Explorer software For information see the Data Explorer User s Guide Section 6 7 Using the Macro Recorder You can also access individual macros directly or assign individual macros to
11. 7 in disaj fF mem D Ee Pas Mma l Heer iki T three Loder P Visas Ute Label Sein w Ca ce Figure 3 21 Chromatogram Peak Label Dialog Box 3 Select Enable Labeling 4 Set the number of decimal points to be displayed 5 Select Label Attributes e Overlapping Allows labels to be displayed when peaks are close together e Peak bounds Displays peak start peak end and baseline e Orientation Specifies Horizontal 45 degree or Vertical labels 6 Select the label content to display for example Spectrum Time Vial Number 7 To create custom labels select User Labels then click User Label Setup See Section 3 5 3 Setting Custom Peak Labels Data Explorer Software User s Guide 3 55 Chapter 3 Peak Detection and Labeling 8 Click OK The trace is displayed The detected peaks that meet the peak labeling criteria are labeled Setting To label spectrum peaks spectrum labels 4 Glick the Spectrum window to activate it 2 From the Peaks menu select Peak Label The Spectrum Peak Label dialog box is displayed Figure 3 22 Pasi Label Opia bera Pan E Ha ppa M is Term Piah Higsa Lanal Tape F Hi D pian diimece hors he ached pas a D biaip diis aan wia peaky adai atar D Deba G Pa bork re akor estan Laba Lonii P dema il E iin itak F ha Site fel Liri Labai Seu n ee ee Figure 3 22 Spectrum Peak Label Dialog Box 3 Select Enable Labeling
12. Data Explorer Software Version 4 Series Software User Guide BS ie applied ns Copyright 2001 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document 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 document Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U S and certain other countries AB Design Applera Biospectrometry CombiSolv Data Explorer Mariner and Voyager are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries HP and Laserjet are registered trademarks of Hewlett Packard Co Microsoft PowerPoint Visual Basic and Windows NT are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Printed in the USA 7 2001 Part Number 4317717 Rev C Table of Contents Table of Contents How to Use This Guide 0ccccccccccceccecccccccccceececceeecaeeees xi Chapter 1 Data Explorer Basics 1 1 1 2 1 3 1 4 OVV SW raie dan sein dc cca aara be dest ail
13. Figure 4 9 Add and Subtract Spectra Dialog Box 4 Select spectra to add by doing one of the following e Right click drag the area of the trace in the Chromatogram window The numbers of the selected spectra are added to the list window A range of spectrum numbers is indicated as X X For example 10 20 indicates spectrum number 10 through spectrum number 20 e Click 2 2 type the spectrum number range to add for example 10 20 then press Enter Hint To sum non contiguous regions of spectra repeat step 4 For example you can add spectra 10 to 20 and spectra 30 to 40 to the list window 5 Click in the Spectra To Be Subtracted list box Data Explorer Software User s Guide 4 21 Chapter 4 Examining Chromatogram Data 4 22 Applied Biosystems NOTE Before you can subtract spectra you must first specify spectra to be added Select spectra to subtract as described in step 4 Select the Add Subtract mode e Average Spectra in each list are averaged before the addition or subtraction occurs e Accumulate Spectra in each list are summed before the addition or subtraction occurs NOTE If you select Accumulate mode include at least the same number of spectra in the Subtract range that you include in the Add range For example if you include spectra 775 to 794 in the Add range make sure to include 20 or more spectra in the Subtract range Click OK The subtracted baseline trace
14. In compounds with more than 100 carbon atoms the height of the first SC isotope peak exceeds the height of the C peak The mass range you analyze and the resolving power of the analyzer determine if you observe resolved isotope peaks in a mass spectrum Figure B 3 represents angiotensin at a resolution of 3 000 Isotopes are fully resolved and sharp peaks are observed in the mass spectrum At lower resolutions the shape of the individual peaks becomes less pronounced or may not be differentiated at all Figure B 4 represents angiotensin at a theoretical resolution of 1 000 Isotopes Figure B 4 Mass Spectrum of Angiotensin I at Resolution 1 000 If isotopes cannot be resolved the highest resolution you can obtain is limited by the width of the isotopic envelope The isotopic envelope is the mass range of the combined isotopes as measured at the half height of the tallest isotope peak in the compound Figure B 5 isotopic envelope Figure B 5 Unresolved Isotopes Data Explorer Software User s Guide B 5 Appendix B Overview of Isotopes B 2 Monoisotopic B 6 and Average Masses When isotopes are clearly resolved Figure B 6 the monoisotopic mass is used for mass labeling and corresponds to the lowest mass peak in the cluster Monoisotopic mass corresponds to lowest mass peak Figure B 6 Monoisotopic Mass When isotopes are not resolved Figure B 7
15. Mass im 7 Figure 7 18 Digest with Several Hundred Peaks Detected Allow Overlapping Labels Enabled 7 18 Applied Biosystems Voyager Data Examples Procedure To detect peaks from complex mixtures 1 Display the spectrum of interest Noise 2 From the Process menu select Noise Filter Smooth filtering smoothing The Noise Filter Smooth dialog box Figure 7 19 is displayed Noise Filter Smooth xi Methods Default X Mass Peak Resolution 500 Cancel Figure 7 19 Noise Filter Smooth Dialog Box 3 Select Default Smoothing or Noise Filter with a Correlation Factor of 0 7 then click OK For more information see Section 5 7 Noise Filtering Smoothing Click in the toolbar or select Peak Detection from Setting detection the Peaks menu thresholds The Spectrum Peak Detection Setup dialog box is displayed with the Basic Settings tab Figure 7 20 displayed Data Explorer Software User s Guide 7 19 Chapter 7 Data Explorer Examples games bisa Hh Lintrerters i cin AA liaete Same Setings Peak Frocenara advanced Saing Gint Precint T fine Paak Irasrap i 4 F Erabi TEP ered Thandi Cini Par fetio 7 pe Fiesa ppsa pariri r imidd irim Paw Mecniatien Bhia eaii iiin fam E m tee te el bec P ver ee Se Figure 7 20 Spectrum Peak Detection Setup Basic Settings Tab 5 Click Use Advanced Settings The Advanced Settings tab is displayed Figure 7 21
16. NOTE Max Peak Area is calculated above the local baseline and can compensate for problems related to a rising global baseline Low Mass Gate spike is identified as the Base Peak Truncate the data to eliminate the Low Mass Gate spike then reapply peak detection settings See Section 5 9 Truncating a Spectrum Peaks detected before deisotoping are not detected after deisotoping Set Max Peak Area and BP Intensity to 0 before deisotoping deisotope then reset thresholds to appropriate settings after deisotoping Data Explorer Software User s Guide 3 9 Chapter 3 Peak Detection and Labeling Problem Suggested Actions Partially resolved peaks If peaks represent two compounds and you want both not detected peaks labeled do either of the following e Set Max Peak Area to 0 then adjust the Base Peak Intensity until peaks are detected e Click the Peak Processing tab then change the default Integration Baseline Setting from Valley to Valley to Valley to Baseline If peaks represent partially resolved isotopes and you want to label and detect the average mass decrease the Mass Resolution setting until the isotopic envelope is detected 4 If you see more than one of the problems listed above in a spectrum you can adjust peak detection parameters for any or all detection ranges referred to as setting parameters locally by doing the following in the order listed
17. Software User s Guide 9 15 Chapter 9 Troubleshooting Table 9 10 Peak Detection and Labeling Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action Peaks are not detected or labeled continued Analyzing masses above 20 000 Da Increase Mass Resolution setting in Peak Detection Setup See Section 3 2 2 Strategy for Voyager Peak Detection Max Peak Area set too high Decrease See Section 3 2 2 Strategy for Voyager Peak Detection Base Peak Intensity set too high Set to 0 Adjust Max Peak Area to optimize peak detection for Voyager data See Section 3 2 2 Strategy for Voyager Peak Detection When creating a custom label for a spectrum you right click drag across a peak to identify the peak and an extracted ion chromatogram is created instead The Chromatogram window was active when you selected Peak Label from the Display menu Make sure the Spectrum window is active before selecting Peak Label from the Display menu 9 16 Applied Biosystems Peak Detection and Labeling Troubleshooting Table 9 10 Peak Detection and Labeling Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action Expected user label not displayed Delta X value includes more than one peak apex Set Delta X value low enough to prevent the peak labeling windows from overlapping For information see
18. Table 1 1 Information Stored In a DAT File Continued Category File Type File Content Settings MSM MS Method settings if data was acquired using an continued Mariner MSM file only NOTE To access the instrument settings used to acquire each spectrum in an MS Method you must first extract the MSM file from the data file then export the BIC files from the MSM file using the Export button in the MS Method editor For more information on exporting a BIC file from an MSM file see the Mariner Workstation User s Guide SET Graphic and processing settings See Section 1 4 2 Customizing Processing and Graphic Settings SET Display LBC Chromatogram label information See Section 3 5 3 Setting Custom Peak Labels LBS Spectrum label information See Section 3 5 3 Setting Custom Peak Labels Process CAL Calibration constants generated by mass calibration For more information see Exporting BIC MSM and CAL files on page 1 36 and Applying new constants to additional files on page 5 16 CTS Processed trace that you access by selecting Processing History from the Display menu For information see Section 2 4 7 Recalling and Rearranging Traces Processing History NOTE You select the name of the trace from the Processing History menu You do not directly select a CTS file To purge or disable CTS files see Setting Processing History options on page 2 23
19. The Centroid peak detection value is derived from a percentage of the peak height which is measured from 0 For information see Section 5 8 2 Using Baseline Correction e Noise filter or smooth Use the method appropriate for your data to remove noise spikes Data Type Method Linear Default or Gaussian smoothing Reflector Noise Reduction For more information see Section 5 7 Noise Filtering Smoothing 3 8 Applied Biosystems Peak Detection e Deisotope reflector data only Peak deisotoping reduces the spectrum to a monoisotopic centroided plot of the monoisotopic masses This is useful in identifying overlapping isotope clusters Make sure peak detection thresholds are set low enough to detect the monoisotopic peak before deisotoping For more information see Section 3 4 Deisotoping a Spectrum Deisotoping provides no benefit on linear data non isotopically resolved or on PSD data pure isotope data If peak detection is not acceptable keep the Use Resolution Dependent Settings option enabled and adjust the parameter associated with the observed problem Problem Suggested Actions High mass peaks not detected Decrease Mass Resolution setting The default Mass Resolution settings are optimized for masses below 20 000 Da Noise detected as peaks Increase the Max Peak Area Decrease the Resolution Peaks of interest are not detected Decrease the Max Peak Area
20. apply Baseline Correction then re detect peaks If you use area based thresholding Max Peak Area during peak detection Baseline Correction is not typically needed Max Peak Area compensates for a rising or falling baseline e With a baseline that is not at 0 Some measurements for example Centroid peak detection value or the Peak Height in the Resolution calculator are derived from a peak height measured from 0 NOTE If you are analyzing Mariner data baseline correction is typically not needed If you baseline correct Mariner data note that due to the shorter flight times and fewer data points associated with Mariner data baseline correction may affect peak shape which in turn affects mass accuracy Correcting the To correct the baseline baseline 4 Display the spectrum of interest 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Baseline Correction The baseline is adjusted and the trace is displayed with a BC trace label Returning to the To return to the original trace see Returning to the original original spectrum spectrum on page 5 3 Data Explorer Software User s Guide 5 47 Chapter 5 Examining Spectrum Data 5 8 3 Using Advanced Baseline Correction This section includes e Description e When to use e Correcting the baseline e General guidelines for setting parameters e Trou
21. described below e Selecting peaks manually on page 8 16 8 14 Applied Biosystems Calibrating a PSD Spectrum Matching peaks if you want the software to compare observed masses in all automatically segment spectra included in the DAT file to reference masses in the selected calibration reference file 1 Click Match If a mass within the tolerance of any of the masses listed in the calibration reference file is found in any spectrum in the DAT file the match is displayed in the Calibration Mass Peak Selection window CAUTION Use the Match function with care Before using ensure that all peaks in all segments are correctly peak detected The Match function examines the complete mass range in all segments in the DAT file NOTE The entire mass range of each spectrum in the DAT file is checked not just the mass range included in the composite spectrum and even if the spectrum is not currently displayed in the Spectrum window For comparison the difference between the reference mass in the calibration reference file and the observed peak mass is displayed Hint You can sort the list of matches by clicking on the column header buttons see Figure 8 5 on page 8 13 You can display complete information about a reference mass by double clicking on the mass If no matches are found an error message is displayed 2 To delete a match from the list select the mass and click Delete Selected Mat
22. on page 1 37 All SET files are user editable However before you edit the default processing graphic SET files see page 1 18 make a copy of the original SET file in case you need to reload the settings for default peak detection Copy the file using Windows NT Explorer Data Explorer Software User s Guide 1 19 Chapter 1 Data Explorer Basics Opening To open customize and save SET files customizing and 1 If you are customizing a default SET file make a copy saving SET files of the original file before opening it See Making a copy of default SET files before customizing on page 1 19 Select Settings from the File Menu Select one of the following e Restore Processing Settings e Restore Graphic Settings e Restore Graphic Processing Settings 4 Inthe Restore dialog box select or type the name of the SET file then click OK 5 Customize processing and graphic settings as needed For information on the contents of processing and graphic settings see What settings contain on page 1 18 6 To save settings select Settings from the File menu then select one of the following e Save Processing Settings As e Save Graphic Settings As e Save Graphic Processing Settings As 7 Inthe Save As dialog box type the name of the SET file then click OK Applying a SET You can apply a SET file two ways file e Select Recent Processing Settings from the File menu then select a SET fi
23. the average mass is used for mass labeling and corresponds to the centroid of the unresolved peak cluster weighted average of all isotope peaks in the cluster Applied Biosystems Monoisotopic and Average Masses Average mass corresponds to centroid of unresolved peak cluster Figure B 7 Average Mass Data Explorer Software User s Guide B 7 Appendix B Overview of Isotopes B 3 Isotopes of Common Elements Table B 1 lists the natural abundance of isotopes for some common elements seen in mass spectrometry Table B 1 Isotopes of Common Elements Natural Natural Isotope Mass abundance Isotope Mass abundance 1H 1 0078 99 985 31p 30 9737 100 2H 2 0141 0 015 325 31 9720 95 02 126 12 98 90 33S 32 9714 0 75 13C 13 0033 1 10 34S 33 9678 4 21 14N 14 0030 99 63 36S 35 9670 0 02 15N 15 0001 0 37 35C 34 9688 75 77 160 15 9949 99 76 37C 36 9659 24 23 170 16 9991 0 04 39K 38 9637 93 2581 180 17 9991 0 200 40K 39 9639 0 012 19F 18 9984 100 K 40 9618 6 7302 23Na 22 9897 100 Br 78 9183 50 69 28Si 27 9769 92 23 81Br 80 9162 49 31 29Si 28 9764 4 67 127 126 9044 100 soSi 29 9737 3 10 1 Meth Enzymol McCloskey J A ed 1990 193 B 8 Applied Biosystems C Data Explorer Toolbox Visual Basic Macros This appendix includes GA Oveni Wavre a a C 2 C 2 Preparing Data Before Accessing Macros scce C 3 C 3
24. white does not print 1 26 2 33 Traces copying from different data file 2 37 to Windows clipboard 1 38 to WMF 1 39 Traces removing active 2 21 from history list 2 22 inactive 2 21 Troubleshooting calibration 9 9 calibration reference file REF not listed 9 10 charge state and isotope 9 17 charge state incorrectly labeled 9 17 conversion 9 5 Deconvolution commands dimmed 5 37 elemental composition limits ignored 6 8 6 11 error messages when you open a file 9 4 9 5 extracted ion chromatogram created when you right click drag to apply custom label 9 15 general 9 3 Isotope calculator 6 19 Troubleshooting continued Link View does not work 9 7 Multiple Charge commands dimmed 5 37 overlaid traces 9 6 overview 9 2 peak detection 9 14 peak detection Voyager 3 9 peak labeling 9 14 printer does not stay set to landscape mode 9 13 printing 9 13 processing 9 6 single charge conversion 5 62 spectrum 9 17 tools 9 6 Truncate spectrum 5 56 TXT file 1 34 U Unresolved peaks deconvoluting and evaluating 7 4 Unzooming 2 14 User labels changing values 3 64 creating 3 61 customizing colors font size 3 61 do not display 3 59 3 66 enabling 3 65 importing from other files 3 65 saving settings 3 64 setting values 3 64 UV Mariner data only see also DAD data diode array detector DAD data diode array detector displaying 5 2 displaying trace 4 2 trace offset 4 30 Data Explorer Software
25. Add or Replace in the Display Trace dialog box For information on Trace Replace mode see Section 2 4 4 Adding Traces from the Same Data File to a Window Data Explorer Software User s Guide 4 23 Chapter 4 Examining Chromatogram Data Hint Add mode is useful when you filter the same trace for different event tags The original trace remains displayed and accessible Each filtered trace up to four total traces is added allowing for visual comparison Filtering To display chromatogram traces for selected event tags event tags 1 Display the data file containing the event tags 2 Click the Chromatogram window to activate it 3 From the Display menu select Traces then select the trace type to filter 4 Ifyou have more than one trace displayed select the trace to filter 5 From the Process menu select Event Tag Filtering NOTE If the Chromatogram window is not active Event Tag Filtering is not displayed on the Process menu If the active chromatogram was not collected with an MS Method that specified event tags the Event Tag Filtering command is dimmed The Event Tag dialog box is displayed Figure 4 10 Chro Event Filter Selection _2 x r System Defined Event Tags Jo Neaetive poang M InSource CID M Generic Event Tags IV Event Tag 1 I Event Tag 5 IV Event Tag 2 I Event Tag 6 IV Event Tag 3 I Event Tag 7 I Event Tag 4 I Event Tag 8 m Multiple Injection Selection I
26. Centroid mass calculated during peak detection 3 69 calculating 3 39 copying from peak list 1 41 definition B 6 determining 5 36 improving accuracy of calculation during peak detection 3 26 labeling 3 57 reverting to original Centroid Percent spectrum instrument 5 22 setting 3 26 Calibration curve peak weighting Centroiding factors 5 10 8 14 centroid data file size smaller than Calibration fit error 5 12 profile 1 33 Calibration reference file REF command not displayed 9 8 ANGIOTENSIN_FRAGMENTS RE F 5 18 contents 5 18 creating 5 18 default 5 18 duplicate m z entries 5 20 5 21 9 10 error message when selecting 8 14 MARINER_NEG REF 5 18 converting profile to centroid data 1 33 creating centroid trace 5 36 during peak detection 3 69 during peak detection improving calculation 3 26 example 5 36 histogram trace 5 36 manual 5 36 MARINER_POS REF 5 18 modifying 5 20 narrow peaks specifying as CGM files 1 31 1 33 Channel displaying DAD data by 1 12 Charge state peak Resolved Isotope mass 5 21 9 9 not listed 9 10 provided 5 18 PSD creating 8 21 PSD specifying Resolved Isotope masses 8 13 selecting for automatic calibration 5 30 selecting for manual calibration 5 9 selecting for PSD calibration 8 13 viewing contents of 5 10 VOYAGER REF 5 18 and isotope spacing 3 53 converting to singly charged 5 59 converting to zero charged 5 40 custom label applied only if specified charge i
27. Continued 3 26 Applied Biosystems Peak Detection Table 3 3 Peak Processing Parameters Spectrum Data Only Continued Parameter Description Charge State Determination Spectrum only Maximum Charge Determines the peak spacing evaluated for the presence of State isotope peaks The expected peak spacing is determined by the Max Charge State plus or minus a tolerance value The tolerance is calculated as proton mass charge state x 15 For example with a Max Charge State of 3 the software checks for peaks that are 0 33 m z apart with a fixed tolerance of 0 05 m z where proton mass equals 1 007276456 Maximum Charge State is 6 for Mariner data and 1 for Voyager data Maximum Isotopes Specifies the maximum number of peaks included in an isotope cluster NOTE Setting this value too low can result in peaks not being included in the appropriate isotope cluster For more information see Max Isotope set too low on page 3 35 Minimum and Specifies the intensity range that a peak must fall within Maximum Intensity relative to the previously evaluated higher mass peak to be included in the current isotope cluster Data Explorer Software User s Guide 3 27 Chapter 3 Peak Detection and Labeling Advanced Settings spectrum data only Table 3 4 describes the parameters in the Advanced Settings tab of the Spectrum Peak Detection Setup dialog box see Figure 3 6 on page 3
28. Copy mass list Managing Files 2 Select the trace window to copy 3 Display the peak list by selecting Output Window from the Display menu then clicking the Chro Peak List or Spec Peak List tab 4 Sort the peak list as needed See Sorting the peak list on page 3 42 From the Edit menu select Copy then select All Peaks Paste the data into an appropriate application for example Microsoft Excel This function copies only the masses for the spectrum and is useful for pasting data directly into spreadsheets or other applications without having to delete header information To copy all centroid or apex mass data from the spectrum peak list to the clipboard 1 Select the Spectrum window to copy from 2 Select Peak Label from the Peaks menu 3 Specify the Mass Type by selecting Apex or Centroid For more information see Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels 4 Click OK 5 From the Edit menu select Copy then select Mass List Hint If you activate the Spec Peak List tab you can copy the mass list by right clicking then selecting Copy Mass List from the popup menu The mass list is copied to the clipboard with a header indicating Apex Mass or Centroid Mass 6 Paste the data into an appropriate application for example Microsoft Excel Data Explorer Software User s Guide 1 41 Chapter 1 Data Explorer Basics 1 42 Applied Biosystems 2 Using Chromatogram
29. PowerPoint 1 38 Applied Biosystems Managing Files NOTE If you paste the image into an application that does not handle Windows Metafile format images for example Microsoft Paint images are distorted Copy trace data To copy raw data x y pairs for the peaks displayed in the active trace to the Windows clipboard 1 Select the trace window to copy Zoom and adjust the trace as needed NOTE Only raw data for the set of peaks displayed in the trace window is copied From the Edit menu select Copy then select Trace Data Paste the data into an appropriate application for example another Data Explorer trace window or Microsoft Excel When pasted into a Data Explorer trace window the trace appears with the filename in parentheses and the trace label from the copied trace CAUTION A trace pasted into an active Data Explorer window contains only the data points for the trace The Sample Info and Instrument settings tabs display data from the original data file These tabs do not include information about the copied trace Copy displayed Use this method to copy the section of the peak list pertaining peaks to the peaks displayed in the active trace window To copy the peak list for all peaks in the active trace see Copy all peaks on page 1 40 Data Explorer Software User s Guide 1 39 Chapter 1 1 40 Copy all peaks Applied Biosystems Data Explorer Basics NOT
30. The first letter of two letter elemental symbols must be capitalized Spaces do not matter Amino Acid One letter or three letter amino acid Sequence codes The first letter of amino acid codes must be capitalized Spaces do not matter NOTE Do not include elemental codes when you type the sequence for example to specify water of hydration Invalid results may be generated DNA ACGT Sequence RNA ACGU Sequence Carbohydrates Any entry listed for carbohydrates from Elemental Composition Limits See Setting limits for other result types on page 6 12 Separate entries with spaces 6 Select the Plus H20 check box if you want to calculate the isotopes for the complete molecule residue plus water of hydration 6 14 Applied Biosystems Using the Isotope Calculator 7 Specify the Add Subtract Group option To enable or disable the option select or deselect the Group Type check box 8 In the Add Subtract Group section select Element or group to add or subtract from the formula before calculating the isotope does not apply if Add Subtract Group is disabled Number of elements or groups to add or remove and the charge state to divide by NOTE When Add Subtract is enabled the Group Count Charge field determines the number of groups to add or subtract and the charge state to calculate When Add Subtract is disabled the Charge field determines the charge state to calculate e Wh
31. Using the Macro Recorder De assigning a To de assign a macro from a button select the macro button in macro from a the Assign Macro dialog box then click De assign button 6 7 4 Running a Macro You can run a macro using a Toolbar button e Menu command Using a toolbar To run a macro using the toolbar button button 4 Open the data file on which you want to run the macro 2 Click the button assigned to the macro you want to run Hint Place the cursor on a macro button to display the name of the macro assigned to the button The macro executes Using the menu To run a macro using the menu command 4 Open the data file on which you want to run the macro 2 From the Tools menu select Macros The Macros dialog box Figure 6 25 is displayed Data Explorer Software User s Guide 6 39 Chapter 6 Using Tools and Applications If the macro contains a syntax error Applied Biosystems Hiii D aa eed ert E Figure 6 25 Macros Dialog Box 3 Select the macro to run from the list 4 Click Run The macro executes If the macro contains a syntax error it may cause the Data Explorer software to close unexpectedly If this occurs restart the Data Explorer software and examine the macro in the Visual Basic Editor See Section 6 7 6 Advanced Macro Editing Macro lines containing syntax errors are displayed in color in the Visual Basic Editor For information on correctin
32. You can display segment spectra by clicking and Indicates Base Peak BP mass and intensity for the tallest peak in the spectrum Displayed as Intensity versus Mass to Charge m z The right axis is scaled to the intensity of the base peak Data Explorer Software User s Guide 1 13 Chapter 1 Context sensitive menus Tabs for data files Data file names 1 14 Applied Biosystems Data Explorer Basics Labels in the chromatogram or spectrum title identify the type of data displayed in the window For a description of labels see Section 2 4 10 Viewing Trace Labels For more information on Chromatogram and Spectrum windows see Chapter 2 Using Chromatogram and Spectrum Windows The commands displayed on the menus in the Data Explorer window depend on the window that is active when you select the menu e Only Mariner related commands are displayed on menus when you open a Mariner DAT file e Only Voyager related commands are displayed on menus when you open a Voyager DAT file e Only spectrum related commands are displayed if you select menus when the Spectrum window is active e Only chromatogram related commands are displayed if you select menus when the Chromatogram window is active For Voyager multiple spectra DAT files UV functions are disabled Chromatogram and Spectrum windows display a tab at the bottom see Figure 1 3 on page 1 11 that allow you to switch between da
33. continued full name not displayed 1 14 inserting traces into 2 37 moving between open 2 8 multiple zooming 2 36 name 1 14 names do not print 9 13 opening 1 30 opening and applying default and selected settings 2 4 opening and running a macro automatically 6 45 opening manually 2 2 opening PSD 8 2 read only 2 7 result opening 2 39 2 40 switching between open 2 8 trace browser 2 8 version of software used to acquire 1 15 working with separate 2 36 zooming multiple 2 36 Data format see DAT format Data points copying 1 39 determining number across a peak 3 21 3 31 5 51 eliminating from spectrum 5 56 Database search macro C 18 DATAEXPLORER VB6 exporting macros from 6 44 importing new macros into 6 43 location 6 35 new supplied macros not available until imported 6 43 not overwritten when new software installed 6 43 DBE definition 6 6 Decimal points number displayed 3 55 3 56 DECONV in spectrum header 2 32 5 40 5 41 Deconvolution see Mass deconvolution Default settings applying from SET file 2 4 DEFAULTBLACK SET 1 19 1 23 Defaults background color 1 23 colors Mariner 1 4 colors Voyager 1 4 peak detection 3 2 processing and graphics settings applying when you open a data file 2 4 REF files 5 6 5 18 SET files 1 19 1 23 8 11 setting for dialog boxes 1 17 trace color 1 23 DEFAULTWHITE SET 1 19 1 23 Deisotoping description 3 45 enhancing peak interpretation 3 9 example 3 50 for
34. e Expand traces Click a trace then click dl in the toolbar to expand the selected trace for closer examination To display all traces click al again e Link traces Click S in the toolbar to link all traces Any zooming actions you perform on one trace affect all traces in that window To unlink traces click again Data Explorer Software User s Guide 2 21 Chapter 2 Using Chromatogram and Spectrum Windows 2 4 7 Recalling and Rearranging Traces Processing History Overview The Chromatogram and Spectrum windows can display up to 8 traces at a time for a data file However up to 16 traces are held in memory You can recall traces previously displayed for an open data file or rearrange the order of traces using the Processing History command You can also set Processing History Options to automatically delete history or to disable the function completely Recalling or To recall or rearrange previously displayed traces rearranging 1 Click the trace position in which you want to recall or rearrange a trace 2 From the Display menu select Processing History A submenu is displayed listing the last 16 processed traces viewed in the window NOTE Unprocessed traces and theoretical traces generated using commands on the Applications menu are not listed 3 Select the trace to display The selected trace is displayed in the active trace position Removing traces To remove traces fro
35. for the converted SPC file to prevent the new conversion from overwriting the existing file Table 9 3 General Troubleshooting Voyager Only Symptom Possible Cause Action Failed to get original calibration message displayed when you open a data file You are opening a Voyager MSF data file collected with version 4 0 or earlier software No action Normal occurrence Data Explorer Software User s Guide 9 5 Chapter 9 Troubleshooting 9 3 Processing Tools and Applications Troubleshooting Table 9 4 Processing Tools and Applications Troubleshooting Mariner and Voyager Symptom Possible Cause Action Failed to calculate result for isotope calculator You may have tried to remove a group that is not present in the formula Can only remove a group that is present in the formula For information see Section 6 2 Using the Isotope Calculator All traces in an overlaid trace are not processed Only the active trace in an overlaid trace is processed Display individual traces select Use same settings for all graphs from Graphic Options then process For more information see e Section 1 5 Setting Graphic Options e Section 2 4 8 Overlaying Traces 9 6 Applied Biosystems Processing Tools and Applications Troubleshooting Table 9 4 Processing Tools and Applications Troubleshooting Mariner and Voyager C
36. macros preparing data and running the macros xii Applied Biosystems How to Use This Guide Conventions This guide uses the following conventions to make text easier to understand Bold indicates user action For example Type 0 and press Enter for the remaining fields e Italic text denotes new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix Notes Cautions A note provides important information to the operator For Warnings and example Hints NOTE If you are prompted to insert the boot diskette into the drive insert it then press any key 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 information essential to the safety of the operator For example WARNING CHEMICAL HAZARD Wear appropriate personal protection and always observe safe laboratory practices when operating your system 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 Data Explorer Software User s Guide xiii How to Use This Guide xiv Related documentation Send us your comments Applied Biosystems The related documents shipped with your system include Mariner Workstat
37. tab are applied to all detection ranges and override the thresholds set for individual detection ranges on the Advanced Settings tab Detection ranges that do not correspond to ranges calculated by resolution dependent peak detection are deleted NOTE To reset to original peak detection settings restore the default SET file for your system For more information see Applying a SET file on page 1 20 Peak Detection 3 2 4 Peak Detection Parameter Descriptions Chromatogram settings This section describes e Chromatogram settings e Basic Settings spectrum data e Peak Processing parameters spectrum data only e Advanced Settings spectrum data only Table 3 1 describes the parameters in the Chromatogram Peak Detection Setup dialog box see Figure 3 3 on page 3 12 Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Table 3 1 Chromatogram Settings Parameter Description Detection Ranges Specifies one or more non contiguous m z ranges for peak detection You can set parameters for each range independently You select a range in the Detection Ranges list box by single clicking the range number To add a detection range do one of the following Select an existing range then click 3 This creates a new range with boundaries ranging from the end of the existing range to the end of the trace If the existing range ends at the end of the trace
38. 1 Strategy for Mariner Peak Detection 3 6 Applied Biosystems This section gives some quick suggestions on how to approach Mariner peak detection For details on peak detection see Section 3 2 3 Setting Peak Detection Parameters Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Strategy When detecting peaks in Mariner data 1 Open the data file and observe the effects of the default peak detection settings If you are analyzing resolved isotopes default settings should yield acceptable peak detection The default resolution setting of 5 000 is optimized for masses below 3 000 Da If you are analyzing proteins decrease the Mass Resolution setting Peak Detection 3 If peak detection is not acceptable leave the Use Resolution Dependent Settings option enabled and adjust the following global threshold parameters in the following order Base Peak Intensity Use this parameter to eliminate peaks with an intensity below the specified threshold This threshold is represented as a percentage of the intensity of the base peak Max Peak Area Use this parameter to fine tune noise discrimination on the baseline or noise spikes on peaks This threshold is represented as a percentage of the area of the peak with the largest area and is calculated above the local baseline To determine an appropriate threshold display the Spectrum Peak list note the area of the peak wit
39. 25 0 0 0 Max Peak Area 0 25 2 2 2 Min Intensity 0 0 0 0 Min Area 0 0 0 Range dependent Additional Additional SET files that have been developed for detection of Voyager SET files different types of data are included in the provided C VOYAGER SETTINGS directory The names of the SET files indicate the type of data the files can be used for Data Explorer Software User s Guide 3 73 Chapter 3 Peak Detection and Labeling 3 74 Applied Biosystems 4 Examining Chromatogram Data This chapter contains the following sections 4 1 OVEIVIEW s sesier tain Anat tae aidia 4 2 4 2 Creating an Extracted lon Chromatogram 4 5 4 3 Creating an Extracted XAC Chromatogram Mariner Data Only cceeceeeeeeeeeeeeeeeeeees 4 13 4 4 Noise Filtering Smoothing 0eeeeeee 4 17 4 5 Adding and Subtracting Spectra 05 4 20 4 6 Displaying MS Method Data Mariner Data Only cceeeeeeeeee eee eeeteeeeeeeees 4 23 4 7 Adjusting the Baseline aeee 4 27 4 8 Using UV Trace Offset 4 30 Data Explorer Software User s Guide 4 1 Chapter 4 Examining Chromatogram Data 4 1 Overview This section includes e Types of Mariner data e Types of Voyager data e Creating macros to combine processing functions e Returning to the original trace Types of You can display different types of chromatogram traces from Mariner data Mariner data files by selecting Traces from the Display menu w
40. 27 21 30 16 20 10 24 0 83 2 152 4 216 6 280 8 345 0 Spectrum Number Figure 4 7 Extracted Absorbance Chromatogram 7 To return to the original trace see Returning to the original trace on page 4 4 4 16 Applied Biosystems Noise Filtering Smoothing 4 4 Noise Filtering Smoothing Description The Noise Filter Smooth command provides three options for reducing noise in chromatogram traces e Noise filter e Smooth by the Gaussian method e Noise removal Procedure To noise filter or smooth a chromatogram trace 1 From the Process menu select Noise Filter Smooth The Noise Filter Smooth dialog box Figure 4 8 is displayed Noise Filter Smooth xi Methods Correlation Factor fos Cancel Figure 4 8 Noise Filter Smooth Dialog Box Data Explorer Software User s Guide 4 17 Chapter 4 Examining Chromatogram Data 2 Select the method to use based upon the type of data you are examining then enter the associated value displayed for the method you select Suggested N Type of Data Method Description Higher resolution Noise Filter Specify a Correlation Factor of 0 to 1 0 data NF Settings from 0 5 to 0 7 yield acceptable results for most data A setting close to 1 0 May affect yields a higher degree of noise reduction peak If applying the Noise Filter with a certain resolution Correlation Factor does not yield the necessary noise removal return
41. 5 3 5 Hints for Calibrating Mariner Data cecseeeeeeeeeeeeeeeeeees 5 24 5 3 6 Hints for Calibrating Voyager Data c ceeeeeeeeeeeeeeeeeeees 5 25 Automatic Calibration ieissar eraren nirre cence eeeeeeeeeeeeneeeneeeeeenaeeees 5 26 5 4 1 Overview of Automatic Calibration cccccccsseeeceeeenneeeeeeees 5 26 5 4 2 Importing and Specifying Automatic Calibration Settings 5 29 5 4 3 Automatically Calibrating Mariner Data Only eessen 5 34 Applied Biosystems 5 5 5 6 5 7 5 8 5 9 5 10 5 11 5 12 Table of Contents E ningo e ALE Ma EAA eheteleni tee dthetcadoudedgundunyesuaneseenaneesenye 5 36 Mass Deconvolution Mariner Data Only ceeeeeeee eee eee eee ee eee 5 37 Noise Filtering SMOothing s eeeeee cece eee eee eee eee ee eee e eee eee ee eee 5 42 Adjusting the Baseline ccccceeee cece ete e tence ee eee seen eeeeeeeeeeeeeeenes 5 45 5 8 1 Using Baseline Offset c ccceeceeeeeeeeeeeaeeeaeeaeeeteeteeeeeeeeeeees 5 45 5 8 2 Using Baseline Correction 0 cccccceeeeeeeeeaeeeeeeeeeeeeteeeeeeeeeeees 5 47 5 8 3 Using Advanced Baseline Correction ceeeeeeeeeeeeeeeeees 5 48 TTUNCATLING a Spectrum a en tcc ce he aa nE EENE a MaMa AREAS A HS EA EAA 5 56 Converting to a Singly Charged Spectrum Mariner Data Only 5 59 AutoSaturation Correction Mariner Data Only eeeeeeeeeee ees 5 62 Adding and Subtracting Raw or Proces
42. 57 XAC 4 14 4 15 XIC 4 7 Ladder Sequencing macro C 2 C 5 Landscape printer orientation lost when you exit 9 13 setting permanently 2 35 LBC or LBS files in DAT format applying 3 65 creating 3 64 description 1 7 extracting from DAT file 1 36 Line mode traces 1 28 Line width setting 1 26 Linking multiple data files for zooming 2 13 traces within a data file 2 21 views 2 13 views does not work 9 7 Local peak detection parameters description spectrum 3 28 setting spectrum 3 28 Loss of H2O determining C 11 Loss of NH3 determining C 10 Low Mass Gate spike eliminating 5 56 5 57 M Macro Recorder advanced editing 6 42 buttons assigning to macros 6 38 DATAEXPLORER VB6 location 6 35 DATAEXPLORER VB6 not overwritten when new software installed 6 43 deleting a macro 6 41 description 6 34 exporting macros from DATA EXPLORER VB6 6 44 functions not supported 6 35 importing macros into DATA EXPLORER VB6 6 43 location of macros 6 35 maximum number of macros 6 34 new supplied macros not available until imported 6 43 recording 6 37 running a macro 6 39 toolbar displaying 6 35 Visual Basic Editor 6 42 Macros provided see Data Explorer Toolbox recording 6 37 running automatically when opening and closing data files 6 45 running manually 6 39 Manual calibration see Calibrating mass manual Manually labeling peaks 3 39 3 52 Mariner data Chromatogram window displaying 2 2 DAD data diode array detector dis
43. 6 5 9 PSD calibration 8 14 Peak resolution see Resolution mass Peak Threshold replaced by Base Peak Intensity 3 20 3 22 Peak weighting factors 5 10 8 14 Peak Width minimum and maximum used 3 21 3 25 set automatically by software 3 21 3 25 Peaks do not appear in spectrum 9 17 Peptide fragmentation macro C 2 C 9 Periodic table 6 10 PerSeptive Biosystems Technical Support see Applied Biosystems Technical Support PKT files 3 40 Points across a peak determining 3 21 3 31 5 51 Polydispersity index determining C 15 Polymer analysis macro C 2 C 15 Positive ion z label 3 58 Precursor mass see PSD analysis Preface xi Previewing traces before printing 2 33 Print preview 2 33 Print Setup 2 36 Printer setting to landscape orientation 2 35 Printing all traces in view 2 34 changing colors to black before 2 33 data cursors 1 27 data file names do not print 2 34 2 37 landscape orientation 2 35 multiple files 2 34 peak list 3 44 traces 2 33 traces do not print 2 34 troubleshooting 9 13 without previewing 2 34 Process menu commands not displayed 9 8 Processing see also Processing settings commands not displayed on menu 9 6 troubleshooting 9 6 Processing History description 2 22 disabling 2 23 options 2 23 using 2 22 Processing settings applying when opening data file 2 4 automatically saved when data file closed 1 18 customizing 1 19 description 1 18 extracting from DAT file 1 36 modifying 1 19 peak det
44. 6 13 6 3 Using the Mass Resolution Calculator 0 6 20 6 4 Using the Signal to Noise Ratio Calculator 6 23 6 5 Using the lon Fragmentation Calculator 6 25 6 6 Using the Elemental Targeting Application 6 31 6 7 Using the Macro Recordev 0 6 34 Data Explorer Software User s Guide 6 1 Chapter 6 Using Tools and Applications 6 1 Using the Elemental Composition Calculator Description Determining identity of fragment masses 6 2 Applied Biosystems This section includes e Determining elemental composition Setting limits 6 1 1 Determining Elemental Composition This section includes e Description e Determining identity of fragment masses e Procedure e Calculating e Results e If no results are displayed e Displaying the theoretical isotope distribution The Elemental Composition calculator determines possible elemental or amino acid compositions for a given mass The application then generates a theoretical isotope pattern using the Mass Resolution specified in Basic Peak Detection settings compares each observed mass and isotope pattern to the theoretical mass and isotope pattern for each possible composition and reports an isotope match score that reflects how closely they match You can also use the Elemental Composition calculator to determine the identity of fragment masses To do so set Peak Labels to display Mass Diff
45. 7 20 Applied Biosystems Voyager Data Examples Spectrum Peak Detection Setup All Traces Basic Settings Peak Processing Advanced Settings m Peak Detection Settings Detection Range s full pal x Range s Lower Bound Upper Bound Range 1 Start End Active Local Range Thresholds BP Intensity 28933 Min Intensity B Max Peak Area 2 Min Area fo Noise Threshold p 4 m Filter Settings Width E a Increment fi 4 OK Cancel Figure 7 21 Spectrum Peak Detection Setup Advanced Settings Tab 6 Set Minimum Area to 0 7 Click the Basic Settings tab see Figure 7 20 on page 7 20 then set Max Peak Area to 0 or 0 1 8 Click Apply Note that many peaks are added to the peak list in the Output window For more information see Section 3 2 Peak Detection Deisotoping 9 From the Peaks menu select Peak Deisotoping The Deisotoping dialog box Figure 7 14 is displayed Data Explorer Software User s Guide 7 21 Chapter 7 Data Explorer Examples Repeat Formula Adduct H Formula CSH 5NO OK Cancel Figure 7 22 Deisotoping Dialog Box 10 For this example spectrum specify H for Adduct and C6H5N0O for Generic Formula 11 Click OK For more information on deisotoping see Section 3 4 Deisotoping a Spectrum Increasing 12 In the Basic Settings tab in Global Thresholds see detection Figure 7 20 on page 7 20 increase Max Peak Area th
46. Accessing the MacroS ccceeeeeeee C 4 C 4 Using the Ladder Sequencing Toolbox C 5 C 5 Using the Peptide Fragmentation Toolbox C 9 C 6 Using the Polymer Analysis Toolbox C 15 C 7 Using MS Fit MS Tag Toolbox C 18 Data Explorer Software User s Guide C 1 C Appendix C Data Explorer Toolbox Visual Basic Macros C 1 Overview Macros provided The following toolbox of Visual Basic macros is provided with Importing macros C 2 provided Modifying the macros Applied Biosystems the Data Explorer software e Ladder Sequencing Use when performing sequencing to label peaks with the appropriate amino acid DNA residue or RNA residue Peptide Fragmentation Use when examining Voyager composite PSD spectra or Mariner in source CID spectra to label immonium ions identify pairs view sequences based on different reference peaks and determine if a selected peak is of a specific fragment ion category e Polymer Analysis Use when analyzing polymer spectra to calculate average molecular weight values MS Fit MS Tag Use when analyzing peptide or peptide fragment spectra to perform a protein database search If the macros listed above are not listed when you try to run them described in Section C 3 Accessing the Macros you must import them into the Data Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLO
47. Applied Biosystems Figure 6 10 Isotope Trace If you have the Replace mode set to Add in the Display Trace dialog box a new trace is added If you have the Replace mode set to Replace the isotope trace replaces the active trace For more information on Replace mode see Setting the Replace mode on page 2 17 To evaluate the trace you can e Overlay theoretical and observed isotope patterns See Section 2 4 8 Overlaying Traces for information Add new traces and calculate the isotope multiple times using different settings then compare or overlay the different calculations To return to the original trace see Returning to the original spectrum on page 5 3 Using the Isotope Calculator Results The results of the calculation are displayed in the Result tab of Output window Figure 6 11 fantaa Celouletion Fmault gt 3 gt C100 H202 Bm SE resolving porer Figure 6 11 Isotope Calculation Results in Output Window If results are not if results are not calculated you may have tried to remove a calculated group that is not present in the formula Data Explorer Software User s Guide 6 19 Chapter 6 Using Tools and Applications 6 3 Using the Mass Resolution Calculator Calculating mass To calculate mass resolution 6 20 resolution Applied Biosystems i Display the spectrum of interest Make sure the Spectrum window is active If you are examining Voyager data baseline
48. Calculator The lon Fragmentation calculator generates a list of possible fragment masses for a peptide sequence you enter It can calculate the masses for e Multiply charged fragments you may see in Mariner data e PSD fragments you may see in Voyager data If the calculated fragments are present in the current data file you can label fragments The calculator includes a list of defined amino acids and residues that you can add to as needed To use the lon Fragmentation Calculator 1 Display the spectrum of interest Click the Spectrum window to activate it 3 From the Applications menu select lon Fragmentation Calculator The lon Fragmentation Calculator dialog box Figure 6 15 is displayed Data Explorer Software User s Guide 6 25 6 26 Chapter 6 Using Tools and Applications lon Fragmentation Calculator xj N terminus Sequence C terminus Hydrogen x AAYYAAYYAAYY Free Acid x Induce Fragmentation Cysteines are modified by unmodified x Options Calculate fragment ion masses as moncisctopic x User defined Amino Acids Peptide Mass MH monoisotopic Print Peptide Mass MH average Label Peaks lons table lons charge Internal fragments table Internal sequence Clear Table Info Figure 6 15 lon Fragmentation Calculator Dialog Box 4 Type or copy the amino acid or residue sequence of interest Use single letter codes Sequence codes are
49. Description Specifies Degree Determines how closely the calculated baseline fits the data Valid entries are 0 0 to 1 0 A value closer to 1 fits the baseline more closely to the data and corrects the midpoint of the noise signal to approximately 0 intensity A value closer to 0 fits the baseline less closely to the data and corrects the midpoint of the noise signal to a value greater than 0 intensity see below For protein and tryptic digest peptide spectra use 0 to 0 5 If the baseline after correction is too high increase the Degree value see below A AAA AAAI Degree 1 Degree 0 Midpoint of noise signal Midpoint of noise signal corrected to 0 corrected above 0 A lower Degree setting allows the baseline correction to occur more quickly and often provides better correction than a higher setting NOTE Flexibility and Degree parameters may require some testing to determine the optimum settings for your data 4 Click OK The baseline is adjusted and the trace is displayed with a AdvBC trace label Data Explorer Software User s Guide 5 53 Chapter 5 Examining Spectrum Data General Refer to the following table to determine how to set Advance guidelines for Baseline Correction parameters and obtain the desired setting baseline correction parameters Condition Set Parameters To Baseline follows a rising peak cluster a Peak Width Average peak wid
50. Mass 9 Troubleshooting This chapter contains the following sections 9 1 OVETVIEW sinuni aide daw dine eee 9 2 9 2 General Troubleshooting eseeeeeeeeeees 9 3 9 3 Processing Tools and Applications Troubleshooting 2 0 0 cece ee eee cette ee eeeeeeeeeeeeeeeneees 9 6 9 4 Calibration Troubleshooting ceeeeees 9 10 9 5 Printing Troubleshooting csseeeeeeeeeeeeeees 9 14 9 6 Peak Detection and Labeling Troubleshooting 0 eee eeeeeeeeeeee ee eeeeeeeeaaeeeeeees 9 15 Data Explorer Software User s Guide 9 1 Chapter 9 Troubleshooting 9 1 Overview 9 2 Applied Biosystems This section includes e General troubleshooting e Processing tools and applications troubleshooting e Calibration troubleshooting e Printing troubleshooting e Peak detection and labeling troubleshooting Troubleshooting information is organized according to likelihood 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 General Troubleshooting 9 2 General Troubleshooting Table 9 1 General Troubleshooting Mariner and Voyager Symptom Possible Cause Action Cannot find data file Did not save the spectrum to a DAT file
51. Match Score 6 6 Isotope Match Score not reported for fragment ion calculations 6 6 isotope min and max values ignored 6 8 6 11 limits setting 6 7 6 9 6 12 Periodic table 6 10 procedure 6 3 results 6 5 Elemental targeting description 6 31 Isotope Match Score 6 33 procedure 6 31 results 6 33 theoretical isotope displaying 6 33 Eliminate Fit Outlier 5 12 E mail address Technical Publications xiv Equations PSD calibration 8 6 resolution based peak detection 3 4 Error calibration fit 5 12 initial 5 12 Event tag filtering Mariner data only not displayed on Process menu 4 24 overview 4 23 performing 4 24 redisplaying original data 4 26 Event tag MS Method Mariner data only see also MS Method Mariner data only events determining tags in file 4 23 displaying 4 23 filtering 4 23 redisplaying original data 4 26 Examples see Data Explorer examples Excel see Microsoft Excel Exiting software 1 3 Expanding traces 2 21 Exporting see also Converting ASCII data 1 34 BIC 1 36 CAL files 1 36 Configuration from DAT file 1 36 entire data file 1 34 macros from DATA EXPLORER VB6 6 44 MSM and CAL from DAT 1 36 results 2 39 RSD and RCD files 2 39 trace to ASCII format 1 34 Extracted absorbance chromatogram XAC Mariner data only creating 4 13 definition 4 3 displaying 4 13 trace label 4 14 4 15 Extracted ion chromatogram CNL corresponds to loss of fragments 4 9 creating example 4 12 definition 4 3 4 9 deter
52. Maximum possible intensity for Mariner data is 11 000 counts spectrum that has not been accumulated or summed and for Voyager data is 66 000 counts NOTE This parameter was previously named Absolute Threshold Minimum Area Specifies the peak area below which peaks are not detected Calculated relative to peak valleys Hint Display the peak list to determine the appropriate area value to enter Noise Threshold Used to determine peak boundaries Click the field then right click drag over a segment of the trace to automatically set For more information see Process that Occurs During Peak Detection Centroiding and Integration on page 3 67 Continued 3 30 Applied Biosystems Peak Detection Table 3 4 Advanced Settings Spectrum Data Only Continued Parameter Description Filter Settings Width Number of data points used in smoothing for peak detection before integration This value is automatically calculated by the software if you select Use Resolution Dependent Settings on the Basic Settings tab For more information see Section 3 1 2 The Resolution Based Peak Detection Routine NOTE If you set Filter Width too high narrow peaks may not be detected Hint If you manually set Filter Width set to a number equal to the number of points across the peak To determine the number of points across a peak you can change the trace display from Line to Vertical Bars Each vert
53. Panel Voyager automatic calibration settings reference masses for 5 28 SET files applying 1 20 applying when opening data file 2 4 contents 1 18 creating for Mariner Sequence Control Panel 5 27 creating for Voyager Sequence Control Panel 5 28 5 29 customizing 1 19 defaults 1 19 1 23 description 1 7 extracting from DAT 1 37 modifying 1 19 restoring 1 20 saving for use with other data files 1 19 saving for Voyager Sequence Control Panel 5 33 viewing contents of 1 18 Settings see also Graphic settings see also Processing settings see also SET files automatically saved when data file closed 1 18 restoring and saving 1 20 Signal intensity summing for one mass 5 2 Signal to noise ratio algorithm 6 23 calculating 6 23 improving 5 2 7 2 7 3 Index 24 Applied Biosystems Single charge conversion description 5 59 example 5 61 multiply charged peaks seen after conversion 5 62 procedure 5 59 troubleshooting 5 62 Single point calibration see Calibrating mass SM in chromatogram header 2 30 4 19 in spectrum header 2 32 5 44 Smoothing chromatogram 4 17 setting method performed by toolbar 5 42 spectrum 5 42 spectrum default 5 43 Software starting and exiting 1 3 Sorting the peak list 3 42 SPC file converting from profile to centroid 1 33 converting to DAT 1 30 results Saving opening and deleting 2 40 Voyager not supported 1 6 Spec peak list Output window 1 15 SPEC window see Spectrum window Spectra accu
54. Prak Procemary da Settee impute baisi Setegs ET Walie ba cree Sine ts iiy Pear Figured D Erabi Gansa Big imasi g 3 Che pe Daa ferent ie DE Hg Hay bery imaorady 5 EEC Figure 3 5 Spectrum Peak Detection Setup Peak Processing Tab NOTE You can enable the BP Intensity Threshold Cursor and click drag it to adjust the Base Peak Intensity 2 Set parameters as needed then click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box For a description of the parameters see Peak Processing parameters Spectrum data only on page 3 26 3 16 Applied Biosystems Peak Detection Setting Advanced To set Advanced Settings that you can apply locally to a Settings selected detection range and that override the thresholds set only 1 Inthe Basic Settings tab select Use Advanced Settings 2 Click the Advanced Settings tab in the Spectrum Peak Detection Setup dialog box NOTE If you select Use Resolution Dependent Settings in the Basic Settings tab Basic Settings override Advanced Settings The Advanced Settings tab is accessible but all parameters are dimmed To make Advanced Settings available for editing select Use Advanced Settings on the Basic Settings tab The Advanced Settings tab is displayed Figure 3 6 Spectrum Peak Detection Setup All Traces Basic Settings Peak Processing Ad
55. Process menu select Baseline Offset The Baseline Offset dialog box Figure 5 14 is displayed Data Explorer Software User s Guide 5 45 Chapter 5 Examining Spectrum Data 5 46 Applied Biosystems Baseline Offset x Baseline Selection Left Baseline Right Baseline 2168 p 85 I Only apply from L to R Baseline midpoint OK Cancel Figure 5 14 Baseline Offset Dialog Box Right click drag the left baseline to offset The selected value is displayed in the Left Baseline field Right click drag the right baseline to offset The selected value is displayed in the Right Baseline field To limit the baseline offset to the area between the two selected points select Only Apply from L to R Baseline Midpoint To perform the baseline offset on the entire x axis deselect Only Apply from L to R Baseline Midpoint Click OK The offset baseline trace is displayed with a BO trace label To return to the original trace see Returning to the original spectrum on page 5 3 Adjusting the Baseline 5 8 2 Using Baseline Correction Description The Baseline Correction function corrects for a curved baseline including a DC offset baseline by eliminating broad artifacts from the data set When to use Baseline correct if you are analyzing data e With a baseline that is not flat and you are using the Base Peak Intensity parameter intensity based thresholding to screen out noise peaks For best results
56. RSD file to open then click OK 4 Click the Sample Info tab in the Output window to display the following information for the result file e Name of the original raw data file the result file was generated from e Processing functions that were performed and saved in the result file Data Explorer Software User s Guide 2 39 Chapter 2 Using Chromatogram and Spectrum Windows Deleting results Use Windows NT Explorer to delete RCD and RSD result for RCD and RSD files files 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only Saving results for To save results for SPC files Mariner data only SPC files 1 2 Process a data file to generate results as needed From the File menu select Result Spectrum or Result Chromatogram then select Save As Enter a file name for the result file in the File Name text box Spectrum files are automatically named with an RST extension Chromatogram files are automatically named with an RCT extension Click OK NOTE You can also save Mariner RCT and RST files from DAT format data files in the Instrument Control Panel For more information see the Mariner Workstation User s Guide Opening results To open results for SPC files 2 40 for SPC files Applied Biosystems 1 Select Open from the File menu The Select Data File to Open dialog box is displayed From the Files of Type drop down list at the bottom of
57. Spectrum The Zero Charge Spectrum Conversion dialog box Figure 5 12 on page 5 41 is displayed Specify the Input Spectrum m z range to include in the calculation by doing one of the following e Type in From and To values e Right click drag over a region of the trace that includes the m z range of interest Mass Deconvolution Mariner Data Only Type values for the following masses for the generated zero charge spectrum e Center Center mass e Half Width Mass from the Center mass to include in the spectrum e Increment Mass increment at which to perform the calculation 0 1 for resolved isotope peaks 1 0 for unresolved isotope peaks gt 1 0 for noisy trace Zemo Chepa Spectrum Coren ES irp Spadin r Parga From pare Ta pais Duigut molha bracia enga Cema iso Aa feo O Adir lon Sedertion Arkit Piara IA Figure 5 12 Zero Charge Spectrum Conversion Dialog Box Select or type the mass of the adduct ion to use in the calculation The mass of a proton H is selected by default Click OK The deconvoluted trace with a DECONV trace label replaces the original trace To return to the original trace see Returning to the original spectrum on page 5 3 Data Explorer Software User s Guide 5 41 Chapter 5 Examining Spectrum Data 5 42 Description Procedure Applied Biosystems 5 7 Noise Filtering Smoothing The Noise Filter Smooth function includes four options for
58. To calculate a signal to noise ratio signal to noise 4 Click the Spectrum window to activate it ratio 2 From the Tools menu select S N Calculator The Signal to Noise Calculator dialog box Figure 6 14 is displayed Signal to Noise Calculator xi Highlight region or enter values Method auto ha Peak fi 296 872 Cancel Figure 6 14 Signal to Noise Dialog Box Data Explorer Software User s Guide 6 23 Chapter 6 Using Tools and Applications 3 Select the method to use then right click drag on peaks in the trace to enter the associated values displayed for the method you select e Auto Right click drag across the apex of the peak for signal to noise calculation The software automatically calculates the average noise across the entire spectrum The noise calculation is not affected by the presence of peaks or a poor baseline e Manual Right click drag across the apex of the peak for signal to noise calculation Right click drag across a flat region of the baseline to use to determine the RMS noise for the calculation NOTE If you enter a baseline value instead of right click dragging the software uses an RMS value of 1 000 to perform the signal to noise calculation 4 Click OK The result is displayed in the Output window 6 24 Applied Biosystems 6 5 Using the lon Fragmentation Calculator Description Using the lon Fragmentation calculator Using the lon Fragmentation
59. Toolbox e Use labeled peaks Calculates average molecular weights using areas of peaks listed in the Spec Peak List It allows calculation of values for a distinct polymer series when two or more species are present Enter an adduct mass Enter the mass of the end group to subtract from the calculation You can leave this field blank if you do not know the end group mass CAUTION Correct labeling of the peaks is essential when using the labeled peaks option 4 Click Calculate 5 Click Copy to Output Window Result Tab to copy results to the Result tab You can then copy from the Result tab to another application such as Notepad as needed Calculations oo i TMN T Md TN oa Mo i i Poalpaispersifvlkder TON TOMM EN Nag i i i For mass range calculations For peak calculations M Peak mass M Peak mass N Peak intensity at each data paint N Peak area i Peak number i Peak number Data Explorer Software User s Guide C 17 Appendix C Data Explorer Toolbox Visual Basic Macros C 7 Using MS Fit MS Tag Toolbox Use the MS Fit MS Tag toolbox when analyzing protein digest peptide or peptide fragment spectra to perform a protein database search Preparing data Before using the MS Fit MS Tag toolbox before accessing 1 Inthe Data Explorer software smooth the data perform a baseline correction then deisotope 2 If needed use Advanced Peak Detection parameters to ad
60. a macro button by selecting the individual macro in the Macros dialog box C 4 Using the Ladder Sequencing Toolbox Use the Ladder Sequencing toolbox when you perform sequencing to label peaks with the appropriate amino acid DNA or RNA residue Labels are determined by calculating the mass differences between the peaks and comparing the values to an internal listing of mass differences and corresponding residues Running the To run the Ladder Sequencing macro macro 4 in the Toolbox Palette dialog box click Ladder Sequencing Toolbox The range displayed in the Spectrum window is reflected in the Mass Range fields NOTE If you zoom on a different region in the Spectrum window the new range is not updated in the Mass Range field but is used for the analysis when you click Label Peaks The range is updated when you click any button Data Explorer Software User s Guide C 5 Appendix C Data Explorer Toolbox Visual Basic Macros C 6 Applied Biosystems Enter the mass tolerance to apply to the analysis Click Get Spec Peak List Remove peaks you do not want included in the calculation by clicking the peak in the list then clicking Delete Selected Peaks Adducts Select the type of spectrum you are examining Peptide DNA or RNA Under Adducts to Remove select the items you do not want included in the interpretation Ladder Sequencing orbas Ea Selection Mess Aarre Dey F Papija w Daa
61. above the low mass solvent ions NOTE For more information on Constant Neutral Loss chromatograms see Section 4 2 2 Creating a Constant Neutral Loss CNL Chromatogram 4 6 Applied Biosystems Creating an Extracted lon Chromatogram Extracted lon Chromatogram x m Mass Range Difference Type Center window 5 Mass Range Difference Mass Window m z ig fo fe fo fe fo fe fo fe for m Extraction Mode Accumulative C Individual OK j Cancel j Figure 4 1 Extracted lon Chromatogram Dialog Box 5 Specify the Extraction Mode e Accumulative Creates one extracted ion chromatogram for all masses entered and sums intensities e Individual Creates one extracted ion chromatogram for each mass entered 6 Click OK The extracted ion chromatogram is displayed in the Chromatogram window Figure 4 2 on page 4 8 with an XIC trace label and the center mass and window or the mass range indicated in the trace label Data Explorer Software User s Guide 4 7 Chapter 4 Examining Chromatogram Data From the To create an extracted ion chromatogram for a mass range Spectrum window from the Spectrum window 1 Click the Chromatogram window to activate it 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 Inthe Spectrum window right click drag over the mass region of interest in the extracted ion chromatogram The width of t
62. and Elemental Composition fields then click OK NOTE You must type a minus sign preceding the charge in the Charge text box for negative charge states NOTE You can save changes to the Calibration Reference File by clicking Save For more information see Modifying a calibration reference file on page 5 20 The Automatic Calibration Settings dialog box is displayed again see Figure 5 7 on page 5 30 7 Repeat step 5 and step 6 to add all needed masses 8 To delete masses do either of the following e Select a reference then click Delete Selected Reference e Click Delete All References 9 Enter Reference Matching Criteria e Minimum Intensity Peaks must be above this intensity to be considered a match Select the unit for Minimum Intensity Relative Intensity or Absolute Counts Mass Tolerance Peaks must be within this tolerance of the theoretical m z to be considered a match Select the unit m z or ppm 5 32 Applied Biosystems Automatic Calibration 10 Select the Peak Weighting Factor If the calibration includes more than two points you can apply the following weighting factors to fit points to the curve e None All peaks are weighted equally Inverse Width Narrower peaks are weighted more than broader peaks e Height More intense peaks are weighted more than less intense peaks 11 Enter Fit Rejection parameters Minimum Peaks to Match Minimum number
63. and Spectrum Windows This chapter contains the following sections 2 1 Opening and Closing Data Files 2 2 2 2 Adjusting the Display Range ceececeeeeeeeeeeeennneeeeeeeeaee 2 11 2 3 Organizing WiNdOWS eceeeeceeee ee aeeeeeeeeeeeeeeeeeeeeeeeeeeaes 2 13 2 4 Manipulating TraCeS ovessa eiaa 2 14 2 5 Working with Multiple Data Files aeeeeeneenn 2 36 2 6 Saving Opening and Deleting DAT Results 2 38 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only 002 2 39 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only ccceeeececeeeeeeeeeeeeeeeeeeeeeeeeeeeaeaaaaaees 2 40 Data Explorer Software User s Guide 2 1 Chapter 2 Using Chromatogram and Spectrum Windows 2 1 Opening and Closing Data Files This section includes e Opening data files e Displaying Mariner UV traces e Displaying Voyager chromatograms e Viewing read only files e Moving between open files e Closing data files 2 1 1 Opening Data Files This section includes e Opening a recently opened file e Opening data files with File Open e Automatically running macros Opening a The File menu lists the last several opened files up to a recently opened maximum of nine To quickly open one of these files select it file from the list NOTE If you access Data Explorer software from the Voyager Instrument Control Panel the most recently sav
64. are saved Overlaying traces To overlay traces in a single data file in a single data file 1 Display the chromatogram or spectrum traces you want to overlay For more information see Section 2 4 4 Adding Traces from the Same Data File to a Window To use settings other than defaults set attributes for the overlay if needed See Setting overlay attributes on page 2 26 Click the trace of interest to activate it NOTE Only the active trace in an overlay is affected by processing tools However all traces are affected by zooming functions From the Display menu select Overlay Traces Hint A toolbar button is available to toggle between Overlay and Undo Overlay mode See Customizing toolbars on page 1 21 for information The s button is located in the Graph category The traces are overlaid The trace names are listed in the trace label NOTE When saving results only the results for the active trace are saved Data Explorer Software User s Guide 2 25 Chapter 2 Using Chromatogram and Spectrum Windows Changing the active trace Sequentially activating overlaid spectra 2 26 Setting overlay attributes Applied Biosystems To change the active trace in an overlay 1 From the Display menu deselect Overlay 2 Click the trace to activate 3 From the Display menu select Overlay activate each trace by clicking When you display overlaid tra
65. by the number of data points specified by the Increment parameter An Increment of 1 is used for chromatogram data e Searches for an upward to downward inflection point greater than the larger of the two specified peak detection thresholds Base Peak Intensity or Minimum Intensity A Minimum Intensity of 2 is used for chromatogram data This inflection point defines the apex region e After the apex region is identified scans to find the downward to upward inflection points These downward to upward inflection points are the valley regions of the peak Data Explorer Software User s Guide 3 67 Chapter 3 Peak Detection and Labeling e For spectral data determines the peak boundaries by one of two means e Ifthe Noise Threshold is greater than zero the software scans from the valley regions toward the apex region using the number of data points defined by the Filter Width If the difference between two consecutive filtered regions is greater than the Noise Threshold the midpoint of the filter region closest to the apex is used as the peak bound e Ifthe Noise Threshold is zero the software scans the valley regions for the minimum points to determine the peak bounds For chromatogram data determines the peak boundaries by e Determining the Noise Threshold by performing an automatic signal to noise calculation on the tallest peak in the chromatogram and using 75 percent of the noise value determined as the thresh
66. case sensitive Click the User Defined Amino Acids button to display the list of allowed residues and their associated codes 5 Select the N terminus and C terminus options for the sequence 6 Specify the cysteine modification and specify the mass type average or monoisotopic for the calculated fragment ion masses Applied Biosystems Using the lon Fragmentation Calculator Setting Options 7 Click Options The lon Fragmentation Options dialog box Figure 6 16 is displayed m lons AA composition ions gg g Ce N term sequencing ions Ma Mb Te D Cancel C term sequencing ions Me My Ty F2 Satellite sequence ions ga sg fF Neutral loss sequence ions P H20 M NH3 M H3P04 I SOCH4 Peeling sequence ions I b H20 Internal fragmentation i Multiple ae Calculate multiple charge states I 7 Set may charge E rm Peak labeling Macs tolerances fi site r nppend new labels to existing User Figure 6 16 lon Fragmentation Options Dialog Box Select the ion types to display and whether or not to generate results for internal fragments If you are analyzing Mariner data select Calculate multiple charge states if desired then specify the maximum charge state to calculate up to 12 Type the Mass Tolerance to use when labeling peaks Masses that fall outside the tolerance are not labeled To add the labels generated by this operation to the list of available user labels select Ap
67. correct the spectrum The Peak Height used by the Resolution calculator is calculated from 0 For information see Section 5 8 2 Using Baseline Correction NOTE Baselines in Mariner data are typically at 0 Baseline correction is not necessary From the Tools menu select Resolution Calculator NOTE If the Spectrum window is not active Resolution Calculator does not appear on the Tools menu In the 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 FWHM of a peak NOTE Measuring resolution at the full width half maximum is the industry standard Use 50 Peak Height for most applications If a peak is not resolved at the Peak Height you set an error message is displayed in the Result tab Using the Mass Resolution Calculator Resolution Calculator x Calculation Parameters Peak Height s4 Select Peak Mass Charge fo FWHM 50 peak height OK Cancel Figure 6 12 Mass Resolution Calculator 5 Specify the peak for which to calculate resolution by doing one of the following e Type in a Mass Charge value e Inthe Spectrum window right click drag over the peak for which you want to calculate resolution The mass is automatically entered in the Resolution Calculator dialog box NOTE If you right click drag ove
68. deisotoping function yields more accurate results than monoisotopic peak filtering See Section 3 4 Deisotoping a Spectrum Charge State Labels the peaks with the selected charge state z If you select a charge state of 0 the software displays mass labels only on peaks for which the software could not determine a charge state CAUTION A zero value in the Spec Peak list does not indicate a charge state of zero It indicates that the software could not determine the charge state Data Explorer Software User s Guide 3 43 Chapter 3 Peak Detection and Labeling Printing the peak list Deleting items from the peak list 3 44 Applied Biosystems To print the peak list 1 Display the peak list as you want it printed 2 Right click the peak list then select Print To delete an item from the peak list select the item right click the spectrum peak list then select Delete Peak The entry is removed from the list and the peak label is removed from the trace To display the deleted item in the peak list again select Peak Detection from the Peaks menu then click OK Deisotoping a Spectrum 3 4 Deisotoping a Spectrum Description During peak deisotoping This section includes e Description e During peak deisotoping e When to use e Requirements e Using the Deisotope function Troubleshooting e Example e Returning to the original spectrum The Deisotope function reduces
69. detected peaks The software iteratively decreases the Max Charge State and repeats the process for all peaks not already identified as members of an isotope cluster To accurately determine charge state Charge State parameters must be set appropriately The following sections illustrate e Max Charge State and Max Isotope set correctly e Max Charge State set too low e Max Isotope set too low Effect of Minimum Intensity Peak Detection Max Charge State When the Max Charge State and Max Isotope are set and Max Isotope correctly in the example shown in Figure 3 7 both are set set correctly to 4 the neurotensin 558 m z isotope cluster contains four peaks at charge state 3 Peaks are 0 33 m z apart Figure 3 7 Spec 1 BP 558 3 824 100 558 3 z3 824 90 en 558 6 23 gt 70 a 60 f amp 50 40 558 9 23 30 j 20 559 3 23 10 9 7 I F 0 554 556 558 560 562 564 Mass m z Figure 3 7 Max Charge State and Max Isotope Set Correctly for Neurotensin Max Charge State When the Max Charge State is set too low the charge state for set too low all peaks may not be correctly determined In this example with the Max Charge State set to 2 only the first and fourth peaks are labeled with charge states Figure 3 8 on page 3 34 This is because the software e Checks for peaks at 0 5 m z from each peak charge state 2 evaluation e Finds no peaks and checks for peaks at 1 m z
70. e Disable Use Resolution Dependent Settings e Click the Advanced tab e Select a detection region that is exhibiting the problem e Adjust the local settings to fine tune detection e Click OK 5 If peak detection is still not acceptable adjust the remaining peak detection parameters as described in Section 3 2 3 Setting Peak Detection Parameters 3 10 Applied Biosystems Peak Detection 3 2 3 Setting Peak Detection Parameters This section includes e Before you begin Setting chromatogram parameters Setting Basic Settings spectrum data Setting Peak Processing parameters spectrum data only e Setting Advanced Settings spectrum data only e Resetting Basic Settings Before you begin Before setting peak detection read e Section 3 2 1 Strategy for Mariner Peak Detection e Section 3 2 2 Strategy for Voyager Peak Detection Setting To set chromatogram parameters for data chromatogram 4 Click the Chromatogram window to activate it parameters 2 Click the trace of interest 3 Click in the toolbar or select Peak Detection from the Peaks menu The Chromatogram Peak Detection dialog box is displayed Figure 3 3 You can click drag the threshold cursor to adjust the Base Peak Intensity For a description of the parameters in the Chromatogram Peak Detection dialog box see Chromatogram settings on page 3 19 Data Explorer Software User s Guide 3 11 Chapter 3 Peak Detection
71. e Ifthe baseline is broad or gently sloping set a larger value for example 10 to 20 times the width of the actual peak width To determine the number of points across a peak change the trace display from Line to Vertical Bars select Graphic Options from the Display menu click Graph Setup and change Line Type Each vertical bar represents one data point For more information see Section 1 4 Customizing the Data Explorer Window continued Data Explorer Software User s Guide 5 51 Chapter 5 Examining Spectrum Data Parameter Description Specifies Flexibility With the Peak Width parameter determines the number of points used to estimate the baseline amplitude at regularly spaced points in the spectrum but is not directly proportional to the number of points used Valid entries are 0 to 1 0 The default value of 0 5 works best for most applications A value closer to 0 reduces flexibility and provides a smoother more generalized baseline correction see below A value closer to 1 provides more localized baseline correction by using a larger number of points to estimate the baseline see below If the baseline rises adjacent to peaks after baseline correcting decrease the Flexibility value and baseline correct again mn O Flexibility 0 Flexibility 1 5 52 Applied Biosystems continued Adjusting the Baseline Parameter
72. extracted ion chromatogram for each mass difference entered 7 Click OK The CNL is displayed in the Chromatogram window Figure 4 5 Data Explorer Software User s Guide 4 11 Chapter 4 Examining Chromatogram Data Example Figure 4 4 shows a TIC that contains three flavonoid compound peaks To determine if the diglycosyl group has fragmented from the parent ion in any of these compounds you can generate a CNL extracted chromatogram TIC Idi T11 9627 5722 90 712 4 651 80 70 e 60 28 T12 6 659 2 30 20 10 0 0 11 40 11 68 11 96 12 24 12 52 12 80 Retention Time Min Figure 4 4 TIC for Flavonoid Mixture Containing Three Peaks Figure 4 5 shows a CNL extracted chromatogram generated for mass difference of 308 146 m z which corresponds to the diglycosyl group CHL IE 45020 0500 0 Faded Z Apiai a amema 30 Pay 1 tt E a A i is 40 py i l m Io 7 L j in fi k J in i tia al Th RN ae j TEM Fiat rthees Time Miah Figure 4 5 CNL Containing Two Peaks The CNL extracted chromatogram contains only two peaks indicating that the diglycosyl group has fragmented from the parent ion in the first two compounds only 4 12 Applied Biosystems Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only 4 3 Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only This section describes how to create an Extracted Absorbance chromatogram
73. included in composite Seg 1 included in composite Seg 2 A 1 L R4 XxX Mp _ included in composite A 7 Seg 3 If first segment acquired i R2 X Mp with PSD Mirror Ratio 1 0 upper limit is Seg 4 A slightly higher than R3 x My precursor ion mass Figure 8 3 Portions of Segment Traces Included in the Composite Spectrum Data Explorer Software User s Guide 8 7 Chapter 8 Viewing Voyager PSD Data 8 2 Applying Fragment Labels Overview Use the lon Fragmentation calculator to apply fragment labels For detailed information on using the lon Fragmentation calculator see Section 6 5 Using the lon Fragmentation Calculator Applying labels To apply fragment labels to PSD spectra 1 From the Applications menu select lon Fragmentation Calculator The lon Fragmentation Calculator dialog box Figure 8 4 is displayed lon Fragmentation Calculator xj N terminus Sequence C terminus Hydrogen x AAYYAAYYAAYY Free Acid z Induce Fragmentation Cysteines are modified by unmodified x Options Calculate fragment ion masses as moncisctopic x User defined Amino Acids Peptide Mass MH average lisbel Peake Peptide Mass MH monoisotopic Print lons table lons charge Internal fragments table Internal sequence Clear Table Info Figure 8 4 lon Fragmentation Calculator Dialog Box 8 8 Applied Biosystems Applying Fragment Labels In the Sequence tex
74. is displayed with the added and subtracted spectrum numbers in the trace label Also if the retention time labels are displayed the retention time range is included in the trace label Displaying MS Method Data Mariner Data Only 4 6 Displaying MS Method Data Mariner Data Only Overview In this section Example applications Displaying acquisition conditions and event tags Setting Trace Replace mode If you acquired a data file using an MS Method and assigned event tags you can display chromatogram traces in Data Explorer filtered by event tag This section includes e Example applications e Displaying acquisition conditions and event tags Setting Trace Replace mode e Filtering event tags e Evaluating filtered traces e Displaying additional filtered traces Assume that you acquired data using two events one specifying a low Nozzle Potential and one specifying a higher Nozzle Potential to induce in source fragmentation You also assigned a Tag 1 event tag to the first event and the In Source CID event tag to the second event You can filter the TIC or any chromatogram data to display separate traces for the different Nozzle Potentials To display instrument settings MS Method segment and event numbers and event tags for a spectrum 1 Display the spectrum of interest 2 Click the Instrument Setting tab in the Output window Before selecting the event tag to display set the Trace Replace mode
75. is used because it is less than the smallest mass difference related to a DNA or RNA base e Labels mass differences plus or minus the specified Tolerance that correspond to DNA ACGT or RNA ACGU bases e Labels the reference peak from which the mass difference was derived with an asterisk e Applies the additional labels you selected under Annotate Spectrum C Displaying the To display the original labels select Peak Label from the original labels Peaks menu in the Data Explorer software then deselect User Labels box C 8 Applied Biosystems Using the Peptide Fragmentation Toolbox C 5 Using the Peptide Fragmentation Toolbox Use the Peptide Fragmentation toolbox when examining Voyager composite PSD spectra or Mariner in source CID spectra to label immonium ions identify fragment ion pairs view sequences based on different reference peaks and determine if a selected peak is of a specific fragment ion category Setup To enter Setup parameters for the Peptide Fragmentation Toolbox macro 1 Inthe Toolbox Palette dialog box click Peptide Fragmentation Toolbox Peptides Fragnentateen Toolbar x SpecPeaklet Setup Pairs Sequence Correlation Meas Mass Tolerance jos Get Spec Peak Figure 3 10 Peptide Fragmentation Setup 2 Inthe Setup tab click Get Spec Peak List Data Explorer Software User s Guide C 9 Appendix C Data Explorer Toolbox Visual Basic Macros 3 Add peaks
76. name and destination for the file to be exported By default the software assigns a TXT extension to the file 4 Click OK Exporting a trace You can export a selected trace to ASCII format for display in to ASCII format Data Explorer software or for use in another application To export trace data to ASCII format 1 With a data file open select the trace window 2 From the File menu select Export then select Chromatogram or Spectrum the menu item is context sensitive A Save As dialog box is displayed 3 Specify the name and destination of the file to be exported By default the software assigns a TXT extension to the file 4 Click OK 1 34 Applied Biosystems 1 6 4 Importing a Trace in ASCII Format Importing a Trace in ASCII Format Managing Files You can import trace data in ASCII format If the file you are importing was originally exported using the Data Explorer software you can import a spectrum trace only into the Spectrum window and a chromatogram trace only into the Chromatogram window However if the data is from another source the software does not know the data type and you must make sure to import the data into the correct window type To import an ASCII trace 1 Open a data file and activate a Chromatogram or Spectrum window NOTE You must open a data file before you can import ASCII data even if the data file is unrelated to the imported data 2 Select Add Remove Trace
77. new traces Setting the To set the Replace mode Replace mode 1 From the Display menu select Add Remove Traces The Display Trace dialog box is displayed Figure 2 7 Display Trace x r Select Traces to Displa I Not Used F Not Used F Not Used I Not Used I Not Used I NotUsed I Not Used Replace Mode C Replace the Active Trace Adda New Trace Corcel Figure 2 7 Display Trace Dialog Box Data Explorer Software User s Guide 2 17 Chapter 2 Using Chromatogram and Spectrum Windows 2 18 2 4 Select the Replace Mode Replace the Active Trace default Replaces the active trace with the newly created trace Add a New Trace Adds the newly created trace to the window The original trace remains displayed and accessible allowing visual comparison of the traces NOTE If Replace Mode is set to Add a New Trace and eight traces are present when you perform a function the active trace is replaced when a new trace is generated Hint A toolbar button is available for switching between Replace and Add mode See Customizing toolbars on page 1 21 for information The button is located in the Graph category If desired you can also add traces by selecting the trace to add from the Select Traces to Display section Click OK Adding a trace To add anew trace to a window Applied Biosystems 1 2 Click the Chromatogram or Spectrum win
78. ranges e PSD peak detection for Voyager data Type of data The resolution based peak detection routine applies to affected spectral data only You can enable or disable the resolution based peak detection routine as described on page 3 14 Process that When enabled the resolution based peak detection routine occurs automatically e Divides a trace into detection ranges based on the expected number of data points across a typical mass spectral peak e Applies a Filter Width that is equal to the number of data points per peak in each detection range Uses a Filter Increment of 1 Data Explorer Software User s Guide 3 3 Chapter 3 Peak Detection and Labeling Detection ranges The software uses the following formula to calculate the expected number of data points in a peak Flight time Expected to which the number of data point corresponds Gale pons 2 x mass resolution x Bin size Where Mass resolution is a user defined value Data type dependent defaults are provided but can be overwritten Bin size is an instrumental constant For Mariner data Bin size 1 ns For Voyager data Bin size is variable based on the digitizer used and the mass range acquired Figure 3 1 is an example of the resolution based detection ranges automatically calculated by the software Intensity Spec 1 BP 148450 6 46504 148449 3 4 7E 4 99033 135842 1 160779 9 198374 52230
79. reducing noise in a spectrum trace Default smoothing e Noise filtering e Smoothing by the Gaussian method e Noise removal To noise filter or smooth the display 1 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 From the Process menu select Noise Filter Smooth The Noise Filter Smooth dialog box Figure 5 13 is displayed Noise Filter Smooth x Methods Default X Mass Peak Resolution 500 Cancel Figure 5 13 Noise Filter Smooth Dialog Box NOTE The smoothing filtering method selected in this dialog box is the method performed when you click in the toolbar Noise Filtering Smoothing 3 Select the method to use based on the type of data you are examining then enter the associated value displayed for the method you select Suggested nara Type of Data Method Description Noisy low resolution Default No associated value is displayed Default data smoothing smoothing is a self adjusting Gaussian filter RSM that uses the Peak Resolution specified in the Peak Detection Setup dialog box to calculate May affect the optimum number of smoothing points to peak apply at every mass point See Figure 3 3 on resolution P 9 eae NOTE Default smoothing is not available for PSD data High resolution data Noise Specify a Correlation Factor of 0 to 1 0 but the Noise Filter NF Settings from 0 5 to 0 7 yield accept
80. s Guide if you did not acquire all segments or did not acquire segments in order of decreasing Mirror Ratio The number above reflects the order of acquisition For example if you listed Segments 1 through 5 for acquisition but acquired Segments 1 3 and 5 you would see three entries above that correspond to the three acquired segments 3 Inthe Spectrum window click the first Not Used trace 4 Inthe PSD Processing dialog box double click the Entry number of the segment to add 5 Repeat step 3 and step 4 to display additional segments 6 To advance through traces select any trace then click j and E Redisplaying the To redisplay the composite spectrum click Generate composite Composite in the PSD Processing dialog box Figure 8 2 spectrum Data Explorer Software User s Guide 8 5 Chapter 8 Viewing Voyager PSD Data How the The software does the following to generate a composite composite spectrum spectrum is e Evaluates all segments in the DAT file to determine if generated there are multiple segments acquired using the same PSD Mirror Ratio e If there are duplicates selects the most recently acquired segment to include in the composite spectrum Using the precursor ion mass and calibration constants from the PSD calibration in the DAT file see PSD calibration equation below determines the region of each segment to include in the composite spectrum as illustrated in Figure 8 3 on
81. select Delete Peak If a label is not displayed or is displayed incorrectly possible causes are The label is too long peaks are too close together or a peak is very close to the right axis If there is not enough room for the label to be displayed the label is suppressed Zooming the region of interest expands the trace and may allow the labels to display To display labels when peaks are close together select Allow overlapping peak labels in the Peak Label dialog box Peak filtering is enabled labeling only peaks that meet the peak list filtering criteria Filter Width is set too high to detect the peak Data Explorer Software User s Guide 3 59 Chapter 3 Peak Detection and Labeling Charge state f no charge is displayed there are a few possible causes not displayed peaks are more than 1 Da apart e Filter width is set too high to detect other isotope peaks e The maximum charge state for charge state determination is set lower than the charge state of the peak e Charge state determination parameters are set such that peaks are determined to have no charge See Section 3 2 5 Charge State Determination and Examples e Peaks are not from the same isotope species NOTE If you set Base Peak Intensity or Max Peak Area too low described on page 3 20 noise peaks can be detected and charge states can be incorrectly labeled 3 60 Applied Biosystems Peak Labeling 3 5 3 Setting Custom
82. software multiplies each mass by 3 and removes the mass of two extra protons The resulting spectrum contains theoretical 1 species If more than one charge state is present each charge state is converted appropriately and the total range of the converted spectrum is approximately 1 2 times the m z of the highest m z present Use this function only on isotopically resolved data that is labeled with the correct charge state Peaks with low signal to noise ratios may be labeled with incorrect charge states Set peak detection thresholds to disregard these peaks before converting the spectrum If you use this function on a spectrum that is not correctly labeled the resulting spectrum may contain charge states other than 1 To convert a multiply charged spectrum to a singly charged spectrum 1 Display the spectrum trace of interest 2 Examine the spectrum Make sure peaks are labeled with the correct charge state and that no noise peaks are labeled with a charge state Adjust peak detection parameters as needed Data Explorer Software User s Guide 5 59 Chapter 5 Examining Spectrum Data 5 60 Applied Biosystems Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing From the Process menu select Single Charge Conversion The Single Charge Spectrum Conversion dialog box Figure 5 20 is displayed Single charge Spectrum Conversion x Adduct Selection Add
83. spacing and charge state 3 53 theoretical generating with Elemental Composition Calculator 6 6 theoretical generating with Elemental Targeting 6 33 theoretical generating with Isotope Calculator 6 18 troubleshooting 9 17 Isotope calculator amino acid codes 6 14 charge state to calculate 6 15 element codes 6 14 evaluating traces 6 18 examples 6 16 result failed to calculate 9 6 results 6 19 using 6 13 Data Explorer Software User s Guide Index 13 Isotope Match Intensity elemental targeting 6 33 Isotope Match Score elemental composition 6 6 elemental composition not reported for fragment ion calculations 6 6 elemental targeting 6 33 K Keywords Windows NT entering 1 31 searching 1 32 viewing 1 32 L Labeling peaks see also Peak labels chromatogram 3 54 customizing label appearance 1 25 factors affecting 3 52 manually 3 39 monoisotopic 3 43 partially resolved 7 11 spectrum 3 56 with amino acid DNA or RNA labels C 5 with area 3 58 with charge state 3 43 3 53 3 58 with time 3 55 with vial number 3 55 Labels see also Data cursor see also Peak labels see also Trace labels AC 5 34 added and subtracted spectra 4 22 AdvBC 5 53 BC 4 29 5 47 BO 4 28 5 46 Index 14 Applied Biosystems Labels continued CNL 4 9 CT 5 36 DECONV 5 40 5 41 filtered trace 4 25 ISO 6 6 6 16 6 33 MC 5 13 NF 4 19 5 44 NR 4 19 5 44 retention time displayed 4 22 RSM 5 44 SC 5 60 SM 4 19 5 44 TR 5
84. the area of all peaks in the isotope cluster Spec 1 gt DI BP 1672 9 260759 100 1190 4 2 4E 4 80 F 70 2 60 E Z 30 1189 4 20 0 0 1184 0 1187 2 1190 4 1193 6 1196 8 1200 0 Mace fm 7 Figure 3 20 Spectrum After Deisotoping 3 50 Applied Biosystems Deisotoping a Spectrum Returning to the To return to the original spectrum original spectrum ifthe original spectrum was an unprocessed spectrum select Spectrum Number from the Display menu The number of the original spectrum is displayed in the Select Spectrum dialog box Click OK e Ifthe original trace was a processed spectrum select Processing History from the Display menu then select the original trace Data Explorer Software User s Guide 3 51 Chapter 3 Peak Detection and Labeling 3 5 Peak Labeling This section includes e Charge state labels e Setting chromatogram and spectrum peak labels e Setting custom peak labels Factors affecting Peak labels are displayed only for detected peaks in the peak peak labeling list displayed in the Output window Thresholds for peak labeling are set independent of peak detection Peaks listed in the peak list are determined by e Peak detection parameters described in Section 3 6 Process that Occurs During Peak Detection Centroiding and Integration e Charge state determination described in Section 3 2 5 Charge State Determination and Examples e Peak list filtering described in Fil
85. then select Delete All Text NOTE Text annotations are included when you print the spectrum They are not saved with the spectrum when you close the data file Data Explorer Software User s Guide 2 29 2 Chapter 2 Using Chromatogram and Spectrum Windows 2 4 10 Viewing Trace Labels Chromatogram trace labels The Data Explorer software includes a label in the trace header to identify the type of data displayed NOTE Trace labels are applied by the software and cannot be removed Labels in the chromatogram title identify the following types of displayed data Chromatogram Trace Label Description BC Baseline corrected BO Baseline offset BP Base Peak mass and intensity CNL Constant neutral loss chromatogram EF Event filtered chromatogram MS Method data only Mass xxx yyy Extracted ion chromatogram for a mass range where xxx is the starting mass and yyy is the ending mass NFX Noise filtered trace where X is the applied Correlation Factor NRX Noise removed trace where X is the number of standard deviations of noise removed SMX Smoothed trace where X is the number of smoothing points applied TAC Total absorbance chromatogram TAC Realigned total absorbance chromatogram XAC Extracted absorbance chromatogram 2 30 Applied Biosystems Manipulating Traces Chromatogram Trace Label Description XAC Realigned extracted absorb
86. to Valley Forces a baseline through all valley points See Figure 3 25 on page 3 70 Trace Settings Use same settings Applies settings to all traces in the active window for all traces in view NOTE In previous versions of Data Explorer software peak detection allowed you to specify Peak Width and Noise Threshold for chromatogram data The software now automatically Uses a minimum peak width that is equal to the Filter Width and a maximum peak width of 10 000 data points Calculates the Noise Threshold by performing an automatic signal to noise calculation on a chromatogram and by using 75 percent of the noise value determined as the threshold Data Explorer Software User s Guide 3 21 Chapter 3 Peak Detection and Labeling Basic Settings spectrum data only Table 3 2 describes the parameters in the Basic Settings tab of the Spectrum Peak Detection Setup dialog box see Figure 3 4 on page 3 14 Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Table 3 2 Basic Settings Tab Parameters Spectrum Data Only Parameter Description Global Thresholds Base Peak Intensity NOTE This parameter was previously named Peak Threshold and Base Peak Relative Specifies a percentage of the base peak intensity as the threshold value To be detected peaks must be above this threshold and above the Max Peak Area value If
87. to label peaks in m z format 4 Set the number of decimal points to be displayed 3 56 Applied Biosystems Peak Labeling 5 Select the Mass Type peak apex or peak centroid 6 Select the Peak Mass Label Type Label Description Mass Labels with Apex or Centroid mass NOTE If you create a custom user label for a mass the user label is displayed instead of the mass For more information see Section 3 5 3 Setting Custom Peak Labels Mass Shifts all peak labels by the value entered either positive or difference negative and can be used to calculate mass differences from a from the reference peak selected Right click drag across the reference peak to enter the negative of peak the value of the peak in the Mass difference text box then click OK NOTE This The reference peak is labeled with zero and all the other labels are label was plus or minus their mass difference from the reference peak previously This label is useful to display neutral losses and adducts on a given called Mass peak Offset Mass Labels peaks with a mass relative to the adjacent labeled peak of difference lower m z between adjacent peaks Data Explorer Software User s Guide 3 57 Chapter 3 Peak Detection and Labeling 7 Select label attributes e Overlapping Allows labels to be displayed when peaks are close together e Peak bounds Displays peak start peak end and baseline e Orientation Specifie
88. to the 8 18 data file Applied Biosystems After matching peaks click Solve and Plot The calibration statistics are displayed in the Result tab of the Output window and the calibration constants are applied to the spectrum displayed If you calibrate more than one time subsequent calibration statistics are added to the end of the list in the Output window Older calibration statistics are listed at the top of the list Use the scroll bar to view newer statistics at the bottom of the list The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Output window To apply the calibration constants to each spectrum all segments in the data file click Apply Calibration All spectra in the data file are calibrated and displayed with an MC trace label The calibration constants are saved with the data file Each spectrum in the data file is calibrated when displayed The software uses different values in the calibration equation calculation based on the type of calibration performed Selecting calibration peaks for optimum mass accuracy Calibrating a PSD Spectrum To improve calibration statistics you can select the same fragment ion from more than one segment Monoamino acid fragments immonium ions below 150 Da are useful for this purpose Because the segments have been collected with different PSD Mirror Ratios the software allows you to add the same mass to th
89. under Remove Mass 5 Click Copy to Output Window Result Tab to copy results to the Result tab You can then copy from the Result tab to another application such as Notepad or print as needed Sequence To list ion sequences 1 Click the Sequence tab Peptede Fragnentaten Toolbor Spec Peak let Seng Pairs Sequence Coredation Clear Qubput Window paatoa noon in Renk Tab Close Figure 3 12 Peptide Fragmentation Sequence Data Explorer Software User s Guide C 11 Appendix C Data Explorer Toolbox Visual Basic Macros C 12 Applied Biosystems If desired click Label Immonium Ions NOTE Label immonium ions before selecting a reference peak and starting the search If you click Label Immonium lons after selecting a reference peak the amino acid labels applied to the spectrum are erased and mass labels are reapplied Select a peak in the Spec Peak List then click Set Selected Peak as Reference The software evaluates mass differences between the reference peak labeled with and all other peaks and labels any differences that correspond to amino acid residues Select a result peak from the results list if desired then continue the sequence search by clicking the Search Down or Up buttons The newly selected peak is now labeled as the reference peak and mass differences minus if you click Down or plus if you click Up are labeled if they correspond to an amino acid residue You ca
90. window right click drag across the peak from which you are measuring the difference This enters a negative value that corresponds to the reference peak in the text box Click OK The reference peak is labeled with zero and all the other peak labels are plus or minus their mass difference from the reference peak For more information on peak labels see Section 3 5 Peak Labeling To create a CNL extracted chromatogram 1 2 Click the Chromatogram window to activate it Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing From the Process menu select Extracted lon or click in the toolbar The Extracted lon Chromatogram dialog box appears Figure 4 3 In the Mass Range Difference Type field select Neutral Loss Creating an Extracted lon Chromatogram 5 Type inthe Mass Difference and Tolerance NOTE Do not right click drag across a peak in the Spectrum window to select the mass difference Extracted lon Chromatogram HE m Mass Range Difference Type Neutral Loss hd m Mass Range Difference Tolerance m z r Extraction Mode Accumulative C Individual Cancel Figure 4 3 Extracted lon Chromatogram Dialog Box with Neutral Loss Selected 6 Specify the Extraction Mode e Accumulative Creates one extracted ion chromatogram for all mass differences entered and sums intensities e Individual Creates one
91. 1 Chapter 3 Peak Detection and Labeling 3 1 Overview This section includes Default peak detection The resolution based peak detection routine 3 1 1 Default Peak Detection 3 2 Overview Applied Biosystems When you open a data file it is automatically peak detected For e Chromatographic data The software uses default settings that have been optimized to yield acceptable peak detection for many sample types For more information see Section 3 7 Default Peak Detection Settings Spectral data The software uses a resolution based peak detection routine to calculate peak detection values that provide optimum peak detection for most sample types For more information see Section 3 1 2 The Resolution Based Peak Detection Routine For many applications the default peak detection settings and settings calculated by the resolution based peak detection routine provide acceptable peak detection If default settings do not provide acceptable peak detection you can adjust the settings as described in e Section 3 2 1 Strategy for Mariner Peak Detection e Section 3 2 2 Strategy for Voyager Peak Detection NOTE If peak detection settings do not detect desired peaks you can manually insert peaks See Section 3 3 2 Inserting Peaks in the Peak List Overview 3 1 2 The Resolution Based Peak Detection Routine This section describes e Type of data affected e Process that occurs e Detection
92. 1 To 100 Figure 2 6 Y Axis Setup Dialog Box Data Explorer Software User s Guide 2 11 Chapter 2 Using Chromatogram and Spectrum Windows 5 Set the parameters described below as needed Parameter Description Scaling Mode Display Relative Autoscales the trace to the largest peak in the selected range Base Peak Autoscales the trace to the base peak in the entire range not the Relative 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 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 trace display to the minimum and maximum Y values Minimum Absolute Max Y Use Limit Sets the minimum value for Y axis scaling 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 6 Click OK 2 12 Applied Biosystems Organizing Windows 2 3 Organizing Windows Linking views Organizing windows Linking Chromatogram or Spectrum windows in different data files allows you to zoom on multiple d
93. 17 Default peak detection values are listed in Section 3 7 Default Peak Detection Settings NOTE If you select Use Resolution Dependent Settings in the Basic Settings tab Basic Settings override Advanced Settings The Advanced Settings tab is accessible but all parameters on the tab are dimmed To make Advanced Settings available for editing select Use Advanced Settings on the Basic Settings tab Table 3 4 Advanced Settings Spectrum Data Only Parameter Description Peak Detection Settings Detection Ranges Specifies the region of the trace defined by x axis lower and upper boundaries to which the settings apply If you select Use Resolution Dependent Settings in the Basic Settings tab described on page 3 23 the software automatically divides the trace into detection ranges If you select Use Advanced Settings the detection ranges calculated by the software are maintained and you can modify the ranges and settings or combine all ranges in the list into one range as described below 3 28 Continued Applied Biosystems Peak Detection Table 3 4 Advanced Settings Spectrum Data Only Continued Parameter Description Detection Ranges continued You can set multiple non contiguous ranges and define parameters for each range independently You select a range in the Detection Ranges list box by single clicking the range number To add a detection range d
94. 18 Replace mode setting 2 18 Advanced baseline correction 5 48 Advanced peak detection parameters description spectrum 3 28 setting spectrum 3 17 AdvBC in spectrum header 2 31 5 53 Air Temperature displaying trace 4 2 Data Explorer Software User s Guide Index 1 Amino acids labeling C 5 Analog signal displaying 4 2 Analyzer Temperature displaying trace 4 2 ANGIOTENSIN_FRAGMENTS REF 5 18 Annotating traces adding text 2 29 deleting text 2 29 text from previous trace displayed 2 29 with ASCII text 2 28 with results 2 28 Apex mass copying from peak list 1 41 labeling 3 57 Applied Biosystems Technical Support 9 2 world wide web address xiv Area peak detection threshold chromatogram 3 20 detection threshold global spectrum 3 23 detection threshold local spectrum 3 30 displaying 3 38 in peak detection algorithm 3 68 labeling chromatogram 3 55 labeling spectrum 3 58 ASC 5 63 ASC in spectrum header 2 6 2 31 5 63 ASCII text annotating traces with 2 28 exporting traces to 1 34 importing traces from 1 35 Assign Macro 6 38 Auto Cal see Calibrating mass automatic Mariner data only Autocolor description 2 27 setting 1 26 Index 2 Applied Biosystems Automatic Calibration On command dimmed 5 34 see also Calibrating mass automatic Mariner data only settings reference masses for Mariner Sequence Control Panel 5 27 settings reference masses for Voyager Sequence Control Panel 5 28 settings
95. 2 16 copying traces from different data files 2 37 customizing 1 17 displayed 1 12 organizing 2 13 2 36 Windows Metafile format copying to 1 39 Windows NT keywords entering 1 31 keywords searching 1 32 keywords viewing 1 32 Print setup 2 36 WMF copying to 1 39 World wide web address Applied Biosystems xiv X X cursors setting 1 27 x y data pairs copying 1 39 XAC in chromatogram header 2 30 see also Extracted absorbance chromatogram XAC X axis chromatogram 2 11 setting range 2 11 spectrum 2 11 XIC see also Extracted ion chromatogram XIC in chromatogram header 2 31 Y y and b ion pairs labeling C 11 Y cursor setting 1 27 Y axis offsetting chromatogram 4 27 offsetting spectrum 5 45 scaling 2 12 spectrum 2 11 Z z labels 3 58 z average molecular weight determining C 15 Zero charge state displayed in peak list 3 40 3 43 Zero charge spectrum for overlapping peaks 5 40 for resolved peaks 5 37 Zooming multiple data files 2 13 2 36 Data Explorer Software User s Guide Index 29 Index 30 Applied Biosystems Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For sales office loca
96. 2 38 saving results 2 38 title when saving 2 38 Results SPC AutoSaturation Correction effect on Mariner only 5 62 copying 2 28 name of raw data file result is derived from 2 38 2 39 2 41 opening RST and RCT files 2 39 2 40 saving RST and RCT files 2 40 Retention time displaying for chromatogram 1 12 on data cursor 1 27 Reverting to instrument calibration 5 22 Right axis turning off 2 12 RNA residues labeling C 5 RSD file deleting 2 39 description 1 10 exporting 2 39 extracting information from 1 36 opening 2 39 RSM in spectrum header 2 32 5 44 RST files see also Results name of raw data file result is derived from 2 38 2 39 2 41 not affected by AutoSaturation Correction 5 62 opening 2 39 2 40 saving 2 40 Running a macro automatically 6 45 manually 6 39 S Sample info tab Output window 1 15 Saturated spectra correcting Mariner data only 5 62 SC in spectrum header 2 32 5 60 Scaling see also Display Range overlaid traces 2 27 to Absolute Value 2 12 to Base Peak 2 12 to min max Y 2 12 Searching for keywords 1 32 Segment spectra PSD calibrating 8 12 creating custom peak labels 8 9 labeling 8 8 lower masses require higher Filter Width setting 8 11 optimum resolution observed near Max Stitch Mass 8 4 resolution trend within 8 11 Data Explorer Software User s Guide Index 23 Sequence Control Panel Mariner automatic calibration settings reference masses for 5 27 Sequence Control
97. 297 655 2094 607 iziii 466 052 3659 309 1707 2 2447 4 W 3927 8 4668 0 7 14 Applied Biosystems Figure 7 13 Calibration Without Baseline Correction or Deisotoping Voyager Data Examples Before calibrating To optimize mass accuracy do the following before calibrating Baseline correcting Deisotoping Ae 2 Display the spectrum of interest From the Process menu select Baseline Correction The spectrum is baseline corrected For more information see Section 5 8 2 Using Baseline Correction From the Peaks menu select Peak Deisotoping The Deisotoping dialog box Figure 7 14 is displayed Repeat Formula Adut Ho Formula CeHSNO OOO OK Cancel Figure 7 14 Deisotoping Dialog Box For this example spectrum specify H for Adduct and C6HS5NO for Generic Formula Click OK Figure 7 15 shows the baseline corrected deisotoped spectrum before calibration For more information on deisotoping see Section 3 4 Deisotoping a Spectrum Intensity Spec 1 gt BC gt DI BP 754 4 981363 5 4E 5 3659 433 1297 722 2094 696 2466 150 642 361 1705 6 2446 2 3186 8 3927 4 hacc imiz 4668 0 Figure 7 15 Baseline Corrected Deisotoped Spectrum Before Calibration Data Explorer Software User s Guide 7 15 Chapter 7 Data Explorer Examples Calibrating To calibrate the deisotoped spectrum 1 From the Peaks menu select Peak Label and select the Mass Labe
98. 3 7 strategy for 3 6 troubleshooting 3 6 Peak detection resolution based see also Peak detection default resolution 3 6 3 24 description 3 3 enabling 3 23 Filter Width value used 3 3 formula used to calculate number of data points 3 4 overriding 3 10 3 14 Peak detection Voyager data see also Peak detection complex digests 7 18 default settings 3 71 deisotoping to aid in peak detection 3 9 high mass peaks not detected 3 9 improving by baseline correcting noise filtering or smoothing 3 8 noise detected as peaks 3 9 partially resolved peaks not detected 3 10 7 11 peaks of interest not detected 3 9 PSD 8 11 strategy for 3 8 troubleshooting 3 9 Peak integration see Integration Peak labels see also Peak labels charge state see also Peak labels filtering see also Peak labels Mass Difference 45 degree angle 3 55 3 58 amino acid C 5 apex 3 56 applying user labels from LBC and LBS files 3 65 area chromatogram 3 55 area spectrum 3 56 3 58 base peak mass 3 55 3 56 baseline changing line width 1 26 baseline displaying 3 55 3 58 Data Explorer Software User s Guide Index 19 Peak labels continued centroid 3 56 chromatogram setting 3 54 custom 3 61 custom creating for fragment spectra 8 9 customizing 1 25 decimal places displayed 3 55 3 56 deleting from trace 3 44 3 59 displaying 3 65 DNA C 5 extracting from DAT file 3 64 factors affecting 3 52 h
99. 35 Data file names do not print for multiple data files Some printers may not print the data file name if you select Print All Views from the File menu with more than two data files open We have observed this on HP LaserJet4 HP LaserJet5 and HP LaserJet6 printers Print views individually or open only two data files before you select Print All Views For more information see Section 2 4 11 Printing Traces 9 14 Applied Biosystems 9 6 Peak Detection and Peak Detection and Labeling Troubleshooting Labeling Troubleshooting This section includes e Peak detection and labeling troubleshooting e Charge state and isotope determination troubleshooting Table 9 10 Peak Detection and Labeling Troubleshooting Mariner and Voyager Symptom Possible Cause Action Peaks are not detected or labeled Peaks are very close together or label is too long e Zoom in on region of interest e Select Allow overlapping peak labels in the Peak Label dialog box See Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels Peak detection parameters in particular Filter Width not set to detect peaks Adjust parameters See Section 3 2 Peak Detection Peak filtering is enabled Only peaks that meet the peak list filtering criteria are labeled Disable peak list filtering See Filtering the spectrum peak list on page 3 42 Data Explorer
100. 4 17 peak list 3 42 Fit Error calibration 5 12 Formulas determining if present in observed spectrum 6 31 determining possible for a mass 6 2 Fragment ions correspond to loss of 4 9 determining if mass difference 4 9 electron state for accurate results 6 5 generating list of masses with lon Fragmentation calculator 6 25 identifying with Elemental Composition calculator 6 2 labeling 6 25 C 9 Peptide fragmentation macro C 9 Fragment spectrum PSD see Segment spectrum PSD FRM files for macros 6 43 FWHM 6 20 Index 12 Applied Biosystems G Gaussian Fitting peak detection 3 26 Gaussian smoothing see Smoothing Global peak detection parameters description spectrum 3 22 overriding for individual detection ranges spectrum 3 30 setting spectrum 3 13 Graphic options accessing 1 24 default settings for white or dark background 1 19 1 23 extracting from DAT file 1 36 graphic compression 1 28 peak labels customizing 1 25 saving and restoring 1 20 saving to a SET file 1 37 setting graph and plot colors with 1 25 turning off right axis 2 12 Graphic settings applying 2 4 automatically saved when data file closed 1 18 description 1 18 extracting from DAT file 1 37 modifying 1 19 saving and restoring 1 20 saving for use with other data files 1 19 H Help see PerSeptive Biosystems Technical Support Histogram centroid 5 36 Horizontal cursor 1 27 Horizontal peak labels 3 55 3 58 How to use this guide xi l Immo
101. 5 moving 1 22 Smoothing button setting method performed 5 42 Tools commands not displayed on menu 9 6 ToolTips 1 12 TR in spectrum header 2 32 5 57 Trace browser 2 9 Index 26 Applied Biosystems Trace labels cannot be removed 2 30 chromatogram 2 30 spectrum 2 31 Trace windows maximizing 2 9 Traces see also Trace labels see also Traces copying see also Traces removing adding 2 16 2 18 annotating 2 28 axes setting 2 11 bar mode 1 28 centering a peak 2 15 centroid creating 5 36 changing colors to black before printing 2 33 colors setting 1 25 comparing 2 38 display range setting 2 11 displaying as vertical bars 5 36 distorted when you copy to another application 1 39 dividing 2 15 do not print 2 34 duplicating 2 15 expanding 2 21 exporting to ASCII format 1 34 filtering see Event tag filtering Mariner data only graphic compression 1 28 importing from ASCII format 1 35 line mode 1 28 line width 1 26 linking 2 21 overlaid troubleshooting 9 6 overlay scaling 2 27 overlaying 2 25 previewing 2 33 printing 2 33 rearranging order 2 22 recalling previously processed 2 22 Traces continued Replace mode setting 2 17 scaling mode setting 2 12 splitting 2 15 switching between 2 8 text customizing 1 25 traces do not print 1 26 2 33 type selecting in Chromatogram window 4 2 use same graphic options settings for all 1 24 UV offset 4 30 vertical bar mode 1 28
102. 57 from adjacent peak user labels 3 62 from selected peak 3 57 Mass list copying to Windows clipboard 1 41 Mass Offset replaced by Mass Difference from Selected Peak 3 57 Mass Resolution Calculator see Resolution mass Mass resolution defaults used in peak detection 3 24 Match Charge State 3 62 Max Stitch Mass definition 8 4 displaying 8 4 optimum focus and resolution observed near this mass 8 4 updated when Precursor mass changed 8 24 Index 16 Applied Biosystems Maximize trace window 2 9 Maximum Charge State description 3 27 set too low 3 33 Maximum Isotopes setting 3 27 setting too low 3 35 MC in spectrum header 2 6 2 32 5 13 5 15 5 17 5 22 5 23 7 17 8 18 Metafile copying to 1 39 Microsoft applications Excel 1 5 Microsoft Office 1 5 Visual Basic Editor 6 42 Word 1 5 Microsoft Excel importing PKT files to 3 41 Minimum Area setting spectrum 3 30 Minimum Intensity in peak detection algorithm 3 67 setting spectrum 3 30 Mn Mw Mz Mw Mn determining C 15 Monoisotopic peak deisotoping a spectrum 3 9 3 45 displaying in peak list 3 43 filtering 3 43 labeling 3 43 mass definition B 6 reducing trace to 3 9 3 45 Moving between open files 1 12 MS Fit MS Tag macro C 2 MS Method Mariner data only applications 4 23 conditions and tags displaying 4 23 Event Tag Filtering command dimmed 4 24 event tag filtering 4 23 instrument settings extracting from DAT file 1 36 MS Method Mariner data only c
103. 6 Applying new You can apply calibration constants from a CAL file to any constants to datafile To apply the new constants from a mass calibration additional files f e to a different file 1 Display the spectrum to calibrate 2 From the Process menu select Mass Calibration 3 Select Import Calibration NOTE If you are importing a calibration into a Voyager data file see Importing a calibration on page 5 25 4 Select the CAL file to use then click Open The software displays the calibrated spectrum with an MC trace label 5 16 Applied Biosystems Manual Calibration 5 To save the calibration to the data file select Mass Calibration from the Process menu then aerate Select The following occurs calibrating Mariner Apply Calibration All spectra in the data file are calibrated and data displayed with an MC trace label The including calibration constants are saved with the data MS Method file Each spectrum in the data file is data calibrated when displayed Voyager Apply Calibration The current spectrum is calibrated and data displayed with an MC trace label The calibration constants are saved with the spectrum Apply to All All spectra in the data file are calibrated NOTE This button is Using the currently displayed calibration and displayed only if you are displayed with an MC trace label The are calibrating a calibration constants are saved with the data Voyager file Ea
104. 8 baseline changing line width 1 26 baseline displaying 3 55 3 58 Basic Settings spectrum 3 13 3 22 charge state determination 3 27 chromatogram 3 68 chromatogram Noise Threshold calculated automatically 3 21 3 68 data cursors turning on and off 3 23 data points across a peak determining number 3 21 3 31 5 51 default 3 2 3 71 Filter Width and Increment used 3 3 Gaussian Fitting 3 26 isotope determination 3 27 isotope partially resolved 7 11 manually inserting peaks in peak list 3 39 Peak detection continued Noise Threshold calculated automatically for chromatogram data 3 21 3 68 overview 3 2 Peak Processing parameters spectrum 3 16 3 26 peak start and end displaying 3 55 3 58 peaks do not appear in spectrum 9 17 process that occurs during 3 67 proteins 3 6 ranges overlapping 3 5 regions setting chromatogram 3 19 3 20 regions setting spectrum 3 28 resetting Basic settings 3 18 spectrum 3 68 troubleshooting 9 14 use same settings for all traces 3 21 3 25 Peak detection parameters chromatogram description 3 19 setting 3 11 3 19 Peak detection parameters spectrum Advanced 3 28 Basic 3 22 global description 3 22 global setting 3 13 individual detection ranges setting for 3 30 local description 3 28 local setting 3 28 Peak Processing 3 26 Peak detection Mariner data see also Peak detection default settings 3 71 noise detected as peaks
105. AT file the command is on the next time you open the DAT file NOTE The Auto Calibrate State command is dimmed unless you specified automatic calibration settings in the data file See Section 5 4 2 Importing and Specifying Automatic Calibration Settings 4 Display the spectrum of interest The spectrum is calibrated and displayed with an AC trace label if calibration is successful Calibration results are displayed in the Output window If calibration fails the spectrum is displayed with an AC failed trace label 5 34 Applied Biosystems Applying new constants to the data file Calibration results Applying auto calibration settings to other files Automatic Calibration To save the calibration constants for each spectrum in the data file select Apply Calibration from the Process menu Automatic calibration results are displayed in the Output window Figure 5 9 Automatic calibration results for Spec 18 lt lt 0914neuro frag001 dat gt gt 1 suceceastul marehes Old calibration constants are 5 1659016e 007 106 41671 ew calibration constants are 5 1es U1ve UUy 115 Ub535 UCCESSrul calibration HAHA Resutt A Cho Peak List A Spec Peak List A Sample info A Instrument Seting 7 Figure 5 9 Automatic Calibration Results For each spectrum in the data file results include e Number of peaks matched e Original and new calibration constants calibrated mass and fit errors e Succes
106. BP 558 3 824 0 3352 23 824 Intensity 2 8 Original trace 404 0 9979 z1 si 0 8965 7 1 7 0838 20 1556 52 9374 166 9946 2398 z4 0 6217 z1 0 100 280 460 640 820 1000 Spec 1 gt SM199 BP 558 4 251 i 558 5317 251 1 Intensity 2 8 20 6 9221 351 5018 ai 100 280 460 640 820 Added traces Spec 1 gt CTIBP 558 3 2539 0 335223 2539 0 2 0 9979 9 a429 21 20 2 a7 i i 52 9374 166 9946 i p 2398 24 9 5217 22 100 280 460 640 820 1001 Intensity 8 Spec 1 gt BO BP 558 3 827 0 3362 23 827 1 40 0 3433 23 a 0 1034 21 7 034466 1240 0 9201 21 23811 z4 0 5194 T 0 100 280 460 640 820 1000 Mass m z Intensity 2 8 Figure 2 9 Added Traces Containing Data For a description of trace labels see Viewing Trace Labels on page 2 30 2 20 Applied Biosystems Manipulating Traces 2 4 5 Removing Traces Removing the To remove the active trace from a window active trace 4 Click the trace to remove 2 Click in the toolbar or right click the trace then select Remove Trace from the menu The trace is removed Removing To quickly remove all inactive traces from the window inactive traces 4 Click the trace to keep displayed to make it the active trace 2 Select Remove Inactive Traces from the Display menu 2 4 6 Expanding and Linking Traces When you have more than one trace displayed in a window you can
107. D IS NOT TRANSFERABLE Data Explorer Software User s Guide A 3 Appendix A Warranty A 4 Applied Biosystems B Overview of Isotopes This appendix contains the following sections BA ISOtOpeS iw es ke ties B 2 B 2 Monoisotopic and Average Masses B 6 B 3 Isotopes of Common Elements B 8 Data Explorer Software User s Guide B 1 Appendix B Overview of Isotopes B 1 Isotopes B 2 Overview Applied Biosystems Many elements in their natural state exist as one of several isotopes An isotope is one of two or more atoms with the same atomic number but a different mass The most abundant isotope of carbon is C but natural carbon also contains 1 C and 4C Because a mass spectrometer measures mass to charge ratios isotopes appear in the mass spectrum Isotopes of low abundance such as 4C do not affect the appearance of a mass spectrum However isotopes that occur in greater abundance such as 1 C which occurs in a natural abundance of approximately 1 1 percent in carbon do affect the appearance of a mass spectrum The mass spectrum of methane Figure B 1 illustrates the impact of an isotope on the appearance of a mass spectrum Methane includes a peak representing the molecular ion at 16 Da CH and a peak representing the isotope at 17 Da 3CH The relative abundance of the ions is about 99 1 Figure B 1 Mass Spectrum of Methane 1 M
108. DAT files 4 From the File menu select Result Spectrum or Result Chromatogram then select Open 2 Select the title of the previously saved results to open then click OK 3 Click the Sample Info tab in the Output window to display information for the result Deleting results To delete results for DAT files for DAT files 4 From the File menu select Result Spectrum or Result Chromatogram then select Delete 2 Select the title of the results to delete then click OK 2 38 Applied Biosystems Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only Exporting results To export results to a RSD or RCD file for RCD and 1 With a data file open in Data Explorer select a RSD files spectrum or chromatogram 2 From the File menu select Export then Result Chromatogram or Result Spectrum A Save As dialog box appears 3 Specify the name and destination of the exported file By default the software assigns a RCD extension for a chromatogram or a RSD extension for a spectrum 4 Click OK Opening results To open results from RCD and RSD files for RCDand 1 Select Open from the File menu RSD files The Select Data File to Open dialog box is displayed 2 From the Files of Type drop down list select Result Spectrum Files RS or Result Chromatogram Files RC 3 Select the RCD or
109. De ma maseka ioma Parat tein dicate Fapta bm Hee Leeman Tari EaR Lea i Pepin Macchi oreen td MS jar tins _ m HEA EATI gona Hm W 50708 ms b bbad i kacat nca pans ee ae 2 Bar Preti 1G MA ee PFi 1 TEE Ter zal mM 1 M1 Tarn PR 1 1 ierti FFF 155 eran Diy Tals Erda Tinya pence re Pre Figure 6 18 lon Fragmentation Results for Synthetic Peptide PPPPPPPPPPPPAR Results Results are displayed in the lons table Lists the masses for each fragment and ion type Internal fragments table Lists possible internal sequences if you enabled this option in the Options dialog box see Figure 6 16 on page 6 27 Click Clear Table Info to clear results You can change options and recalculate ion fragmentation results Data Explorer Software User s Guide 6 29 Chapter 6 Using Tools and Applications Labeling peaks Click Label Peaks The ion peaks specified in the Options dialog box are labeled on the trace if they are present Figure 6 19 Hint To screen out labels decrease the Label Tolerance in the Options dialog box Spec 1 BP 1410 8 62787 6 3E 4 Intensity 0 29 0 311 2 593 4 875 6 1157 8 1440 0 Mass m z Figure 6 19 Labeled lon Fragmentation Peaks for Synthetic Peptide PPPPPPPPPPPPAR Hint The Label Peaks function creates User Labels in the data file To view select Peak Label from the Peaks menu then select User Label Se
110. Default smoothing applied where X is the resolution value from peak detection used to calculate the optimum number of smoothing points to apply at every mass point SC Mariner data only Converted to single charge SMX Smoothed where X is the number of smoothing points applied Stitched PSD Composite PSD spectrum TR Truncated spectrum 2 32 Applied Biosystems Manipulating Traces Figure 2 12 illustrates a smoothed spectrum with an SM5 spectrum trace label Trace label Spec 1 gt QM5 BP 558 3 657 400 S3443 657 1 2 go 5 60 40 59620 20 219 9331 837 4029 z2 0 E N r T r 99 0 279 2 459 4 639 6 819 8 1000 0 Mass m z Figure 2 12 Spectrum Trace Label 2 4 11 Printing Traces Printing traces To print traces 1 Display the traces to print To obtain a clear printout you can set the Trace Color and other attributes to dark colors before printing traces by selecting Default from the Display menu then selecting White Background NOTE If you previously modified the colors associated with this command as described in Section 1 5 1 Changing Background Color selecting this command may not set a white background and black traces When you manually set 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
111. E Copy Displayed Peaks copies all fields and headings However some data applications may not work correctly if headings are present because the first row contains text and not data For information on copying the peaks list without headings see Section 3 3 3 Saving the Peak List To copy the peak list for the displayed peaks to the Windows clipboard 1 Set peak detection as needed to create a peak list See Section 3 2 Peak Detection 2 Select the trace window to copy Zoom and adjust the trace as needed NOTE Only peak list information for the set of peaks displayed in the trace window is copied 3 From the Edit menu select Copy then select Displayed Peaks 4 Paste the data into an appropriate application for example Microsoft Excel Use this method to copy the peak list for all peaks in the active trace To copy only the section of the peak list pertaining to the peaks displayed in the active trace window see Copy displayed peaks on page 1 39 NOTE Copy All Peaks copies all fields and headings However some data applications may not work correctly if headings are present because the first row contains text and not data For information on copying the peaks list without headings see Section 3 3 3 Saving the Peak List To copy the peak list for all peaks in the active trace to the Windows clipboard 1 Set peak detection as needed to create a peak list See Section 3 2 Peak Detection
112. Full Base Peak 0 25 Intensity Max Peak Area 0 25 Filter Width 3 Integration V to B Baseline Setting Data Explorer Software User s Guide 3 71 Chapter 3 Peak Detection and Labeling The following table lists the default settings in the SET files provided for spectra Voyager Spectrum Mariner Parameter Spectrum Linear Reflector PSD MARINER S VOYAGER ET VOYAGER REFLECTOR VOYAGER LINEAR SET PSD SET SET Basic Settings spectrum data Base Peak Intensity 0 25 0 0 0 Max Peak Area 0 25 2 2 2 Use Resolution Dependent ON ON ON OFF Settings Mass Resolution 5 000 2 000 10 000 N A Peak Processing Integration Baseline Setting V to V V to B V to B V to B Centroid 50 50 50 50 Max Charge State 6 1 1 1 Max lsotopes 6 5 10 5 Min Intensity 5 10 10 10 Max Intensity 100 100 100 100 3 72 Applied Biosystems Default Peak Detection Settings Voyager Spectrum Mariner Parameter Spectrum Linear Reflector PSD MARINER S VOYAGER ET VOYAGER REFLECTOR VOYAGER LINEAR SET PSD SET SET Advanced Detection Range Resolution Resolution Resolution Five dependent dependent dependent ranges Filter Width Resolution Resolution Resolution Range dependent dependent dependent dependent Increment Resolution 1 1 1 dependent Noise Threshold 0 0 0 0 Base Peak Intensity 0
113. Graphics and Processing Settings option to apply to new files you are opening settings are not applied to files in the list that are already open e Use Settings from Data File Applies the last settings used on the data Use Default Settings Applies settings from the appropriate default SET file for your system See Default processing and graphic settings on page 1 18 Use Selected Set File Opens the Restore Graphics and Processing Settings dialog box where you select the SET file to open For information on customizing a SET file see Section 1 4 2 Customizing Processing and Graphic Settings SET 6 Click Finish to open the selected files The Data Explorer window displays the selected data files with the processing and graphic settings you selected Figure 2 2 and Figure 2 3 2 4 Applied Biosystems CHRO plate with 7 different samples 34012 dat TIC 150 oe Bl a of i 0 0 36 2 724 108 6 144 8 181 0 Spectrum Number 1 9E 4 Intensity Opening and Closing Data Files CHRO plate with 7 different samples 3A010 dat TIC 148 2 105 28 cal AE A AN 35 8 71 6 107 4 Spectrum Number f 0 143 2 179 0 Intensity 1 9E 4 IMSPEC plate with 7 different samples 3A012 dat Spec 1 ASC BP 121 1 66 ba al PARE a 305 2391 3 501 5 635 4 803 8 429 4 619 6 809 8 2 g S 2 oe Gi 0 49 0 239 2 1000 0 CHRO plate with 7 different sam
114. K Data Explorer Software User s Guide 2 23 Chapter 2 Using Chromatogram and Spectrum Windows 2 4 8 Overlaying Traces This section includes e Overlaying traces from different data files e Overlaying traces in a single data file e Changing the active trace e Sequentially activating overlaid spectra Setting overlay attributes 2 Overlaying traces To overlay traces from different data files from different 4 Copy chromatogram or spectrum traces you want to data files overlay into a trace window For more information see Section 2 5 2 Copying Traces from Multiple Data Files to a Window Hint The copied traces display the original trace label and filename 2 To use settings other than defaults set attributes for the overlay if needed See Setting overlay attributes on page 2 26 3 Click the trace of interest to activate it NOTE Only the active trace in an overlay is affected by processing tools However all traces are affected by Zooming functions 4 From the Display menu select Overlay Traces Hint A toolbar button is available to toggle between Overlay and Undo Overlay mode See Customizing toolbars on page 1 21 for information The E button is located in the Graph category The traces are overlaid The trace names are listed in the trace label 2 24 Applied Biosystems Manipulating Traces NOTE When saving results only the results for the active trace
115. LL OF WHICH ARE EXPRESSLY DISCLAIMED Warranties THE REMEDIES PROVIDED HEREIN ARE BUYER S SOLE limitations AND EXCLUSIVE REMEDIES WITHOUT LIMITING THE GENERALITY OF THE FOREGOING IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTACT 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 CONSEQUENTIAL OR SPECIAL DAMAGES SUSTAINED BY BUYER OR ANY OTHER PERSON OR ENTITY WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE OPERATION OF ANY EQUIPMENT OR SOFTWARE 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 AN
116. Noise Removal more than one time with the same Standard Deviation setting will not improve noise removal 3 Click OK The trace is displayed with an NF SM or NR trace label 4 To return to the original trace see Returning to the original trace on page 4 4 Data Explorer Software User s Guide 4 19 Chapter 4 Examining Chromatogram Data 4 5 Adding and Subtracting Raw Spectra Within a Data File Use the Add Subtract Spectra command to manipulate raw spectra within a single data file NOTE To manipulate processed spectra spectra from different data files or spectra acquired under slightly different instrument calibrations use Trace Arithmetic For more information see Section 5 12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files Dual Spectral Trace Arithmetic Adding and To add and subtract spectra subtracting 4 Click the Spectrum window to activate it spectra Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Add Subtract Spectra The Add and Subtract Spectra dialog box is displayed Figure 4 9 4 20 Applied Biosystems Adding and Subtracting Raw Spectra Within a Data File Add and Subtract Spectra x m List Spectra To Be Added List Spectra To Be Subtracted m Add Sub Mode Average C Accumulate OK Cancel
117. OLT HE M x TESSEECCAD mass Calibration TITAL MILO mie 3 1 Stations BAAS statistics D AGA dara bak sOfting CECHI LIT Figure 5 5 Calibration Statistics in Output Window If you calibrate more than once subsequent calibration statistics are added to the end of the list in the Output window Older calibration statistics are listed at the top of the list Automatically You can automate peak matching by clicking Match Peaks matching peaks instead of right click dragging individual peaks and selecting the mass If any masses within the tolerances of any of the masses listed in the calibration reference file are found in the spectrum the matches are displayed in the Calibration Mass Peak Selection window with an Initial Error reported 5 14 Applied Biosystems Manual Calibration NOTE If you are calibrating Mariner data see Ensuring that masses match during calibration on page 5 24 Applying new To apply the calibration constants to the data constants to the data file myers Click The following occurs calibrating Mariner data Apply All spectra in the data file are calibrated and including Calibration displayed with an MC trace label The calibration MS Method constants are saved with the data file Each data spectrum in the data file is calibrated when displayed Voyager data Apply The current spectrum is calibrated and Calibration displayed with an MC trace label The calibration consta
118. Peak Labels This section includes e Description e Customizing colors font and size e Creating custom peak labels e Applying user labels from LBC or LBS files e Displaying user labels e User labels not displayed Description A custom peak label displays the label name you enter If you specify a custom label for a spectrum it is displayed instead of the mass value Customizing To set colors font and size for custom labels or peak labels colors font and see Section 1 5 Setting Graphic Options size Creating custom To create custom peak labels peak labels 4 Glick the Chromatogram or Spectrum window to activate it 2 From the Peaks menu select Peak Label The Peak Label dialog box is displayed see Figure 3 22 on page 3 56 3 Click User Label Setup The User Label Setup dialog box appears Figure 3 23 NOTE The Chromatogram User Label Setup dialog box displays X Value and X Tolerance instead of Peak Mass and Mass Error Data Explorer Software User s Guide 3 61 Chapter 3 Peak Detection and Labeling 3 62 Applied Biosystems Spectrum User Label Setup x m User Label Settings Originated from Filename r Label Type Mass Mass difference from adjacent peak m Label List Match Charge State T Wx Column header buttons Figure 3 23 User Label Setup Dialog Box Select the Label Type spectra only e Mass Labels with Apex or Centroid mass
119. RER VB6 The Visual Basic macros described in this document are provided in a file called DataExplorer VB6 in the directory that contains the Mariner or Voyager program file New macros you create using the Macro Recorder in the Data Explorer software are also added to this file You can modify the macros in this file as desired However before you make changes make a copy of the DataExplorer VB6 file for example make a copy called DataExplorer BAk If the changes you make are not acceptable you can copy or rename the backup file over the modified file Preparing Data Before Accessing Macros References The following references are selected by default in the required DataExplorerProject in the Visual Basic Editor and are required for the macros in the DataExplorer VB6 file to successfully run e Visual Basic For Applications e Data Explorer 4 0 Type Library e OLE Automation e Microsoft Forms 2 0 Object Library e Microsoft Internet Controls To view references 1 Open the DataExplorerProject select VisualBasicEditor from the Tools menu in the Data Explorer window 2 Select References from the Tools menu in the Visual Basic Editor C 2 Preparing Data Before Accessing Macros Before you access the toolbox open data files of interest and Smooth the data if necessary to reduce noise e Set optimum peak detection and labeling e Set the display range or zoom as needed e Resize and organize the displays as needed
120. Resutt A Chro Peak List J Spec Peak List Sample info J Instument Seting A Elemental Analysis _ Elemental Target a window NMN 3 For Help press F1 Figure 1 3 Parts of the Data Explorer Window Data Explorer Software User s Guide 1 11 Chapter 1 Data Explorer Basics Toolbar The toolbar contains buttons that access Data Explorer functions For a description of a toolbar button place the cursor on the button A brief description of the button ToolTip is displayed below the button For information on adding or removing toolbar buttons see Customizing toolbars on page 1 21 Chromatogram Refer to the following tables for descriptions of the types of and Spectrum data that you can display in the Chromatogram CHRO and windows Spectrum SPEC windows Mariner data Table 1 3 Voyager data Table 1 4 Table 1 3 Mariner Data Displayed in Chromatogram and Spectrum Windows Window Mariner Data CHRO Displays e Total lon Chromatogram Includes the entire mass range saved in the data file e Extracted lon Chromatogram XIC optional Includes only the signal response from a mass window or range e Constant Neutral Loss Chromatogram CNL optional Extracts only the response from peaks that are separated by a selected mass difference Optionally displays the following from Diode Array data DAD e Total Absorbance Chromatogram TAC e Channel Ch e Extracted Absorban
121. Section 3 5 3 Setting Custom Peak Labels Delta X value is outside acquisition range for the data file Set Delta X value within acquisition range for the data file For information see Section 3 5 3 Setting Custom Peak Labels Peak label placed on peak shoulder instead of peak apex Filter Width Increment set higher than 1 Set Increment to 1 See Increment on page 3 31 Table 9 11 Peak Detection and Labeling Troubleshooting Voyager Only Symptom Possible Cause Action Noise detected as peaks Max Peak Area set too low Increase See Section 3 2 2 Strategy for Voyager Peak Detection Low Mass Gate spike identified as Base Peak linear data Software identifies the most intense peak ina trace as the base peak and does not ignore artifacts Truncate the data See Section 5 9 Truncating a Spectrum Data Explorer Software User s Guide 9 17 Chapter 9 Troubleshooting Table 9 11 Peak Detection and Labeling Troubleshooting Voyager Only Symptom Possible Cause Action Partially resolved peaks not detected Mass resolution set too high to detect average mass Decrease Mass Resolution setting See Section 3 2 2 Strategy for Voyager Peak Detection Base Peak Intensity not adjusted correctly Adjust See Section 3 2 2 Strategy for Voyager Peak Detection Table 9 12 Charge State and Isotope Dete
122. Tools and Applications Troubleshooting Mariner Only Symptom Possible Cause Action Failed to calculate result for mass deconvolution Did not select at least two peaks for same charge envelope Select at least two peaks for example 1 and 2 For more information see Section 5 6 Mass Deconvolution Mariner Data Only Multiple Charge Mass Deconvolution commands dimmed on Process menu Your system does not include the optional mass deconvolution software Contact Applied Biosystems to purchase the option Centroiding Mass Calibration Multiple Charge commands not displayed on Process menu Spectrum window not active Activate Spectrum window then select Process menu Resolution command not displayed on Tools menu Spectrum window not active Activate Spectrum window then select Tools menu Only the active trace zooms in Overlay mode Zooming may not behave as expected on overlaid traces Click in the top right corner of the window to restore the view Data Explorer Software User s Guide 9 9 Chapter 9 Troubleshooting 9 4 Calibration Troubleshooting Table 9 6 Calibration Troubleshooting Mariner and Voyager Symptom Possible Cause Action Auto Calibration is turned on but current spectrum is not auto calibrated Current spectrum is not calibrated until the next time the spectrum is displayed Advance to the nex
123. User s Guide Index 27 V Valley to Baseline integration chromatogram 3 21 3 70 spectrum 3 26 3 70 Valley to Valley integration chromatogram 3 21 3 70 spectrum 3 26 3 70 Version of software used to acquire data 1 15 Vertical bars displaying centroid traces 5 36 traces do not print 2 34 Vertical cursor 1 27 Vertical peak labels 3 55 3 58 Vial number displaying in Chromatogram window 3 55 Viewing read only files 2 7 Views linking 2 13 Visual Basic Editor 6 42 Visual Basic macros provided see also Data Explorer Toolbox accessing C 4 Ladder Sequencing macro C 5 Ladder sequencing macro C 2 modifying C 2 MS Fit MS Tag macro C 2 overview C 2 Peptide fragmentation macro C 2 C 9 Polymer analysis macro C 2 C 15 preparing data for C 3 References required C 3 Voyager MS files converting to DAT not supported 1 30 Voyager data Chromatogram window displaying 1 13 2 7 examples 7 11 isotope resolution limits 3 53 peak detection strategy 3 8 Index 28 Applied Biosystems Voyager Sequence Control Panel automatic calibration settings reference masses for 5 28 VOYAGER REF provided 5 18 viewing masses in 5 10 VOYAGERPSD SET 8 11 W Warranty exceptions A 3 for computers with altered configuration A 1 Weight average molecular weight determining C 15 White background changing colors 1 20 default settings 1 23 SET file 1 19 Windows activate 2 8Applied adding traces from same data file
124. Voyager DE Biospectrometry Workstations NOTE Application systems that automatically acquire and process data such as Mariner High Throughput Analysis Option CombiSolve and Proteomics Solution 1 Option require specific versions of Data Explorer software Features Data Explorer software includes a suite of tools and processing options to allow you to graphically and interactively manipulate chromatographic and mass spectral data For example you can e Smooth and noise filter data e Automatically and manually calibrate spectral data e Set peak detection parameters and custom labels for regions of the trace Detected peaks can be evaluated for charge state determination according to user defined parameters e Determine elemental composition theoretical isotope distributions resolution signal to noise ratio and fragment ions e Perform target compound analysis elemental targeting e Customize windows toolbars and traces e Create scripts and macros to automate your work using the Macro Recorder and Visual Basic Editor 1 2 Applied Biosystems Overview Starting and To start the Data Explorer software from the Windows NT exiting the desktop double click the Data Explorer icon on the desktop software he Data Explorer window opens The Data Explorer window is blank with only a few menus displayed until you open a data file Z Figure 1 1 shows the Data Explorer main window with a Data Expl
125. XAC for DAD Mariner data e From the Chromatogram window From the Spectrum window From the To create an extracted absorbance chromatogram XAC for a Chromatogram selected wavelength window or range window 1 Click the Chromatogram window to activate it 2 Display a TAC or Channel chromatogram by selecting Traces from the View menu then selecting the chromatogram 3 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 4 Select Extracted Absorbance from the Process menu or click in the toolbar 5 Inthe Extracted DAD Chromatogram dialog box Figure 4 6 click one of the following e Center Window then enter the wavelengths of interest from 190 nm to 950 nm and the window for wavelengths to include e Range then enter the From and To values for wavelengths from 190 nm to 950 nm Data Explorer Software User s Guide 4 13 Chapter 4 Examining Chromatogram Data 4 14 Applied Biosystems Extracted Absorbance Chromatogram fi EG m Wavelength Range Ditference Type Center Window hal r Wavelength Range Difference Wavelength Window nm fe fo fe fo fe fox fe fo fe for m Extraction Mode Accumulative C Individual OK Cancel Figure 4 6 Extracted Absorbance Chromatogram Dialog Box Specify the Extraction Mode e Accumulative Creates a single trace combining intensities of all specified wavelengths Indi
126. a 1 Display the spectrum to truncate 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Truncate Spectrum The Truncate Spectrum dialog box Figure 5 17 is displayed Truncate Spectrum xi Mass Range to Keep Range Start Range End fo fo OK Cancel Figure 5 17 Truncate Spectrum Dialog Box 4 Type the starting and ending m z values for the range of the spectrum to retain or right click drag over the range in the spectrum 5 Click OK 5 56 Applied Biosystems Truncating a Spectrum The data in the spectrum is truncated to the selected range and is displayed with a TR trace label The range displayed on the axis of the truncated trace is the range of the original data file and may be wider than the range of the truncated spectrum 6 To return to the original trace see Returning to the original spectrum on page 5 3 Example Figure 5 18 and Figure 5 19 illustrate the effects of truncating a Voyager spectrum and eliminating the Low Mass Gate peak Figure 5 18 includes a Low Mass Gate spike Because the Low Mass Gate spike is the most intense peak in the spectrum it is identified as the base peak and All other peaks in the spectrum are scaled as a percentage of the base peak e A default peak detection threshold Base Peak Intensity is set to 1 percent of the base peak One percent of an intense p
127. a precursor mass that you incorrectly typed before acquiring The precursor ion flight time associated with the incorrect mass is stored in the DAT file and will yield invalid fragment masses even if you correct the precursor mass You must specify the correct precursor mass and reacquire the data to obtain correct mass and flight time values to yield a valid fragment mass calibration Data Explorer Software User s Guide 8 23 Chapter 8 Viewing Voyager PSD Data 8 24 Changing Applied Biosystems If the precursor mass taken from the data file is not correct 1 Display the Segments tab see Figure 8 2 on page 8 4 by doing either of the following e Inthe PSD Calibration dialog box click the Segments tab e From the Process menu select PSD Processing 2 Click Change Mass and enter a new mass to use for calibration 3 Click OK 4 Click Plot to display the composite spectrum for the new mass The following occurs Anew composite spectrum is generated as described in How the composite spectrum is generated on page 8 6 and displayed e The PSD calibration for the data file is updated with the new precursor ion mass e The new composite spectrum is displayed NOTE The Max Stitch Masses displayed in the PSD segment list in the PSD Processing dialog box are not updated until you apply the new calibration 5 To save the updated calibration information in the data file click Apply Precursor
128. a spectrum to a centroided plot by deconvoluting the monoisotopic peaks from the current peak list Peak deisotoping is an advanced peak filtering method that can determine the relative abundance of multiple components with overlapping isotope distributions The deisotoping algorithm uses the elemental composition that you specify to improve the determination of the monoisotopic mass by considering the centroid masses of all peaks in the isotopic envelope For each detected peak in a spectrum the software inspects the peak list for the higher theoretical masses and areas associated with additional expected peaks in a theoretical isotopic cluster relative to the peak in question Data Explorer Software User s Guide 3 45 Chapter 3 Peak Detection and Labeling 3 46 When to use Applied Biosystems If the expected higher theoretical peak masses and areas are present in the peak list e The peak in question is considered to be a monoisotopic peak e The intensities of the higher mass peaks that correspond to the expected isotope ratios are combined with the intensity of the peak in question additional intensity that may be related to a contaminant or an overlapping isotope envelope is not combined and will be evaluated in the next iteration e The peak in question is represented in the trace as a centroid bar with increased amplitude The total intensity of the centroid bar represents the total area of each fitted
129. abels are applied to the peak and are separated by commas Charge State Charge state that the peak must have in order to apply user labels Data Explorer Software User s Guide 3 63 Chapter 3 Peak Detection and Labeling Chromatogram Parameter Description Label Text of the label to display X Value Retention time or Spectrum Number of the peak to label The default units for X Tolerance correspond to the units of the x axis X Tolerance Retention time or Spectrum Number that the peak must fall within to apply user labels 10 11 3 64 Applied Biosystems To enter Peak Mass X Value or Mass Difference you can type values or right click drag over a peak A Mass Tolerance or X Tolerance of 1 is automatically entered when you right click drag Charge state is also automatically entered when you right click drag a spectrum peak Click OK The User Label Setup dialog box reappears To change any information double click the Label field in the User Label Setup dialog box The User Label Entry dialog box for the label opens Enter the new values then click OK Hint You can sort the label list by any field by clicking the column header button see Figure 3 23 on page 3 62 To save the label settings as a LBC or LBS file click Save As type a file name then click Save Click OK The Peak Label dialog box reappears Applying user labels from LBC or LBS fil
130. able Removal method results for most data A setting close to 1 0 ae better May affect yields a higher degree of noise reduction eae peak If applying the Noise Filter with a certain resolution Correlation Factor does not yield the necessary noise removal return to the original trace see Returning to the original spectrum on page 5 3 and apply the Noise Filter again with a higher Correlation Factor setting Applying the Noise Filter more than one time with the same Correlation Factor setting does not improve noise removal Noisy low resolution Gaussian Specify a Filter Width in data points odd data chromatogram Smooth integers only The maximum number of data or Voyager SM smoothing points is 2001 Points less than 1 PSD data Filter Width from the edge of the spectrum are May affect not smoothed peak resolution Data Explorer Software User s Guide 5 43 Chapter 5 Examining Spectrum Data Type of Data Suggested Method Description High resolution data Noise Removal NR Does not affect peak resolution Specify the number of standard deviations of noise to remove The software automatically calculates the average white noise for all frequencies across the spectrum then removes the specified number of standard deviations of noise This method slightly affects peak intensity and removes peaks with a signal to noise ratio less than the specified standard de
131. aces is added allowing for visual comparison 7 To return to the original trace see Returning to the original trace on page 4 4 Evaluating Note the following when evaluating filtered traces filtered traces e If you select more than one tag to display all spectra containing the specific combination of tags are included in the filtered trace For example assume the MS Method used to acquire the data specifies Tag 1 for event 1 and both Tag 1 and Tag 2 for event 2 If you select Tag 1 to filter spectra from event 1 are included in the filtered trace but not spectra from event 2 e A filtered trace displays data points for the selected tags only However if you double click the filtered trace in an area that does not appear to contain data a spectrum is displayed in the Spectrum window e Any actions you perform on a filtered trace such as summing include only the spectra displayed in the filtered trace Spectra from areas of the filtered trace that do not appear to contain data are not included NOTE In a summed trace the trace label includes the spectrum numbers for all spectra in the summing range even if they are not in the filtered trace However only the spectra with the appropriate tags are included in the summed spectrum Displaying To display additional filtered traces additional filtered 4 Select the original unfiltered trace traces SAA 2 From the Process menu select Event Tag Filt
132. ader 2 30 in spectrum header 2 31 5 46 BP Relative label on data cursor 1 27 BPI see also Base peak intensity in chromatogram header 2 30 in spectrum header 1 13 2 32 C CAL files see also Calibration constants applying 5 16 8 20 description 1 7 exporting during calibration 5 16 exporting from DAT 1 36 importing 5 16 8 20 importing error displayed 9 11 9 12 PSD creating 8 20 PSD overview of creating 8 10 saving 5 16 Calculator tools elemental composition 6 2 lon Fragmentation 6 25 isotope 6 13 mass resolution 6 20 signal to noise ratio 6 23 Calibrating mass automatic Mariner data only see also Calibration constants applying settings to other data files 5 29 batches of related samples 5 29 calibration reference file REF 5 17 calibration settings 5 29 commands dimmed on menu 9 11 constants calculating A and B 5 34 error fit 5 12 error initial 5 12 overview 5 26 peak weighting factor 5 33 results 5 35 Data Explorer Software User s Guide Index 3 Calibrating mass automatic Mariner data only continued reverting to instrument calibration 5 22 troubleshooting 9 9 turning on 5 34 when to use 5 28 Calibrating mass manual see also Calibration constants baseline correcting and deisotoping to optimize mass accuracy 7 14 calibration reference file REF 5 17 commands dimmed on menu 9 11 constants applying A and B to new file 5 16 error fit 5 12 error initial 5 12 fit outliers
133. ak apex or peak centroid to use for calibration Click OK 3 From the Process menu select Mass Calibration then select PSD Calibration The PSD Processing dialog box is displayed with the Calibration tab selected Figure 8 5 Hint You can also access this dialog box by selecting PSD Processing from the Process menu then clicking the Calibration tab 8 12 Applied Biosystems Calibrating a PSD Spectrum PSD Piecing Lae Caia mar This bha aa a hi aat at Meo Sd a lie Agtererce mpk bry enj prun cpa Dimm Gaien ara Pied be naa F biana bein prr z ir a a tarps Cah Prem arghia bent fire Eria matched Column P T header IESE ERE 133 wia EERS buttons oa Tee EL Fae iii Ame TT EE uarn Figure 8 5 PSD Processing Dialog Box with Calibration Tab Displayed Select a PSD Calibration Reference File that you generated as described in Section 8 3 4 Creating PSD Calibration Reference REF Files A calibration reference file called Angiotensin_Fragments REF is provided with the software NOTE Use a calibration reference REF file that specifies the peak type for reference masses as Resolved Isotope Mass even if they are not resolved isotopes The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the softw
134. ance chromatogram XIC xxx yyy Extracted ion chromatogram for a selected mass where xxx is the center mass and yyy is the specified window NOTE Extracted ion chromatograms were previously labeled with Mass instead of XIC Figure 2 11 illustrates an extracted ion chromatogram with XIC chromatogram trace label a Trace label 1103 Intensity 2 S Figure 2 11 Chromatogram Trace Label Spectrum trace Labels in the spectrum title identify the following types of data labels displayed Spectrum Trace Label Type of Processing of mmm nnn Mariner data only Added accumulated or subtracted spectrum from spectrum mmm to nnn AC Automatically mass calibrated AdvBC Advanced baseline correction ASC Mariner data only AutoSaturation corrected trace BC Baseline corrected BO Baseline offset Data Explorer Software User s Guide 2 31 Chapter 2 Using Chromatogram and Spectrum Windows Spectrum Trace Label Type of Processing BPI Base peak intensity CT Centroid DAD Diode array data DECONV Zero charge deconvoluted trace Mariner data only DI Deisotoped trace ISO Isotope MC Manually mass calibrated NF Noise filtered trace NRX Noise removed trace where X is the number of standard deviations of noise removed RSMX does not apply to Voyager PSD data
135. and Labeling Diiw ei Peet eteeten brisi AD Siac Pra Decin Marge Paria Ea Farge Lempaa Upper Poured Range Sii ine CEP inani hs z Pis gih q i nagan Beenie lsir YE alep io Hiirte PP Make ia aliy D pears p rgi je al ipon m a ow wa Figure 3 3 Chromatogram Peak Detection Setup Dialog Box 4 Select a detection range and set parameters as needed 5 To apply settings to all traces select Use same settings for all traces in view To set parameters independently for all traces ina window deselect Use same settings for all traces in view 6 Click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box 3 12 Applied Biosystems Peak Detection Setting Basic Basic Settings should provide acceptable peak detection for Settings most applications After you apply these parameters no spectrum data further adjustment should be required If further adjustment is required select Use Advanced Settings and adjust parameters as needed To set Basic Settings parameters for spectrum data 1 Click the Spectrum window to activate it 2 Click the trace of interest 3 Click in the toolbar or select Peak Detection from the Peaks menu The Spectrum Peak Detection Setup dialog box opens with the Basic Settings tab Figure 3 4 displayed NOTE If you applied Advanced Settings to a data file they override the settings on the Basic Se
136. and for all hardware and software installed by the buyer and for all other products the applicable warranty period begins the date the component is delivered to the buyer For software installed by the buyer or any person other than Applied Biosystems the applicable warranty period will begin the date the product 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 above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation outside of the environmental or use specifications or not in conformance with the instructions for the instrument system software 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 of the instrument or the software not authorized by Applied Biosystems THE FOREGOING 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 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 A
137. and then importing it Right click the peak list to copy You can import peak list values into Microsoft Excel to create a spreadsheet To import and save the peak list in Microsoft Excel 1 In Excel select Open from the File menu The Open dialog box is displayed 2 Select All Files from the Files of Type drop down list at the bottom of the dialog box Select the directory and file to import then click Open The Text Import wizard is displayed 3 Follow the prompts from the Text Import wizard accepting the default settings provided The peak list is converted to an Excel spreadsheet 4 Select Save As from the File menu The Save As dialog box is displayed 5 Select Microsoft Excel Workbook xls in the Save as Type drop down list at the bottom of the dialog box 6 Type a file name or modify the default name delete quotation marks if present and the PKT extension 7 Click Save The file is saved with an XLS extension For more information on using Excel refer to your Microsoft Excel User s Guide Data Explorer Software User s Guide 3 41 Chapter 3 Peak Detection and Labeling 3 3 4 Sorting Filtering 3 42 and Printing the Peak List Sorting The peak list is displayed in order by index number You can the peak list _ sortthe list by any field by clicking the column header buttons Figure 3 16 Click column header buttons to sort by different fields Figure 3 16 S
138. are automatic calibration settings You use the Automatic Calibration function in Data Explorer to prepare Automatic Calibration settings contain reference masses and other matching information and to calibrate the data You can also use the Automatic Calibration function in Data Explorer to prepare Automatic Calibration settings contain reference masses and other matching information for use by the Mariner Sequence Control Panel described in the Mariner Workstation Version 4 0 Supplement You can save a SET file that contains the settings and specify the SET file in the Sequence Control Panel If you create and specify a macro to automatically calibrate the Sequence Control Panel can use the calibration settings contained in the SET file Data Explorer Software User s Guide 5 27 Chapter 5 Examining Spectrum Data 5 28 When to use Automatic calibration for Voyager data When to use Applied Biosystems Use automatic calibration for Mariner data when you Have many spectra to calibrate e Know in advance what reference masses to use e Know in advance that the quality of the reference mass signals is acceptable For information on manual calibration see Section 5 3 Manual Calibration The Automatic Calibration function in Data Explorer is used by the Voyager Sequence Control Panel described in the Voyager Biospectrometry Workstation User s Guide to calibrate data as it is acquired You use the Au
139. are mistakes these narrow peaks as isotopically resolved and ignores the reference mass For more information on creating calibration reference files see Section 5 3 3 Creating or Modifying a Calibration Reference File REF Data Explorer Software User s Guide 8 13 Chapter 8 Viewing Voyager PSD Data NOTE If the calibration reference file is stored ona network drive an error message may display when you select the calibration file when performing a calibration If an error message is displayed copy the file to a local drive on your computer using Windows NT Explorer 5 Enter Reference Matching Criteria e Minimum Intensity Peaks must be above this intensity to be considered a match Select the unit for Minimum Intensity Relative Intensity or Relative Area e Mass Tolerance Peaks must be within this tolerance of the theoretical mass to be considered a match Select the unit m z or ppm 6 Select the Peak Weighting Factor If the calibration includes more than two points you can apply the following weighting factors to fit points to the curve e None All peaks weighted equally Inverse Peak Width Narrower peaks are weighted more than broader peaks e Height More intense peaks are weighted more than less intense peaks 7 Match observed peaks in the spectrum with reference masses in the calibration reference file using one of the following procedures e Matching peaks automatically
140. arge state 1 Spec 1 BP 275 6 1085 400 235 6 21 663 90 80 70 60 5 s0 40 30 20 i0 736 6 71 t i is pran 0 baT 2354 235 8 236 2 236 6 237 0 Mace imiz Figure 3 12 Minimum Intensity Set Correctly If you increase the Minimum Intensity setting to 85 in the example shown in Figure 3 12 the software cannot determine a charge state for the first peak Figure 3 13 This is because e The increased threshold suppresses the detection of the 236 6 m z peak When the software evaluates the peak at 235 6 m z it does not find a peak at the appropriate peak spacing for charge state 1 and therefore determines that the peak has no calculated charge Spec 1 BP 275 6 1085 400 235 6 663 90 80 70 a 60 50 40 30 20 10 t T T 0 235 0 235 4 235 8 236 2 236 6 237 0 Macr fens Figure 3 13 Minimum Intensity Set Incorrectly Charge state See Table 9 12 Charge State and Isotope Determination determination Troubleshooting Mariner Only on page 9 18 troubleshooting 3 36 Applied Biosystems Peak List 3 3 Peak List This section describes e Displaying the peak list e Inserting peaks in the peak list e Saving the peak list Sorting filtering and printing the peak list 3 3 1 Displaying the Peak List After peak detection centroiding and integration the software creates a peak list for the chromatogram Mariner data only and each spe
141. arged 70 i 2 a species a Ss 50 40 30 149 0313 20 181 0265 40 ico p MRES _391 2708550 6242 107 0 487 8 868 6 1249 4 1630 2 2011 0 Mass m z Figure 5 22 Spectrum After Single Charge Conversion Data Explorer Software User s Guide 5 61 o Chapter 5 Examining Spectrum Data NOTE Charge states other than 0 or 1 in the converted trace indicate that a peak in the original spectrum is labeled with an incorrect charge state Set peak detection thresholds to disregard these peaks and convert the spectrum again CAUTION A zero value in the Spec Peak list does not indicate a charge state of zero It indicates that the software could not determine the charge state 5 11 AutoSaturation Correction Mariner Data Only 5 62 Function Hardware requirements Applied Biosystems NOTE Autosaturation correction is not supported for Mariner RST or DAD data The AutoSaturation Correction function mathematically corrects for signal saturation of the Mariner detector system to provide optimum mass accuracy The AutoSaturation Correction feature is turned on by default and automatically corrects the data when you open a Mariner data file in Data Explorer Leave this feature turned on for normal operation The AutoSaturation Correction function requires special hardware If the system used to acquire the data does not include the required hardware the AutoSaturation Cor
142. ariner CAL 9 12 Applied Biosystems Calibration Troubleshooting Table 9 8 Calibration Troubleshooting Voyager Only Symptom Possible Cause Action Error displayed when you import a calibration CAL file corrupted Create new CAL file See Exporting BIC MSM and CAL files on page 1 36 Importing a CAL file generated from a Mariner data file Import a Voyager CAL Importing a CAL file generated from a data file collected in a different instrument mode Linear Reflector or PSD Import a CAL generated from a data file collected in the same instrument mode Importing a CAL file generated on a different instrument Import a CAL generated on the same instrument Data Explorer Software User s Guide 9 13 Chapter 9 Troubleshooting 9 5 Printing Troubleshooting Table 9 9 Printing Troubleshooting Mariner and Voyager Symptom Possible Cause Action Traces do not print Line width is set to 0 or 1 Change the line width See Section 1 5 Setting Graphic Options Line color is set to white Change the color See Section 1 5 Setting Graphic Options Landscape printer setup lost when you close Data Explorer Landscape option set using Printer Setup in Data Explorer Set landscape printing using Printer Settings in Windows Control Panel See Dedicating a printer to landscape orientation on page 2
143. ata files NOTE When different data files are linked zooming functions performed on one data file are applied to all linked files Processing and peak centering functions are applied to the active file only To link data files 1 Open the data files you want to link 2 In the first data file click the window Chromatogram or Spectrum that you want to link to another data file then select Link View from the View menu NOTE Clicking in the toolbar links traces not views 3 Repeat step 2 if you want to link both windows 4 Repeat step 2 and step 3 for the remaining data files NOTE You must select Link View for each window and each data file you want to link You can organize all open windows by clicking buttons in the toolbar Tile Horizontal e Tile Vertical e Cascade Windows You can also move and resize windows by click dragging Data Explorer Software User s Guide 2 13 Chapter 2 Using Chromatogram and Spectrum Windows 2 4 Manipulating Traces This section includes e Zooming centering and customizing a trace e Duplicating a trace e Dividing the active trace e Adding traces from the same data file to a window e Removing traces e Expanding and linking traces e Recalling and rearranging traces Processing History e Overlaying traces e Annotating traces e Viewing trace labels e Printing traces 2 4 1 Zooming Centering and Customizing a Trace Zoom
144. ata points Data Explorer Software User s Guide 3 25 Chapter 3 Peak Detection and Labeling Peak Processing parameters spectrum data only Table 3 3 describes the parameters in the Peak Processing tab of the Spectrum Peak Detection Setup dialog box see Figure 3 5 on page 3 16 Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Table 3 3 Peak Processing Parameters Spectrum Data Only Parameter Description Integration Baseline Settings NOTE You can set peak labels to display peak start peak end and baseline marks See Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels Valley to Baseline Draws a vertical line from all valleys to a horizontal baseline The level of the horizontal baseline is determined using the minimum peak valley point left or right for each peak See Figure 3 25 on page 3 70 Valley to Valley Forces a baseline through all valley points See Figure 3 25 on page 3 70 Spectrum Parameters Centroid Specifies the percentage of peak height used to determine the location of the centroid mass For example with a Centroid of 10 the software examines the top ten percent of signal Enable Gaussian Fitting Increases the accuracy of the centroid calculation for spectral peaks with limited data points fewer than 6 data points across the peak in particular Mariner data below m z 400
145. ation The Manual Mass Calibration dialog box is displayed Figure 5 2 Manual Calibration Reread Warn E sieti Fists pee ware heey E T Fii pempig Lact rrm i poy Calta monn x l TARE Teed Uo rot EHN S00 13011 mama THEID 41m TITS P Figure 5 2 Manual Mass Calibration Dialog Box Select a calibration reference file For information on creating a reference file see Section 5 3 3 Creating or Modifying a Calibration Reference File REF Enter reference matching and calibration criteria e Minimum Intensity Peaks must be above this intensity to be considered a match Select the unit for Minimum Intensity Relative Intensity or Absolute Counts e Mass Tolerance Peaks must be within this tolerance of the theoretical m z to be considered a match Select the unit m z or ppm Data Explorer Software User s Guide 5 9 Chapter 5 Examining Spectrum Data 6 Select the Peak Weighting Factor If the calibration includes more than two points you can apply the following weighting factors to fit points to the curve None All peaks weighted equally e Inverse Width Narrower peaks are weighted more than broader peaks e Height More intense peaks are weighted more than less intense peaks Manually matching 7 To manually select the reference mass for a peak peaks right click drag over the peak of interest The Select or Create Reference Peak Information dialog box Figure 5 3 is displayed a
146. ation Correlation Data Explorer Software User s Guide C 13 Appendix C Data Explorer Toolbox Visual Basic Macros 4 Click Find Correlation Correlations for the selected peak are listed NOTE Yp Sp and Tp represent phosphotyrosine phosphoserine and phosphothreonine respectively 5 Click Copy to Output Window Result Tab to copy results to the Result tab You can then copy from the Result tab to another application such as Notepad as needed 6 You can remove a peak from the Spec Peak list by selecting a correlation in the results list then clicking Delete Selected Peaks from Spec List Displaying the To display the original labels select Peak Label from the original labels Peaks menu then deselect User Labels C C 14 Applied Biosystems Using the Polymer Analysis Toolbox C 6 Using the Polymer Analysis Toolbox Use the Polymer Analysis Toolbox to determine the following values that define the molecular distribution of a polymer M Number average molecular weight e My Weight average molecular weight e M _z average molecular weight e M M Polydispersity Index The Polydispersity Index represents how widely dispersed the polymeric distribution is A lower value for example 1 02 indicates a narrowly dispersed polymer A higher value for example 3 0 indicates a widely dispersed polymer Using Polymer 1 inthe Toolbox Palette dialog box click Polymer Analysis Analysis Toolbox T
147. automatic calibration calibration You specify auto calibration settings reference masses matching criteria and fit rejection parameters Auto calibration settings are saved as part of processing settings in a DAT file e If Auto Calibrate is enabled in the Data Explorer software Mariner data only or if the Automatic Calibration function is accessed by the Voyager Sequence Control Panel Voyager data only the software does the following for each spectrum it displays or processes e Compares peaks in the trace to peaks listed in the Masses to Match list in auto calibration settings 5 26 Applied Biosystems Automatic calibration for Mariner data Automatic Calibration e Matches all peaks that meet the specified Reference Matching criteria If the number of peaks matched is greater than or equal to the specified Minimum Number of Peaks to Match and the resulting fit errors are less than or equal to the specified Max Outlier Error calibration is successful e If any points exceed the specified Max Outlier Error the software eliminates the outliers one by one worst to best until all points are within the specified Max Outlier Error If the number of matching peaks falls below the Minimum Number of Peaks to Match when the software eliminates an outlier the calibration fails The Automatic Calibration function in Data Explorer is useful for quickly calibrating all spectra in a Mariner data file after you prep
148. bleshooting e Returning to the original spectrum Description The Advanced Baseline Correction feature corrects the baseline by e Iteratively estimating baseline amplitude at regularly spaced intervals throughout the spectrum e Smoothly connecting the calculated baseline points e Removing the calculated baseline from the spectrum IF BRBRPA Figure 5 15 Advanced Baseline Correction 5 48 Applied Biosystems Adjusting the Baseline When to use Use advanced baseline correction if you are analyzing data with an offset in the spectrum particularly data with a strong sloping baseline at low mass NOTE Because this function is iterative it may take several seconds to complete and typically takes longer for narrower peaks Correcting the To correct the baseline baseline 4 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 From the Process menu select Advanced Baseline Correction The Advanced Baseline Correction dialog box Figure 5 16 is displayed Advanced Baseline Correction Baseline Correction Baseline Correction Parameters Peak Width E 2 Flexibility 5 i Degree Figure 5 16 Advanced Baseline Correction Dialog Box Data Explorer Software User s Guide 5 49 Chapter 5 Examining Spectrum Data 3 Enter parameters as described below These parameters interact with each other and require experimentatio
149. calibration reference files are named with an REF extension When you create PSD calibration reference files include a _PSD suffix when you name files to help you distinguish them from non PSD calibration reference files Hint Calibration reference files are ASCII text files You can delete unwanted fragment ion entries using Microsoft Notepad Calibrating a PSD Spectrum 8 3 5 Changing the Precursor Mass When to change precursor mass When analyzing the composite spectrum you may find that the observed fragments and sequence are not consistent with the precursor mass used to acquire the DAT file For example you acquired the data with a precursor mass of 1 000 5 Da and while examining the data in Data Explorer you realize that the data may correspond to a precursor mass of 1 000 Da or 1 001 Da You can change the precursor mass in the Data Explorer software and regenerate the composite spectrum to observe whether the mass accuracy of the fragment ions improves with a different precursor mass specified Before changing the precursor mass note the following points e The Change Mass function changes the value for Mp in the PSD calibration equation described on page 8 6 It does not change the value for tp e Use the Change Mass function only if the precursor mass used to acquire the data does not correspond to the sequence you are observing in Data Explorer e Do not use the Change Mass function to correct
150. ce Chromatogram XAC Can be displayed as Intensity versus Retention Time or Spectrum number by selecting Traces from the Display menu then selecting X Axis In then selecting Spectrum Number or Time 1 12 Applied Biosystems Parts of the Data Explorer Window Table 1 3 Mariner Data Displayed in Chromatogram and Spectrum Windows Continued Window Mariner Data SPEC Displays the spectrum for the selected time in the TIC or TAC trace By default displays spectrum 1 The trace label includes DAD for spectra selected from TAC Indicates Base Peak BP mass and intensity for the tallest peak in the spectrum Displayed as Intensity versus Mass to Charge m z The right axis is scaled to the intensity of the base peak Table 1 4 Voyager Data Displayed in Chromatogram and Spectrum Windows Window Voyager Data CHRO Window is not displayed by default Optionally displays Total lon Current TIC for multiple spectra DAT files if you select Restore Chromatogram from the View menu NOTE DAD functions are not supported for Voyager data SPEC Depends on the type of data file you open e Single spectrum files Displays the spectrum and labels the plot as spectrum 1 e Multiple spectrum files Displays the spectrum for the selected time in the TIC trace if displayed By default displays spectrum 1 e PSD files Displays the composite spectrum and labels it as Stitched PSD
151. ces a can sequentially and NOTE If you overlay processed spectra and then display a different spectrum number the processed spectrum is lost For example assume you smooth Spectrum 3 in a trace then overlay it with another trace If you select Spectrum 3 then click to display Spectrum 2 then click to return to Spectrum 3 Spectrum 3 is no longer smoothed To set overlay attributes P 2 Display the individual traces to overlay Select Graphic Options from the Display menu The Graph and Plot Options dialog box is displayed NOTE If traces are overlaid when you select Graphic Options you can set attributes for the active trace only Click View Setup To apply the graphic options to all traces select the Use same settings for all traces check box NOTE If the Use same settings for all traces check box is selected when traces are overlaid the attributes are applied to the active trace only When you display individual traces again settings are applied to all traces Manipulating Traces NOTE You must select the Use same settings for all traces check box before selecting options for traces If you do not settings are applied only to the active trace 5 In View Setup select Overlay Trace scaling e Display Relative Autoscales each trace to the base peak in the display range BP Relative Autoscales each trace to the base peak in the trace e Absolute Value Ma
152. ch To clear the entire list click Delete Entire List 3 Complete the calibration as described in Solving and plotting on page 8 18 Data Explorer Software User s Guide 8 15 Chapter 8 Viewing Voyager PSD Data Selecting peaks For optimum mass accuracy select peaks as described in manually Selecting calibration peaks for optimum mass accuracy on page 8 19 To manually select the reference mass for a peak 1 Right click drag over the peak of interest The Reference Mass dialog box Figure 8 6 is displayed and lists all masses in the calibration reference file The entry highlighted is the nearest match in the calibration reference file to the selected peak that is within the mass tolerance specified Select or Create Reference Peak Information Es Nae Theoretical Mass Charge Elemental Composition 000 537 fi a pea ec Cancel Resolved Isotope Mass N Save s C Average Mass Charge ElementalComp 650 341000 Resolved y5 1 1 739 389000 Resolved a6 17 1 1 756 416000 Resolved a6 1 1 767 384000 Resolved b6 17 1 1 784 411000 Resolved b6 1 1 E 1001 000 Resolved a8 1 1 1046 542000 Resolved b8 H20 1 1 1165 591000 Resolved b9 1 1 1181 658000 Resolved p 9 1 1 1183 601000 Resolved b9 H20 1 1 1250 680000 Resolved al0 1 1 ZANRRNNN Resolved P 1 B Figure 8 6 Reference Mass Dialog Box 8 16 Applied Biosystems Calibrating a PSD Spe
153. ch spectrum in the data file is multispectrum data calibrated when displayed file 5 3 3 Creating or Modifying a Calibration Reference File REF This section includes e Definition e REF files provided e Calibration reference file contents e Creating and saving a calibration reference file e Modifying a calibration reference file e Specifying mass type for highly charged narrow peaks Data Explorer Software User s Guide 5 17 Chapter 5 Examining Spectrum Data 5 18 Definition REF files provided Calibration reference file contents Creating and saving a calibration reference file Applied Biosystems A calibration reference file REF is a list of masses and corresponding information from which you can select reference masses during calibration The following default calibration reference files are provided with the software e MARINER_POS REF e MARINER_NEGREF e VOYAGER REF e ANGIOTENSIN_FRAGMENTS REF e IMMONIUM_IONS REF A calibration reference file is a tab delimited text file that contains the following columns of information e Reference mass e Charge defaults to 0 if you do not specify e Type Resolved Average or Unknown defaults to Resolved if you do not specify Name optional e Elemental composition optional NOTE If you view the reference file in a text editor such as Notepad Editor the order of the information listed in the re
154. cid Limits Dialog Box Change the Min Number and Max Number for the compound for which you are setting limits then click OK Change limits for other entries as needed then click OK to return to the Elemental Composition Calculator Using the Isotope Calculator 6 2 Using the Isotope Calculator Description Use the Isotope calculator to generate a theoretical isotope distribution You can compare or overlay the theoretical distribution with your observed distribution Using the Isotope To use the Isotope calculator Calculator 4 Display the spectrum containing the observed isotope distribution 2 Click the Spectrum window to activate it 3 From the Applications menu select Isotope Calculator The Isotope Calculator dialog box Figure 6 9 is displayed Forma Eksais Forais F Eene O O Armoire Serpe Adal 2 hage urd Arp Cu Din OpY pir i Led Hai Phe DAA Segara jT a FI papara jas Oer CE x Aabi ia Ferien Feta Tye a rf w rE mti E idi C Sdiri Fiat PVE My Fimtan Tsp fi Cikaiiia aal Figure 6 9 Isotope Calculator Dialog Box Data Explorer Software User s Guide 6 13 Chapter 6 Using Tools and Applications calculate 4 Select the Formula or Sequence for the type of isotope to 5 Select a formula from the list or type in a new formula Valid entries for each formula type are Formula Type Valid Entries Elemental Any element from the Periodic Table
155. cluster If the expected higher theoretical peak masses and areas are not present in the peak list the peak in question is represented in the trace as a centroid bar with its original amplitude Figure 3 17 illustrates how peaks that are and peaks that are not part of an isotope cluster are represented in a deisotoped trace For most applications particularly peptide analysis the deisotoping function yields more useful results than monoisotopic peak filtering described in Section 3 3 4 Sorting Filtering and Printing the Peak List because the deisotoping function e Can successfully identify the monoisotopic peaks in overlapping clusters e Does not consider noise peaks that exhibit the mass but not the area of an expected isotope peak e Amplifies the intensity of monoisotopic masses at high m z due to contribution from other peaks in the cluster which allows the peak detection thresholds to eliminate chemical noise without eliminating the high m z peaks of interest enables improved peak matching in calibration and provides better results in database searching which relies on monoisotopic masses Deisotoping a Spectrum Peaks in original trace Deisotoped trace Increased amplitude of first peak indicates it is a monoisotopic peak If first three peaks are part of Peak that is not the same isotope cluster J pan ofcl sier Contribution to expected Increased amplitude of first isotope ratio peak indica
156. color for the active trace is saved 2 Click the window Chromatogram or Spectrum to print Data Explorer Software User s Guide 2 33 Chapter 2 Using Chromatogram and Spectrum Windows 3 To print with the x axis along the longest length of the paper select Print Setup from the File menu then select Landscape orientation NOTE The Landscape printing orientation you set in Data Explorer is lost when you close Data Explorer To permanently set the printer see Dedicating a printer to landscape orientation on page 2 35 4 From the File menu select a print option Print File with Instrument Settings Prints displayed trace windows from the active file along with the settings used to obtain the data e Print Spectrum or Chromatogram Trace Prints the active trace e Print Spectrum or Chromatogram View Prints all traces in the Spectrum or Chromatogram window Print All Views Prints traces for all open data files NOTE If you select Print All Views when more than two data files are open certain printers may not print the data file name To ensure data file names are printed print views individually or only open two data files before you select Print All Views 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 on certain printers See Section 1 4 Customizing the Data Explorer Window f
157. ctrum 2 Do any of the following e Click OK to accept the highlighted reference mass for matching e Select a different reference mass and click OK e Type new reference mass information in the Name Theoretical Mass Charge and Elemental Composition text boxes and select the mass type Click OK to accept the reference mass for matching NOTE You must type in a minus sign for negative charge states e Type new reference mass information in the appropriate text boxes click Save or Save As to add the information to the calibration reference file then click OK to accept the reference mass for matching The PSD Calibration dialog box is displayed again see Figure 8 5 on page 8 13 with the observed mass and the reference mass you selected displayed in the Matched Peak list 3 Repeat step 1 through step 2 until the desired peaks are in the matched list For optimum mass accuracy take at least one mass from each segment if possible The software does not allow you to add duplicate masses collected with the same PSD Mirror Ratio However if a mass exceeds the Mass Tolerance specified in Reference Matching Criteria but has the same PSD Mirror Ratio the software allows you to add it 4 Complete the calibration as described in Solving and plotting on page 8 18 Data Explorer Software User s Guide 8 17 Chapter 8 Viewing Voyager PSD Data Solving and plotting Applying new constants
158. ctrum in the data file Displaying To display the peak list select Output Window from the View menu then click the Chro Peak List or Spec Peak List tab at the bottom of the window Figure 3 14 Data Explorer 0914neuro_frag001 dat ioj x File Edit View Display Process Peaks Tools Applications Window Help e Se n7 i eins 8 tek lmeo oe e aee CHRO 0914neuro_frag001 dat 2 H 12 4 18 6 a Spectrum Number MSPEC 0914neuro_frag001dat oj x 2 Spec 1 gt SM5 BP 33 gn i Z amp 100 1490317 338 3388 i 837 40292 6 71 990 279 2 459 4 639 6 819 8 1000 0 Click the Peak List tab Mass mz at the bottom of lu SPEC 091 Ln CHRO 091 the Output window 107 10132 109 11173 T 1120171 For Help press F1 Figure 3 14 Peak List in Output Window Data Explorer Software User s Guide 3 37 3 Chapter 3 Peak Detection and Labeling Contents of peak list Chromatogram peak list Spectrum peak list 3 38 Applied Biosystems The peak lists contain the following information Each entry represents one chromatographic peak in the trace and includes Index a sequential number assigned to each entry in the peak list Spectrum or retention time minutes determined by the command selected from the Display menu Lower bound spectrum or minutes Upper bound spectrum or minutes Peak height calculated relative to zero Peak area cal
159. culated relative to local baseline Each entry represents one spectral peak in the trace and includes Index a sequential number assigned to each entry in the peak list Centroid or Apex mass reflects the Mass Type selected in the Peak Label dialog box Lower bound m z Upper bound m z Charge z Peak height Relative intensity calculated relative to zero not to baseline Peak area calculated relative to local baseline Peak List 3 3 2 Inserting Peaks in the Peak List Description If chromatogram or spectrum peaks are not detected and labeled by the selected detection parameters you can manually detect and label peaks by inserting peaks in the peak list Procedure To insert peaks 1 Display the chromatogram or spectrum trace of interest 2 From the Peaks menu select Insert Peaks The Insert Peaks dialog box is displayed Figure 3 15 Figure 3 15 Insert Peaks Dialog Box 3 To enter the Left Edge and Right Edge of the peak to insert right click drag over the region of the trace to calculate or type in masses 4 Click Calculate The peak is inserted in the peak list displayed in the Peak list tab in the Output window and centroid information is listed in the Result tab For more information see Output window on page 1 15 Data Explorer Software User s Guide 3 39 3 Chapter 3 Peak Detection and Labeling Inserted peaks are Removed from the list when you close th
160. d Color NOTE For consistency all Mariner and Voyager screen examples in the following sections of this User s Guide are shown with a white background 1 4 Applied Biosystems File Formats and Types 1 2 File Formats and Types This section describes e Software applications compatibility e Data DAT file format 1 2 1 Software Applications Compatibility You can use the Data Explorer Macro Recorder function to create Visual Basic scripts to automate tasks You can also use the Visual Basic Editor directly to create more complex programs customized to suit your needs For more information see Section 6 7 Using the Macro Recorder Additionally you can convert data to ASCII format for import into other software applications or import ASCII results For more information see Section 1 6 3 Converting to and Exporting ASCII Data and Section 1 6 4 Importing a Trace in ASCII Format 1 2 2 Data DAT File Format DAT file format Data generated by Mariner and Voyager systems is stored in DAT file format The DAT file format incorporates all information about how a data file was acquired and processed into a single file This format improves data processing and data storage efficiency Data files can contain spectra from a single acquisition or from multiple acquisitions for example multiple spectral data from a Voyager acquisition or multiple injection results from a Mariner CombiSolv run Data Explorer So
161. d area of the peak from the left to the right intersection of the centroid baseline then dividing by the area of the same region Data Explorer Software User s Guide 3 69 Chapter 3 Peak Detection and Labeling Integration The Data Explorer software integrates chromatographic and spectral peaks to calculate the peak area using one of two methods to determine the peak baseline Figure 3 25 e Valley to baseline Drops a vertical line from all valleys to a horizontal baseline The level of the horizontal baseline is determined using the minimum peak valley point left or right for each peak Valley to valley Forces a baseline to all valley points 0 02 0 01 0 00 1 0 1 6 2 0 1 0 15 20 Valley to Baseline Valley to Valley Figure 3 25 Valley to Baseline and Valley to Valley Integration 3 70 Applied Biosystems Default Peak Detection Settings 3 7 Default Peak Detection Settings Default SET files provided This section includes e Default SET files provided e Additional Voyager SET files provided Default peak detection settings are contained in the following SET files e MARINER SET e VOYAGERLINEAR SET e VOYAGERREFLECTOR SET e VOYAGERPSD SET The following table lists the default settings in SET files provided for chromatograms Chromatogram Settings in All SET malemetet Files Mariner and Voyager Basic Settings chromatogram data Detection Range
162. d by Default Data Explorer Software User s Guide 2 5 Chapter 2 Using Chromatogram and Spectrum Windows The data is labeled accordingly Type of Data Spectrum Trace Labels Mariner data If you open a data file you previously calibrated in Data Explorer all spectra in the data file are calibrated and displayed with an MC or AC trace label If AutoSaturation Correction is turned on all spectra in the data file are corrected and displayed with an ASC trace label For more information see Section 5 11 AutoSaturation Correction Mariner Data Only Voyager data If you open a data file you previously calibrated in Data Explorer the spectrum is calibrated and displayed with an MC trace label For multispectrum data files you can display chromatograms For more information see Section 2 1 3 Displaying Voyager Chromatograms Automatically You can set macros to automatically run when you open or running macros close files For information see Running Macros Automatically When Opening and Closing Files on page 6 45 2 1 2 Displaying Mariner DAD Traces When you use a diode array detector DAD to acquire Mariner data you can display the following types of DAD traces e DAD chromatogram With the Chromatogram window active select Traces from the Display menu then select DAD TAC or DAD Channel e DAD spectrum Double click a DAD chromatogram For more information see Types o
163. described in the following sections Click OK Setting Graphic Options View Setup Graph 1 Setup Spec 1 BP 3752 3 512 m Graph Title Setup _ Axis Title Setup Font EME Color lightRed v Size 12 a Eont faia IV Bold F Italics T Underline Size fio a Line or vertical Plot Setup Bold I Italics I Underline bar traces i 5 a m Axis amp Peak Bound Setup Pas Peak Line Type Lines z F Show Bight Y Axis Line width p jing wich ps bounds Trace jo J Line wieth fO Trace Color Bue m Axis Tick Label Setup p Peak Label Setup I Show Tick Labels Color Red bd Color eack Eont arial Eont faia Size fio Size fo g T Bold T Italics T Underline Bold Italics I Underline m Data Cursor Data cursor a Show Data Cursor Line Style Dot z Type Color Medium Gray Y Y Label Font arial XX Label Absolute Size 10 z Width DE sat ete el peering G raph IC Graphic Compression compression Local Max C Average C Sum Cancel Apply Figure 1 4 Graphic Options Dialog Box Graph Setup Tab Setting colors You can set colors manually or automatically Manually To manually select the color of graph features axis peak bounds tick labels data cursor and plot features traces peak labels 1 Select Graphic Options from the Display menu 2 Set colors in the Graph Setup tab see Figure 1 4 Data Explorer Softwar
164. dow Click in the toolbar NOTE You can also add traces by selecting Add Remove Traces from the Display menu The software adds a trace to the window You can add up to seven traces Figure 2 8 Hint Resize the window to view all added traces Manipulating Traces ae f 100 Original trace 0 9976 z1 0 2 911744 1 9695 20 1555 Intensi NeB2e sees Spec 1 ASC MC BP 558 3 938 0 3317 z 52 9372 166 9990 23 2397 z4 0 4232 938 4 99 0 279 2 100 805 404 204 Intensity aa 459 4 y 639 6 Not Used 819 8 0 1000 0 100 0 T 99 0 279 2 100 804 Added traces 404 204 Intensity 459 4 639 6 Not Used 819 8 0 1000 0 100 0 T 99 0 279 2 100 804 605 405 205 Intensity 459 4 639 6 Not Used 819 8 0 1000 0 100 0 99 0 279 2 459 4 639 6 Mass m z 819 8 0 1000 0 Figure 2 8 Adding Traces Four Traces Shown up to Four More Can Be Added When you perform a function that adds a new trace the label of the trace changes from Not Used to the label for the type of trace created Figure 2 9 Data Explorer Software User s Guide 2 19 Chapter 2 Using Chromatogram and Spectrum Windows Figure 2 9 shows the original trace and three added traces that now contain a smoothed spectrum SM a centroided spectrum CT and a baseline offset spectrum BO Spec 1
165. e Extracted ion chromatogram CNL Context sensitive menus 1 14 Converting see also Exporting DAT to ASCII 1 34 profile data to centroid 1 33 SPC to DAT 1 30 Voyager MS files 1 30 Copying all peaks 1 40 apex mass list 1 41 centroid mass list 1 41 data from other data files 2 37 data to Windows clipboard 1 38 displayed peaks 1 39 mass list 1 41 peak list 1 40 results 2 28 to metafile 1 38 trace data 1 39 2 37 trace image 1 38 Correcting baseline chromatogram 4 29 spectrum 5 47 spectrum advanced 5 48 Correlation factor noise filtering 4 18 5 43 CT file suffix 1 33 in spectrum header 2 32 5 36 CTS files 1 7 disabling 2 23 purging 2 23 Cursors see Data cursors 1 27 Custom labels see Graphic options peak labels customizing see Peak labels custom Customer feedback Technical Publications e mail address xiv Customizing colors text and line width 1 25 creating your own software applications 1 5 data compression 1 28 Data Explorer window 1 17 defaults 1 17 graphic options 1 24 peak labels 3 52 processing settings 1 17 Data Explorer Software User s Guide Index 7 Customizing continued SET files 1 17 toolbars 1 22 D DAD displaying Channels 1 12 4 2 displaying TAC 1 12 displaying traces 2 6 extracted absorbance chromatogram XAC 4 13 in spectrum header 2 32 spectrum displaying 4 2 5 2 TAC in chromatogram header 2 30 XAC in chromatogram header 2 30 Dark background changing colors 1 20 d
166. e SET file then click OK For information see Section 1 4 2 Customizing Processing and Graphic Settings SET Data Explorer Software User s Guide 1 37 Chapter 1 Data Explorer Basics Saving LBS To save spectrum LBS or chromatogram LBC peak label and LBC files files froma DAT RSD or RSC file see Section 3 5 3 Setting Custom Peak Labels 1 6 6 Copying from Data Files Overview You can copy the following types of data from data and result files to the Windows clipboard Trace lmage Copies the graphic image of the trace in the active window e Trace Data Copies the raw data for the trace displayed in the active window You can also use this command by right clicking then selecting Copy Trace Data For more information see Section 2 5 2 Copying Traces from Multiple Data Files to a Window e Displayed Peaks Copies peak list entries for the peaks displayed in the active window e All Peaks Copies peak list entries for all peaks in the active view e Mass List Copies all centroid or apex masses from the peak list for the active Spectrum window Copy trace image To copy the trace as it is displayed in the active window to the Windows clipboard 1 Select the trace window to copy Zoom and adjust the trace as needed 2 From the Edit menu select Copy then select Trace Image 3 Paste the image into an application that handles Windows Metafile format images for example Microsoft
167. e User s Guide 1 25 Chapter 1 Automatically using Auto Color 1 26 Setting line widths Applied Biosystems Data Explorer Basics When you manually set colors note 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 when you close the data file Automatically assigning trace colors is useful when overlaying traces To automatically assign trace colors 1 Select Graphic Options from the Display menu 2 Select Auto Color in the View Setup tab The software assigns and displays trace colors when the traces are overlaid When you use Auto Color e The active trace color stays at its original setting e Other trace colors are set based on the active trace color For example if the active trace is yellow other traces are assigned the colors pale blue pale green and medium gray which are the colors listed after yellow in the Trace color list excluding white NOTE White is not used in Auto Color because white may not print on certain printers You can control trace appearance by setting line widths To set line widths 1 Select Graphic Options from the Display menu 2 Set line width in the Plot Setup section of the Graph Setup tab see Figure 1 4 on page 1 25 NOTE Line Widths of 0 or 1 or lines set to the color white may not print on certa
168. e data file reprocess the data or set peak detection parameters so that the inserted peak is no longer detected e Assigned a charge state of 0 to indicate the charge state is unknown CAUTION A zero value in the Spec Peak list does not indicate a charge state of zero It indicates that the software could not determine the charge state 3 3 3 Saving the Peak List 3 40 Saving asa stand alone PKT file Applied Biosystems You can save the contents of the chromatogram and spectrum peak lists as stand alone peak list files PKT Stand alone peak list files can be used in other applications such as Microsoft Notepad Editor or Microsoft Excel To save a peak list as a stand alone PKT file for use in other applications 1 Click the trace of interest 2 Display the peak list of interest in the Output window by clicking the Chro Peak List or the Spec Peak List tab 3 Right click the Output window then select Save As The Save Peak Table As dialog box is displayed 4 Select a directory and type a file name 5 Click Save The software automatically assigns a PKT extension and saves the peak list as a tab delimited text file Importing and saving in Excel Peak List NOTE Inserted peaks are included when you save a PKT file NOTE Peak list headings are not included when you save a PKT file If you require headings copy the peak list directly to Excel instead of saving it as a PKT file
169. e items with the same m z value to the reference file if any other attribute of the reference compound is different for example charge state or name Each mass in the list is considered during calibration If the mass list contains duplicate entries the calibration may return an invalid number of matches 6 To modify an entry click the reference mass in the list to select it modify the entry as needed then click Update The modified entry is saved in the REF file 7 Click Save 8 Inthe Save As dialog box select a location and type a name for the file then click OK The name of the reference file used for mass calibration is stored in the DAT file and is displayed in the Calibration Reference File field when you open the Manual Calibration dialog box again NOTE Only the name and path of the reference file are stored in the DAT file Contents of the file are not stored Modifying a Hint You can also modify a calibration reference file when calibration you manually select reference masses during calibration reference file See step 8 on page 5 11 To add or delete information in a calibration reference file 1 From the Process menu select Mass Calibration then select Edit Create Reference File The Edit Create Reference Peak Information dialog box Figure 5 6 on page 5 19 is displayed 2 Click Browse then select a calibration reference file 5 20 Applied Biosystems Specifying mass ty
170. e list multiple times Hint A calibration reference file called Immonium_lons REF is provided with the software To optimize mass accuracy across the entire PSD composite spectrum multi point calibration e Select reference peaks from a wide range of segments to ensure that high and low Mirror Ratios are represented Include peaks with masses that are substantially below the Max Stitch Mass for example up to 50 percent lower than the Max Stitch Mass e Avoid selecting peaks with masses that are above the Max Stitch Mass e Avoid selecting peaks with signal to noise ratios less than 20 Data Explorer Software User s Guide 8 19 Chapter 8 Viewing Voyager PSD Data 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files 8 20 Creating PSD CAL files Applying new constants to additional files Applied Biosystems To generate a PSD CAL file T Acquire a standard for example angiotensin in the Instrument Control Panel in PSD mode For more information see the Voyager Biospectrometry Workstation User s Guide Open the DAT file in the Data Explorer software Calibrate as described in Section 8 3 2 Calibrating From the File menu select Export then select Calibration Name and save the file Hint Include a_PSD suffix when you export a PSD CAL file to help you distinguish them from non PSD CAL files For example type Cal_PSD as the file name The c
171. e to view from the Window menu e Use tabs e Use the Activate File dialog box Using tabs To move between open files using tabs click the tab at the bottom of the Data Explorer window Figure 2 4 to activate the file INCHRO We k MSPEC Neu F llel Es UycHRo eur MISSREC Neu Ly CHRO cyt j SPEC cyte For elp press F1 Click a tab to Double click select an open data file an active title bar to maximize a window Figure 2 4 Tabs for Open Data Files If the window for the tab you click is already open clicking the tab activates the window If the window for the tab you click is minimized clicking the tab activates the title bar for the window Double click the active title bar to display the window Using Activate To move between Spectrum windows of open data files using File Activate File 1 Select Activate File from the Windows menu 2 Select a file from the Current Data File list 2 8 Applied Biosystems Opening and Closing Data Files Select File to Activate x Current Data Files h 7 different samples 34008 dat h 7 different samples 34009 dat h different samples 34010 dat h different samples 34011 dat h 7 different samples 34012 dat rm 6 C Maximize Activate oe Figure 2 5 Select File to Activate Dialog Box 3 Select Maximize To maximize the Spectrum window of the selected file Activate To activate the Spectr
172. eak yields a high default threshold and many peaks in the spectrum are not detected Intensity Spec 1 BP 1018 0 64531 6 5E 4 3772 607 2973 632 3481 4303918 423 0 1900 2800 3700 4600 5500 Mass m z Figure 5 18 Complete Spectrum with Low Mass Gate Spike Figure 5 19 shows the spectrum truncated to eliminate the Low Mass Gate spike The peak at 3772 Da is identified as the base peak the Base Peak Intensity is set to a lower value and more peaks in the spectrum are detected Data Explorer Software User s Guide 5 57 Chapter 5 Examining Spectrum Data Spec 1 gt TR BP 3771 9 17814 Ps 3772 607 1 8E 4 2973 632 3481 430 3918 423 1175 558 1671 727 Intensity r 4137 174 0 1001 0 1900 8 2800 6 3700 4 4600 2 5500 0 Mass m z Figure 5 19 Truncated Spectrum Low Mass Gate Spike Eliminated 5 58 Applied Biosystems Converting to a Singly Charged Spectrum Mariner Data Only 5 10 Converting to a Singly Charged Spectrum Mariner Data Only Description Requirements Converting NOTE Single charge conversion is not supported for Mariner DAD data The Single Charge Conversion function generates a theoretical centroided singly charged spectrum This function uses isotopic spacing in detected spectral peaks to generate the theoretical spectrum For example if you use this function on a spectrum that includes 3 peaks the
173. ection parameters spectrum 3 26 saving and restoring 1 20 saving for use with other data files 1 19 Profile data converting to centroid 1 33 traces do not print 1 26 2 35 Properties see File properties Protein database search macro C 18 Proteins detection 3 6 Proton mass value 3 32 PSD analysis see also Calibration mass PSD Voyager data only see also Voyager Biospectrometry Workstation Users Guide CAL file creating 8 20 calibrating 8 10 Change Mass function 8 23 Data Explorer Software User s Guide Index 21 PSD analysis continued composite spectrum displaying 8 2 8 5 composite spectrum how it is generated 8 6 fragment labels applying 8 8 optimum resolution observed near Max Stitch Mass 8 4 peak detection algorithm 3 5 peak detection checking 8 10 Peptide fragmentation macro C 9 precursor mass changing 8 23 PSD overview of creating 8 10 REF file creating 8 21 segment labels applying 8 8 segment traces displaying 8 3 PSD calibration equation 8 6 Precursor mass impact of changing 8 23 PSD data in Voyager displaying 1 13 PSD mode constants 8 6 equation 8 6 R Raw data copying x y pairs 1 39 RCD file deleting 2 39 description 1 10 exporting 2 39 extracting information from 1 36 opening 2 39 RCT files see also Results name of raw data file result is derived from 2 38 2 39 2 41 opening 2 39 2 40 saving 2 40 Read only files viewing 2 7 Index 22 Applied Biosystems Realign UV t
174. ed NOTE For information on acquiring PSD spectra see the Voyager Biospectrometry Workstation User s Guide Displaying the NOTE You cannot display the PSD data you are currently composite acquiring in the Instrument Control Panel in the Data spectrum Explorer software until you stop the PSD experiment in the Instrument Control Panel Error messages are displayed if you try to open the file For more information see the Voyager Biospectrometry Workstation User s Guide To display PSD data 1 Open the PSD data file of interest in the Data Explorer software as described in Section 2 1 Opening and Closing Data Files NOTE PSD data files are named with a DAT extension When you acquire PSD data files include a _PSD suffix when you name PSD data files for example Test_PSD to help you distinguish them from non PSD data files The composite spectrum is generated as described in How the composite spectrum is generated on page 8 6 and displayed with a Stitched PSD trace label Figure 8 1 8 2 Applied Biosystems Displaying PSD Data Date Explorer p 2ar_psd0001 dat _ Oj x File Edit View Display Process Peaks Tools Applications Window Help jsajjar iss stem leoe oe aeee Em ASPEC p12ar_psd0001 dat Stitched PSD BP 1404 7 65142 1404 655 1212 845 194 288 1017 978 359 949 Intensity T SPEC pia For Help press F1 Figure 8 1 PSD Spectrum
175. ed Managing Files To check that the SPC and CGM files are in the same directory 1 3 Select Open from the File menu The Open dialog box is displayed From the Files of type drop down list select All Files A list of all files contained in the directory is displayed Check that the SPC and CGM files are present In the Data Explorer window 1 2 3 Open or click the SPC file to convert Select Convert from the File menu Select New Data Format The Convert to DAT Format dialog box is displayed Figure 1 5 Convert to DAT Format x Do you want to convert C Mariner Debbie 41001 spc to new format DAT Set Property Cancel Figure 1 5 Convert to DAT Format Dialog Box To add file property information for example Title Author or Keywords to the file click Set Property enter the appropriate information then click OK For more information see Viewing file properties on page 1 32 Data Explorer Software User s Guide 1 31 Chapter 1 Data Explorer Basics The Convert to DAT Format dialog box reappears 5 Click OK A message box is displayed showing the file name of the newly created DAT file 6 Close the SPC file and open the DAT file before processing If desired manually delete the SPC file and associated files Viewing file File properties are accessible in Windows NT Explorer and properties provide general information about a data file
176. ed Voyager data file is displayed in the Data Explorer window This data file and other recently acquired data files are listed in the Data Explorer File menu the next time you open Data Explorer Opening data files You can open up to eight data files using the File Open dialog with File Open _ box 1 Select Open from the File menu The Select Files dialog box Figure 2 1 appears 2 2 Applied Biosystems Opening and Closing Data Files Lowa Fe j Dabrg aiad s ol mmj w pram l bas ja Wicara icto at Fei oem Odatga Fi a ape aj Fameg Gape md hamog Teig Va pe Teig bra lh Pale C kelasi paip C tir Sasmi b File ail Tiri aqha shear lien ae operat Figure 2 1 Select Files Dialog Box 2 Click the down arrow to display the Files of type list then select the file extension to display 3 Select up to eight data files to open then click Add or Add All Add All is not active if the number of selected files exceeds eight NOTE You can also select files by double clicking the file name in the file list The files are listed in the Files Selected box Displaying 4 To display acquisition comments before opening the data acquisition file right click a file name in the top pane of the dialog comment box select Properties then click the Summary tab Data Explorer Software User s Guide 2 3 Chapter 2 Using Chromatogram and Spectrum Windows Specifying settings 5 Select a Restoring
177. eded 4 Click the first trace in the Spectrum window to make it the active trace NOTE The Dual Spectral Trace Arithmetic command is dimmed unless the first trace in the Spectrum window is the active trace 5 From the Process menu select Dual Spectral Trace Arithmetic The Dual Spectral Trace Arithmetic dialog box is displayed Figure 5 23 5 64 Applied Biosystems Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files Dual Dual Spectral Trace Arithmetic 2 x m Mass Tolerance C Dalton fo ppm fio Operation Selection Type Add Result Trace Placement AddNew Trace Replace Active Trace OK Cancel Figure 5 23 Dual Spectral Trace Arithmetic Dialog Box 6 Setthe Mass Tolerance within which data points from the different traces will be considered as the same mass 7 Select Add or Subtract for Operation 8 Select Add New Trace or Replace Active Trace for the result trace 9 Click OK Absolute intensities of the two traces are added or subtracted and the result trace is displayed with ADD or SUB in the trace header Data Explorer Software User s Guide 5 65 Chapter 5 Examining Spectrum Data 5 66 Applied Biosystems 6 Using Tools and Applications This chapter contains the following sections 6 1 Using the Elemental Composition Calculator 6 2 6 2 Using the Isotope Calculator
178. efault settings 1 23 SET file 1 19 DAT format converting DAT file to ASCII 1 34 converting from SPC 1 30 converting profile to centroid 1 33 extracting information from 1 6 1 36 file properties adding to DAT file 1 31 file properties searching 1 32 file properties viewing 1 32 information stored in 1 6 overview 1 5 results saving opening deleting 2 38 Data cursors Base Peak Intensity spectrum 3 23 displaying retention time on 1 27 horizontal 1 27 labels 1 27 4 2 printing and suppressing 1 27 Index 8 Applied Biosystems Data cursors continued turning on and off in graphic options 1 27 turning on and off in peak detection 3 23 vertical 1 27 Data Explorer moving between open files 2 8 starting and exiting the software 1 3 window customizing 1 17 window parts of 1 11 Data Explorer examples Mariner data 7 2 Voyager data 7 11 Data Explorer Toolbox accessing C 4 Ladder Sequencing macro C 2 C 5 modifying C 2 MS Fit MS Tag macro C 2 overview C 2 Peptide fragmentation macro C 2 C 9 Polymer analysis macro C 2 C 15 preparing data for C 3 Visual Basic References required C 3 Data file activate 2 8 cannot find 9 3 closing 2 10 closing and running a macro automatically 6 45 comparing open files 2 36 2 38 converting from profile to centroid 1 33 converting to ASCII 1 34 copying traces from 2 37 decreasing size 1 33 default settings applying 2 4 error message displayed when opening PSD 8 2 9 3 Data file
179. eight 3 55 3 56 horizontal 3 55 3 58 immonium ions C 9 manually applying 3 39 3 52 manually inserting peaks 3 39 monoisotopic 3 43 not displayed 3 59 3 66 9 14 9 15 overlapping 3 55 3 58 peak start and end displaying 3 55 3 58 spectrum number 3 55 3 56 spectrum setting 3 56 time 3 55 3 56 troubleshooting 9 14 turning on and off in chromatogram 3 52 user defined 3 61 vertical 3 55 3 58 Vial number 3 55 Peak labels charge state filtering 3 43 incorrect 9 20 not displayed 9 19 parameters used 3 53 requirements 3 53 selecting 3 58 user labels 3 62 Index 20 Applied Biosystems Peak labels filtering charge state 3 43 monoisotopic peak 3 43 Peak labels Mass Difference From Adjacent Peak regular labels 3 57 From Adjacent Peak user labels 3 62 From Selected Peak 3 57 Peak list charge state displaying selected 3 42 Chro and Spec 3 40 contents 3 38 copying apex masses only 1 41 copying centroid masses only 1 41 copying to Windows clipboard 1 40 copying with headings 1 40 copying without headings 3 41 deleting peaks 3 44 3 59 description 3 37 displaying 3 37 3 40 filtering 3 43 importing and saving in Excel 3 41 inserting peaks manually 3 39 monoisotopic peaks displaying 3 42 printing 3 44 saving as a file 3 40 sorting 3 42 when created 3 37 zero charge state displayed 3 43 Peak matches clearing list 5 13 8 15 sorting 5 11 Peak matching automatic calibration 5 32 manual calibration 5
180. elected above The software includes these other peaks in the calculation to improve signal to noise ratio and the accuracy of the calculation e Regardless of mode the software uses all data points in this range to construct the deconvoluted spectrum 9 Select or enter the mass of the adduct ion to use in the calculation The mass of a proton H is selected by default 10 To display both the zero charge spectrum trace and the deconvolution results select Generate Zero Charge Spectrum To display only the deconvolution results do not select Generate Zero Charge Spectrum Data Explorer Software User s Guide 5 39 Chapter 5 Examining Spectrum Data 5 40 11 12 13 Click OK The result is displayed in the Output window and the zero charge spectrum is displayed with a DECONV trace label if selected NOTE The numerical result displayed in the output window generally is more accurate than the computer generated spectrum To return to the original spectrum click in the toolbar The number of the original spectrum is displayed in the Select Spectrum dialog box Click OK Converting to To convert to a zero charge spectrum zero charge 2 Applied Biosystems Activate the Spectrum window Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing From the Process menu select Multiple Charge then select Convert to Zero Charge
181. eliminating 5 12 importing Voyager requirements 5 25 multi point calibration 5 8 overview 5 5 peak matches clearing list 5 13 peak matches sorting 5 11 peak matching automatic 5 6 peak matching manual 5 6 peak weighting factor 5 10 procedure 5 8 results 5 13 reverting to instrument calibration 5 22 single point calibration 5 8 troubleshooting 9 9 when to use 5 6 Index 4 Applied Biosystems Calibrating mass PSD Voyager data only all segment masses considered 8 15 calibration reference file REF 8 21 constants applying A and B to new file 8 20 error message when selecting calibration reference file 8 14 internal calibration using known monoamino acid fragment ions 8 10 internal calibration values used 8 7 multi point calibration 8 10 peak detection checking before calibrating 8 10 peak matches clearing list 8 15 peak matching automatic 8 15 peak matching manual 8 16 peak weighting factor 8 14 Precursor mass changing 8 23 procedure 8 12 results 8 18 reverting to instrument calibration 5 22 selecting peaks for optimum mass accuracy 8 19 selecting peaks near Max Stitch Mass 8 19 single point calibration 8 10 Calibration see Calibrating mass Calibration constants see also Calibrating mass applying to new file 5 16 8 20 calculating 5 34 displayed in Output window 5 14 8 18 exporting 1 36 extracting from DAT file 1 36 importing from another source 5 16 8 20 PSD 8 20 Centering a peak 2 15
182. en Opening and Closing Files aeee 6 45 Data Explorer Software User s Guide vii Table of Contents Chapter 7 Data Explorer Examples 7 1 7 2 Mariner Data Examples esr esisin ANE ARAA AR 7 2 7 1 1 Improving Signal To Noise Ratio seeen 7 2 7 1 2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks 7 4 7 1 3 Determining if a Peak is Background Noise ssec 7 8 Voyager Data Examples ccccsceeece eee eeee eee REEERE AINE EKARA 7 11 7 2 1 Detecting and Labeling Partially Resolved Peaks 0 7 11 7 2 2 Processing Before Calibrating to Optimize Mass Accuracy 7 14 7 2 3 Detecting Peaks from Complex Digests csceeeeees 7 18 Chapter 8 Viewing Voyager PSD Data 8 1 8 2 8 3 Displaying PSD Data iiis nieren siete ata aa ela eei iene ta ee eee 8 2 Applying Fragment Labels eeeeeee eee eee ee erence eee tees eeeeeeeeeeees 8 8 Calibrating a PSD Spectrum c eee ee cece eee ence eee eee ee eee eeeeeeeneee 8 10 8 3 1 Checking Peak Detection cccceeeececeeeeeeeeeeteeeteeteeeeeeeeeeees 8 11 8 3 2 GaliDratinG sisses noirm n a a a aa oia aeania 8 12 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files 0 eeceeeeeeeeeeeee eee rennene 8 20 8 3 4 Creating PSD Calibration Reference REF Files 8 21 8 3 5 Changing the Precursor MASS eeeeeeeeeeeeeeeeeeeeeeeaeaaes 8 23 Chapter 9 Troubleshooti
183. erence From Selected Peak then specify the mass of the intact molecule For more information see Setting spectrum labels on page 3 56 Using the Elemental Composition Calculator Procedure To determine elemental composition 1 Display the spectrum containing the peak of interest 2 Click the Spectrum window to activate it 3 From the Applications menu select Elemental Composition The Elemental Composition Calculator dialog box Figure 6 1 is displayed blan Indore Taga Har tchag Ae EE T a Toke P Peale om AH 1D Mati Tape F Hea arta Wise Peak Appin F Rend Peni Te ET COT E ET djrarend Passera tow Peete Ekren Loman ae ee Figure 6 1 Elemental Composition Calculator Dialog Box Data Explorer Software User s Guide 6 3 Chapter 6 Using Tools and Applications 4 Enter m z values in the m z ratio list by doing any of the following e Right click drag over a peak in the spectrum to add the m z and the associated charge state e Double click the line to display the Elemental Target Mass dialog box type the m z and the associated charge state then click OK to add a line to the list then double click the line to display the Elemental Target Mass dialog box type the m z and the associated charge state then click OK 5 Specify the Tolerance to use The Tolerance value entered is directly used as the window for elemental comparisons and is also multiplied by 2 and u
184. ering then select the tags to display 4 26 Applied Biosystems Adjusting the Baseline 4 7 Adjusting the Baseline This section includes Using Baseline Offset Using Baseline Correction 4 7 1 Using Baseline Offset Use the Baseline Offset command to offset the y axis in a chromatogram to improve the appearance of a trace or correct a sloping baseline To use Baseline Offset 1 Activate the window on which you want to perform the offset NOTE You can select a Chromatogram or Spectrum window If you do not activate the correct type of window before performing the next step the software does not select values when you click drag on the trace For example you must select a Chromatogram window before starting baseline offset on a chromatogram Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing From the Process menu select Baseline Offset The Baseline Offset dialog box Figure 4 12 is displayed Data Explorer Software User s Guide 4 27 Chapter 4 Examining Chromatogram Data 4 28 Applied Biosystems Baseline Offset xj Baseline Selection Left Baseline Right Baseline fan 68 0 95 T Only apply from L to R Baseline midpoint OK Cancel Figure 4 12 Baseline Offset Dialog Box Right click drag the left baseline to offset The selected value is displayed in the Left Baseline field Right click drag the right baseline to offs
185. es Displaying user labels Peak Labeling You can apply labels you previously created and saved in LBC or LBS files Open the LBC or LBS file by clicking H in the User Label Setup dialog box Select the file then click Open The labels are imported into the DAT file and displayed in the label list You can modify the settings delete or add labels and save the changes in a new LBC or LBS file NOTE Changes you make to user labels imported into a DAT file are not saved in the LBC or LBS file from which they originated Click Save As then resave the file if you want the changes saved to the originating file Select both Enable Labeling and User Labels in the Peak Label dialog box to display user labels Peaks that do not meet the peak detection criteria are not labeled NOTE If you specify user labels with an X Tolerance that is outside the acquisition range for the data file the user labels are not displayed Be aware that the default X Tolerance of 1 may be outside the acquisition range when the chromatogram is displayed in retention time Data Explorer Software User s Guide 3 65 Chapter 3 Peak Detection and Labeling User labels not ifa user label is not displayed or is displayed incorrectly displayed possible causes are e User labels are not enabled e The peak does not fall within the specified Tolerance The label is too long or peaks are too close together If there is not en
186. es Continued Category File Type File Content Process RCD Chromatogram results file exported from DAT files See continued Section 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only RSD Spectrum results file exported from DAT files See Section 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only 1 10 Applied Biosystems Parts of the Data Explorer Window 1 3 Parts of the Data Explorer Window This section describes e Overview Toolbar e Chromatogram and Spectrum windows e Tabs for data files e Data file names e Output window Overview Figure 1 3 shows the Data Explorer window with Mariner data lb Bate apforer 091 4neuro_fragO01 dat ioj x File Edit View Display Process Peaks Tools Applications Window Help Isuse lam wise tek llewe ee Bete lee ef i Toolbar McHrRo 091 4neuro_frag001_dat Chromatog ram ee 9010 2 window 50 l i 80 8 6 92 9 8 10 4 11 0 Spectrum Number Data file name BRE gt Spec 1 AS Moricas 558 3 696 Ne 219 9381 23 _ 696 4 Spectrum SONG 25153 14 9806 103 1656 0 5239 22 window 2 N 279 2 459 4 639 6 819 8 1000 0 o Mass m z Tabs for Ji mecka S open data files Resolution at mass 558 27740 is 3497 for Spec 1 AS MC gt BC gt SM7 Output lt lt noi4neuro fragO01 dat gt gt lt 1 gt Tet
187. escribed on page 3 28 to detect peaks for the selected detection range Enable BP Intensity Threshold Cursor When enabled allows you to click drag the cursor to set the Base Peak Intensity Threshold Peak Detection Use Resolution Dependent Settings not available for Voyager PSD data When selected the software automatically determines detection ranges and uses an appropriate Filter Width and an Increment of 1 For more information see Section 3 1 2 The Resolution Based Peak Detection Routine Use Advanced Settings When selected the software ignores the Global Threshold values on the Basic Settings tab and uses the threshold values set on the Advanced tab See Advanced Settings spectrum data only on page 3 28 Continued Data Explorer Software User s Guide 3 23 Chapter 3 Peak Detection and Labeling Table 3 2 Basic Settings Tab Parameters Spectrum Data Only Continued Parameter Description Peak Resolution Mass Resolution Value used to determine the Filter Width used for detection For more information see Section 3 1 2 The Resolution Based Peak Detection Routine Default values for different types of data are e Mariner data 5 000 which is optimized for masses below 3 000 m z Decrease this value if you are analyzing proteins e Voyager linear data 2 000 which is optimized for masses below 20 000 m z Decrease this value if
188. et The selected value is displayed in the Right Baseline field To limit the baseline offset to the area between the two selected points select Only Apply from L to R Baseline Midpoint To perform the baseline offset on the entire x axis deselect Only Apply from L to R Baseline Midpoint Click OK The offset baseline trace is displayed with a BO trace label To return to the original trace see Returning to the original trace on page 4 4 Adjusting the Baseline 4 7 2 Using Baseline Correction Description The Baseline Correction feature is a function that corrects for a curved baseline including a DC offset baseline by eliminating broad artifacts from the data set When to use Baseline correct if you are analyzing data e With a baseline that is not flat and you are using the Base Peak Intensity parameter intensity based thresholding to screen out noise peaks For best results apply Baseline Correction then re detect peaks If you use area based thresholding Max Peak Area during peak detection Baseline Correction is not typically needed Max Peak Area compensates for a rising or falling baseline e With a baseline that is not at 0 to improve peak detection of small peaks Correcting the To correct the baseline baseline 4 Display the spectrum of interest 2 From the Process menu select Baseline Correction The baseline is adjusted and the trace is displayed with a BC trace label Return
189. eth Enzymol McCloskey J A ed 1990 193 869 Isotopes As the number of carbon atoms in a compound increases the possibility of the compound containing a 18 C instead of a 2C also increases A compound with ten carbon atoms includes a molecular ion M and an isotopic ion M 1 one mass unit greater than the molecular ion which is approximately 11 percent of the abundance of the molecular ion The possibility of including two 1 C atoms in the same molecule also increases with increasing number of carbon atoms Therefore M 2 ions become more visible In a compound with ten carbon atoms such as decahydro naphthalene CoH Figure B 2 relative heights of M M 1 and M 2 peaks are 100 11 0 5 Figure B 2 Mass Spectrum of Decahydro Naphthalene Isotopic pattern All compounds containing carbon include molecular ions and in mass spectra isotopic ions that are 1 and 2 mass units higher than the molecular ion At higher masses the isotopic pattern of a mass spectrum is more pronounced as the relative abundance of isotopes increases In angiotensin Figure B 3 with a molecular weight of 1 296 Da Cg2Hg90 4N7 a peak containing one 18C isotope is approximately 70 percent of the pure C peak Data Explorer Software User s Guide B 3 Appendix B Overview of Isotopes B 4 Isotope limited resolution Applied Biosystems Figure B 3 Mass Spectrum of Angiotensin I at Resolution 3 000
190. ether to add or subtract the specified number of elements or groups NOTE If you specify Subtract the group to subtract must be present in the formula you specify in step 5 For more information see Add and subtract examples on page 6 16 9 Select the Resolution option e Da Resolves peaks that are separated by the number of daltons you enter e Resolving Power Resolves peaks using the resolving power M AM you enter e PPM Resolves peaks within the number of PPM parts per million you enter Data Explorer Software User s Guide 6 15 Chapter 6 Using Tools and Applications 10 Select the calculation mode FWHM Resolves peaks using the full peak width at peak half height e 10 Valley Resolves peaks to a 10 percent valley 11 Setthe Threshold Signal intensity below this percent intensity is not included in the calculation or display NOTE Setting Threshold too high distorts the isotope pattern 12 Click Calculate The theoretical isotope distribution is displayed in the Spectrum window with an ISO trace label and the elemental formula or the amino acid DNA or RNA sequence The result is displayed in the Result tab in the Output window For more information see Evaluating traces on page 6 18 Add and subtract The following examples illustrate how the software adds and examples subtracts groups to or from an elemental formula and how it applies the cha
191. everal small peaks around a very large peak after deisotoping the software has successfully identified a monoisotopic peak but the isotope cluster does not exactly match the theoretical elemental composition that you specified This may be caused by the following e The signal to noise ratio of the spectrum is poor and the calculated peak areas are not accurate e The specified elemental composition is not correct for the compound For example if the peptide contains sulfur and sulfur was not in the specified formula a small peak may occur 2 Da higher than the expected monoisotopic mass There are two overlapping isotope clusters but the intensity of one cluster is stronger than the intensity of the other Increase the peak detection threshold after deisotoping to remove the residual peaks from the peak list Data Explorer Software User s Guide 3 49 Chapter 3 Peak Detection and Labeling Example Figure 3 19 and Figure 3 20 illustrate the effects of deisotoping Before deisotoping Figure 3 19 the spectrum includes an isotope pattern with four detected peaks Spec 1 BP 1672 9 29103 100 1190 4 4406 2 a g Figure 3 19 Spectrum Before Deisotoping After deisotoping Figure 3 20 the trace includes two labeled centroid bars that represent monoisotopic masses indicating that the original trace represents two isotopic envelopes The intensity of a centroid bar is proportional to
192. f Mariner data on page 4 2 and Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only on page 4 13 2 6 Applied Biosystems Opening and Closing Data Files 2 1 3 Displaying Voyager Chromatograms To display chromatograms for multispectrum Voyager DAT files 1 Open the DAT files as described in Opening Data Files on page 2 2 Spectrum traces for the DAT files are displayed 2 To display a Chromatogram window click a Spectrum window to activate it then select Restore Chromatogram from the View menu A Chromatogram window is displayed for the data file NOTE Diode array support and UV display are disabled for Voyager chromatogram traces 2 1 4 Viewing Read Only Files For quick scanning of archived data files you can view read only files using the File Open dialog box When you open a read only file a message indicates that the file is read only and prompts you to open the file All functions that write information to disk are disabled when you view a read only file CAUTION Although you can process read only files you cannot save any changes you make When you close a read only file any changes you made are lost Data Explorer Software User s Guide 2 7 Chapter 2 Using Chromatogram and Spectrum Windows 2 1 5 Moving Between Open Files You can have more than one file open at a time You can move between open files in three ways Select the open fil
193. feature creates a Visual Basic script of your actions as you record the macro then executes the script when you run the macro Macros provided with the Data Explorer software are described in Appendix C Data Explorer Toolbox Visual Basic Macros This section includes e Before using the macro recorder e Recording a macro e Assigning macros to buttons e Running a macro e Deleting a macro e Advanced macro editing e Importing or Exporting Macros in DATAEXPLORER VB6 e Running macros automatically when opening and closing files 6 7 1 Before Using the Macro Recorder Importing macros provided Maximum number 6 34 of macros Applied Biosystems Macros are provided with the Data Explorer software but are not available for use until you import them into the Data Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 You can create an unlimited number of macros However there are only 10 buttons in the toolbar to which you can assign macros at a given time You can change the macro assigned to a button Using the Macro Recorder Location of All macros you record are stored in a file called macros DATAEXPLORER VB6 in the C MARINER PROGRAM or C VOYAGER directory Displaying the Ifthe macro toolbar Figure 6 22 is not displayed macro toolbar 1 Select Toolbar from the View menu 2 Select Macros then click Close Al gt fel Figure 6 22 Macro T
194. ference file is different than the order of the information displayed in the Reference Mass dialog box see Figure 5 3 on page 5 10 NOTE You can also create a reference file using the lon Fragmentation calculator For more information see Creating a calibration reference file REF on page 6 30 To create and save a calibration reference file 1 From the Process menu select Mass Calibration then select Edit Create Reference File Manual Calibration The Edit Create Reference Peak Information dialog box Figure 5 6 is displayed Edit Create Reference Peak Information xj Calibration reference file Browse Close Name Theoretical m z Charge Elemental Composition a E po Resolved Isotope Mass Insert Average Mass Reference Mass Type Name Charge Elemental Comp Figure 5 6 Edit Create Reference Peak Information Dialog Box Type the Name and Theoretical m z for a reference compound then select the charge state Optionally enter the Elemental Composition for the compound Specify the mass type Resolved Isotope Mass or Average Mass If you are adding masses for highly charged compounds see Specifying mass type for highly charged narrow peaks on page 5 21 Click Insert Repeat step 2 through step 4 for remaining compounds Data Explorer Software User s Guide 5 19 Chapter 5 Examining Spectrum Data CAUTION The software allows you to add multipl
195. from each peak charge state 1 evaluation Finds one peak pair at 558 3 m z and 559 3 m z and labels these peaks as charge state 1 e Does not find a match for peaks at 558 6 m z or 558 9 m z so these peaks are considered to have no calculated charge and are not labeled with a charge state Figure 3 8 on page 3 34 Data Explorer Software User s Guide 3 33 Chapter 3 Peak Detection and Labeling Spec 1 BP 558 3 824 400 558 3 z1 824 90 20 558 6 gt 70 a 60 f g 50 40 558 9 30 20 59 3 z1 10 Q d T 7 eel T ae 554 556 558 560 562 564 Mass m z Figure 3 8 Max Charge State Set Too Low and Max Isotope Set Correctly The grouping of the first and fourth peaks into an isotope cluster is apparent when you turn on monoisotopic peak list filtering Figure 3 9 The software labels the first peak but removes the mass and charge state labels from the 559 3 m z peak indicating that it is part of the 558 3 m z isotope cluster Note that turning on monoisotopic peak list filtering does not affect the mass labels on the 558 6 m z or 558 9 m z peaks because these peaks have no calculated charge and are not part of the determined isotope cluster Spec 1 BP 558 3 824 100 558 3 z1 824 90 Hs 558 6 70 60 f 50 Z 558 9 30 20 10 0 T T apt 7 pet A 554 556 558 560 562 564 Mass m z Figure 3 9 Max Charge State Set Too Low Monois
196. ftware User s Guide 1 5 Chapter 1 Data Explorer Basics Mariner SPC file in Mariner software versions earlier than version 3 0 data files format are stored in SPC format You can view and process SPC files in Data Explorer or you can convert these files to DAT format For information about the differences between the DAT and the SPC formats see the Mariner Workstation User s Guide Voyager MS file In Voyager software versions earlier than version 5 0 data format files are stored in MS MSF MSA and MSB format You can view and process MS MSF MSA and MSB files in Data Explorer You cannot convert them to DAT format NOTE Voyager SPC format files are not supported in the Data Explorer software Extracting You can also store parameter settings in separate files by information from extracting information from a DAT file as needed for use with _DAT files ther files For more information see Section 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files The types of information stored in a DAT file are described below Table 1 1 Information Stored In a DAT File Category File Type File Content Settings BIC Instrument settings for controlling the operation of the mass analyzer For more information see the Mariner Workstation User s Guide e Voyager Biospectrometry Workstation User s Guide 1 6 Applied Biosystems File Formats and Types
197. g errors refer to the online help available in the Visual Basic Editor Using the Macro Recorder 6 7 5 Deleting a Macro To delete a macro 1 From the Tools menu select Macros The Macros dialog box Figure 6 26 is displayed z Figure 6 26 Macros Dialog Box 2 Select the macro to delete from the list 3 Click Delete NOTE Other buttons on this dialog box are for advanced editing Refer to the online help available within the Visual Basic Editor Data Explorer Software User s Guide 6 41 Chapter 6 Using Tools and Applications 6 7 6 Advanced Macro Editing Accessing the You can access the Visual Basic Editor to enhance or edit a Visual Basic script created by the Macro Recorder in Data Explorer or to Editor create a new script Access the Visual Basic Editor from Data Explorer in three ways e Select Visual Basic Editor from the Tools menu e Select Macros from the Tools menu select the macro to edit then click Edit e Click a in the Macro toolbar The DATAEXPLORER VBS6 file is displayed in the Visual Basic Project Explorer window Macro scripts created using the Macro Recorder in Data Explorer are stored in Module folders in the Project Explorer window Displaying To display a list of the scriptable objects available within Data scriptable objects Explorer select Object Browser from the View menu in the Visual Basic Project Explorer window For information on using t
198. ge the Max Peak Area from 1 the default to 0 and the Base Peak Intensity from 0 the default to 1 e Click the Peak Processing tab and change the default Integration Baseline Setting from Valley to Valley to Valley to Baseline 3 Click OK Figure 7 11 shows the partially resolved peaks that are now labeled Intensity 4117 663 1577 8 0 4174 4 4280 8 4387 2 4493 6 4600 0 Mace im Figure 7 11 Partially Resolved Peaks Labeled Data Explorer Software User s Guide 7 13 Chapter 7 Data Explorer Examples 7 2 2 Proces sing Before Calibrating to Optimize Mass Accuracy Calibrating without baseline correcting and deisotoping This section includes e Calibrating without baseline correcting and deisotoping e Before calibrating e Calibrating For optimum mass accuracy baseline correct and deisotope a spectrum before calibrating Figure 7 12 shows a spectrum before calibration 100 90 Intensity 967 0 80 70 466 150 g 40 3659 433 30 20 g 642 361 Spec 1 BP 757 1 53319 1297 722 2094 696 AEn 4707 2 2447 4 oe 3927 8 4668 0 Figure 7 12 Spectrum Before Calibration Figure 7 13 shows a spectrum that has been calibrated without initial baseline correction and deisotoping Mass accuracy is not acceptable 100 DO 70 60 50 40 30 20 10 Intensity a mam Spec 1 gt MC gt MC BP 757 1 53319 1
199. ges and Filter Width settings in ranges to optimize detection For more information see Detection Ranges on page 3 28 Fragment ion peaks in segments collected with lower PSD Mirror Ratios are broader and include more data points Use a higher Filter Width setting for these segments Within a segment resolution increases with increasing mass as the flight time of the fragment ions approaches the flight time of the precursor Use a lower Filter Width setting for higher resolutions For more information see Section 3 2 Peak Detection Smoothing f a segment appears noisy smooth the trace for example use 25 point Gaussian smoothing before calibrating Data Explorer Software User s Guide 8 11 Chapter 8 Viewing Voyager PSD Data 8 3 2 Calibrating This section includes e Calibrating e Matching peaks automatically e Selecting peaks manually Solving and plotting e Applying new constants to the data file e Selecting calibration peaks for optimum mass accuracy Calibrating To calibrate a PSD spectrum 1 Click the Spectrum window to activate it Select the spectrum of interest NOTE You cannot calibrate a result spectrum or a composite spectrum you have accessed from the Processing History command on the Display menu For more information see Section 2 4 7 Recalling and Rearranging Traces Processing History 2 From the Peaks menu select Peak Label then select the Mass Label Type pe
200. gory to the Graph toolbar Data Explorer Software User s Guide 1 21 Chapter 1 1 22 Undocking toolbars Creating toolbars Applied Biosystems Data Explorer Basics 4 To remove a button from a toolbar click drag the button from the toolbar NOTE The Customize dialog box must be displayed to click drag a button from a toolbar 5 Click OK to close the Customize dialog box The toolbar at the top of the Data Explorer window is divided into sections A section is preceded by a double vertical bar You can move undock each section of the toolbar within the Data Explorer window by click dragging the double bar at the left side 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 You can display or hide each section individually add or remove buttons on the toolbar and rearrange the order of buttons displayed To do so you must have the Customize dialog box open as described in the previous section You can create new toolbars by click dragging buttons to a window area where there is no toolbar You can then add buttons to the new toolbar as described in Customizing toolbars on page 1 21 Setting Graphic Options 1 5 Setting Graphic Options This section includes e Changing background color e Customizing options e Reverting to previous graphic options NOTE Changes you make to Graphic Optio
201. h the greatest area and the areas of unwanted peaks and estimate the percentage to enter as the threshold Specifying a Max Peak Area Threshold is particularly useful if the spectrum includes a rising baseline that would cross a Base Peak Intensity Threshold and eliminate peaks of interest in one portion of the trace 4 If peak detection is still not acceptable adjust the remaining peak detection parameters as described in Section 3 2 3 Setting Peak Detection Parameters Data Explorer Software User s Guide 3 7 Chapter 3 Peak Detection and Labeling 3 2 2 Strategy for Voyager Peak Detection This section gives some quick suggestions on how to approach Voyager peak detection For details on peak detection see Section 3 2 3 Setting Peak Detection Parameters Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Strategy When detecting peaks in Voyager data 1 Open the data file and observe the effects of the default resolution dependent settings Hint To improve peak detection you can calculate the resolution on a tall resolved peak in the middle of the spectrum If the resolution result differs by more than 50 from the default resolution setting fine tune the Resolution setting and reapply peak detection For more information see Section 6 3 Using the Mass Resolution Calculator 2 To aid in peak interpretation do all of the following e Baseline correct
202. he Visual Basic Editor refer to the online help in the Visual Basic Editor Numbering When you create Macro scripts the Visual Basic Editor uses a sequence numbering sequence that starts at 0 If you specify a number of 1 in the Visual Basic Editor it indicates the second item in a sequence not the first For example if you create a script to sort the Peak List in the Output window and you want to sort by Area column 8 in the Peak List specify sorting on column 7 in the script 6 42 Applied Biosystems Using the Macro Recorder 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 You can import macros into or export macros from the DATAEXPLORER VB6 project for use in the Data Explorer software Importing when When you install a new version of the Data Explorer software new versions of The DATAEXPLORER VB6 file is not overwritten This Data Explorer allows you to maintain any macros you have developed software installed in the DATAEXPLORER VB6 file e New macros are supplied in stand alone ASCII format BAS and FRM files in C MARINER PROGRAM MACROS or C VOYAGER MACROS and are not incorporated into the DATAEXPLORER VB6 file automatically To make the new macros available for use import them into the DATAEXPLORER VB6 file Importing NOTE You can also import by opening NT Explorer then click dragging the files into the DataExplorerProject displayed in the Visual Basic Editor window To import macr
203. he box you draw defines the precise mass range used in the extracted ion chromatogram The extracted ion chromatogram is displayed in the Chromatogram window Figure 4 2 with the mass range indicated in the trace label Extracted ion chromatogram center window XIC 579 240 1 641 240 4 400 626 651 2134 5 20 70 2 60 2 50 E 40 30 20 10 0 o 600 620 640 660 680 700 Spectrum Number Figure 4 2 Extracted lon Chromatogram 4 To return to the original trace see Returning to the original trace on page 4 4 4 8 Applied Biosystems Creating an Extracted lon Chromatogram 4 2 2 Creating a Constant Neutral Loss CNL Chromatogram This section includes e Overview e Applications e Labeling spectrum peaks with mass difference optional e Procedure e Example Overview To rapidly screen for the presence of mass differences Applications corresponding to loss of specific fragments you can create a Constant Neutral Loss CNL extracted chromatogram When you generate a Constant Neutral Loss Extracted ion chromatogram the software constructs a chromatogram that contains signal only for spectra that contain peaks separated by the specified mass difference To construct a Neutral Loss Extracted ion chromatogram the software e Compares each mass in the peak list to every other mass in the peak list and derives mass differences e Takes the total signal of any pairs of peaks that generated
204. he range displayed in the Spectrum window is reflected in the X Display Range fields NOTE If you zoom on a different region in the Spectrum window the new range is not updated in the X Display Range field but is used for the analysis when you Click Calculate The range is updated when you click Calculate 2 Baseline correct the trace Peak intensities and areas used in the polymer calculations are calculated from a 0 y axis value not from the calculated baseline of the peak Data Explorer Software User s Guide C 15 Appendix C Data Explorer Toolbox Visual Basic Macros C 16 Applied Biosystems 3 Select the mode for the analysis e Use the entire mass range Calculates average molecular weights using all peak intensities within the X Display Range It does not distinguish between different polymeric species that may be present in the mass range Enter an adduct mass Poles Analysis Toolboa Ei Display Flange From b Ds To EZ Da Use etie masa range i iise Latesked paai Enter Addu bore Matt Da Enia End Gasca Mass a 0 D Araiye Rasut Figure 3 14 Polymer Analysis Toolbox NOTE If the predominant ion series is M Na enter 23 for the adduct ion If the predominant ion series is M Ag enter 108 for the adduct ion If you are analyzing an unknown or a mixture leave this field blank the calculation is performed without subtracting the adduct ion mass Using the Polymer Analysis
205. hooting Mariner Only on page 9 18 Requirements To label isotope peaks with charge states the following must be true e Mass must be within the range in which the system can resolve the isotope peaks e lt 4 000 Da on a Mariner system e lt 5 000 Da on a Voyager DE PRO system e lt 6 000 Da on a Voyager DE STR system Data Explorer Software User s Guide 3 53 Chapter 3 Peak Detection and Labeling e Isotopes must be resolved e Peak detection parameters must be set to detect all of the peaks in the isotope cluster e Charge determination parameters must be set appropriately e Charge state labels must be enabled 3 5 2 Setting Chromatogram and Spectrum Peak Labels This section includes e Customizing colors font or size e Setting chromatogram labels e Setting spectrum labels e Deleting labels e Labels not displayed e Charge state not displayed Customizing To set colors font and size for peak labels see Section 1 5 colors font and Setting Graphic Options size Setting To label chromatogram peaks chromatogram 4 Click the Chromatogram window to activate it labels 2 From the Peaks menu select Peak Label The Chromatogram Peak Label dialog box is displayed Figure 3 21 3 54 Applied Biosystems Peak Labeling Chesmatageem Maat labai Peah Late Ciani FE Erabi Labeling Deore Poihi fi catia sina M Deeslapping lobes FF Pask Beaune lenis Ferrea Lite Conis
206. ibration The current spectrum is calibrated and data displayed with an MC trace label The calibration constants are saved with the spectrum Apply to All All spectra in the data file are calibrated NOTE This button is Using the currently displayed calibration and displayed only if you are displayed with an MC trace label The are calibrating a calibration constants are saved with the data Voyager file Each spectrum in the data file is multispectrum data calibrated when displayed file If you apply the calibration the next time you open the data file the MC trace label is not displayed Data Explorer Software User s Guide 5 23 Chapter 5 Examining Spectrum Data Ensuring that masses match during calibration 5 24 Applied Biosystems 5 3 5 Hints for Calibrating Mariner Data Mariner TOF Analyzer parameters affect flight times of ions If you acquired Mariner data using different TOF Analyzer parameters and did not calibrate the data in the Instrument Control Panel to compensate for the altered parameters masses in the data file may be significantly different from the reference masses For information on calibrating in the Instrument Control Panel see the Mariner Workstation User s Guide Before performing automatic matching set the Mass Tolerance and Minimum Intensity appropriately to match the expected reference masses After automatic matching check the list of matched peaks to make s
207. ical bar represents one data point For more information see Section 1 4 Customizing the Data Explorer Window Increment Number of data points the filter moves across Use 1 for all applications This value is automatically set to 1 if you enable Use Resolution Dependent Settings on the Basic Settings tab For more information see Section 3 1 2 The Resolution Based Peak Detection Routine Data Explorer Software User s Guide 3 31 Chapter 3 Peak Detection and Labeling 3 2 5 Charge State Determination and Examples 3 32 Isotopes Charge state determination Charge state parameter examples Applied Biosystems NOTE Isotope resolved peaks in Voyager data are typically singly charged See Section 3 7 Default Peak Detection Settings for recommended settings This section includes e Charge state determination e Charge state parameter examples e Charge state determination troubleshooting For information on isotopes see Appendix B Overview of Isotopes Charge state is determined by evaluating the relative isotope peak spacing The expected peak spacing is determined by the Max Charge State plus or minus a tolerance value The tolerance is calculated as proton mass charge state x 15 where proton mass equals 1 007276456 During charge state determination the software starts with the most intense peak and the peak spacing determined by the Max Charge state then evaluates all
208. importing 5 29 settings specifying 5 29 Automatic macros 6 45 AutoSaturation Correction Mariner data only description 5 62 effect on Mariner RST files 5 62 effect on mass accuracy 5 62 Average mass definition B 6 labeling partially resolved isotopes 3 10 7 11 Averaging data graphic compression 1 28 Averaging spectra 4 22 Axes numbers do not match spectrum numbers 4 25 offsetting Y 4 27 scaling 2 11 turning off right axis 2 12 B Background color changing 1 23 default Mariner 1 4 default Voyager 1 4 Background signal evaluating 7 8 subtracting 4 20 Bar mode traces 1 28 BAS files for macros 6 43 Base mass labeling peaks 3 55 3 56 Base peak intensity definition 4 2 scaling to 2 12 threshold for peak detection chromatogram 3 20 threshold for peak detection spectrum 3 22 3 30 Baseline changing line width 1 26 displaying on trace 3 55 3 58 Baseline correction chromatogram 4 29 spectrum 5 47 spectrum advanced 5 48 Baseline offset chromatogram 4 27 spectrum 5 45 Basic peak detection parameters description chromatogram 3 19 description spectrum 3 22 resetting 3 18 setting chromatogram 3 11 setting spectrum 3 13 Basics of the Data Explorer software 1 1 1 2 BC in chromatogram header 2 30 4 29 in spectrum header 2 31 5 47 BIC files see also Instrument settings description 1 6 exporting from DAT 1 36 exporting from RSD and RCD 1 36 Binning data graphic compression 1 28 BO in chromatogram he
209. in Data Explorer not the m z range in the original data file No action Normal occurrence Text annotation from a previous trace displayed on current trace Annotation stays in the view until you delete it even if you advance to the next spectrum Delete the annotation See Section 2 4 9 Annotating Traces Table 9 2 General Troubleshooting Mariner Only Symptom Possible Cause Action Failed to open chromatogram data message displayed when you open a data file You are opening a data file collected with version 2 1 software and the CGM file not located in the same directory as the SPC file Place the CGM and SPC files in the same directory 9 4 Applied Biosystems General Troubleshooting Table 9 2 General Troubleshooting Mariner Only Continued Symptom Possible Cause Action Spectra labeled with spectrum numbers that do not correspond to the axis You are viewing event filtered MS Method data Spectra in an event filtered trace are numbered contiguously 1 2 3 regardless of their relation to the overall acquisition However the axis of the trace reflects the numbering of the overall experiment No action Normal occurrence Conversion of SPC to DAT failed A DAT file with the same file name you are specifying for the converted SPC file is open 1 Close the open DAT file 2 Use a different name
210. in Data Explorer Advancing To advance through segment traces click and through segment Segments are displayed in the order in which they were traces 2cquired NOTE If these buttons are not displayed in the toolbar you can add them See Section 1 4 3 Customizing Toolbars Displaying To display multiple segment traces multiple segment 4 Glick F in the toolbar three times to add three Not traces Used traces 2 From the Process menu select PSD Processing Data Explorer Software User s Guide 8 3 Chapter 8 Viewing Voyager PSD Data 8 4 Applied Biosystems The PSD Processing dialog box is displayed Figure 8 2 and lists all segments contained in the PSD DAT file in the order in which they were acquired with associated Mirror Ratios and Max Stitch Masses Column header 1 10 Figure 8 2 PSD Processing Dialog Box The Max Stitch Mass is equal to the Precursor Mass times the Mirror Ratio 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 Optimum focus and resolution is achieved for fragment ions close to this mass Displaying PSD Data NOTE The entry number in the PSD Segment list above may not correspond to Segment number specified in the Segment list for acquisition described in the Voyager Biospectrometry Workstation User
211. in printers If traces do not print change the line width or color Setting Graphic Options Setting data To enable data cursors and set cursor labels and attributes cursors 4 Select Graphic Options from the Display menu 2 Inthe Data Cursor section of the Graph Setup tab see Figure 1 4 on page 1 25 select Show Data Cursors then select one of the following cursors from the Type drop down list e X Single vertical cursor e Y Single horizontal cursor e X Y Vertical and horizontal cursors X X Two vertical cursors X Y X Two vertical cursors and one horizontal cursor NOTE If data cursors are displayed they are printed when you print traces To suppress data cursor printing deselect Show Data Cursors before printing To set the cursor mode select the appropriate label type as described below Label Type Options Y Label Absolute Displays the number of counts BP Relative Displays the Intensity value relative to the base peak in the trace Includes BP marker on the cursor label Display Relative Displays the Intensity value relative to the largest peak in the current display range X X Absolute Displays the Spectrum Number or Retention Time Label chromatogram or the Mass spectrum X Relative Available if you have two X cursors displayed For the first cursor displays the Spectrum Number or Retention Time chromatogram or the Mass spectrum For
212. ine other isotope peaks Set Filter Width correctly See Advanced Settings spectrum data only on page 3 28 Data Explorer Software User s Guide 9 19 Chapter 9 Troubleshooting Table 9 12 Charge State and Isotope Determination Troubleshooting Mariner Only Continued Symptom Possible Cause Action Spectrum peaks not labeled with charge state when charge state labels are selected Mass of original molecule above 4 000 Da not range in which the Mariner system can resolve the isotope peaks No action Normal occurrence Charge State parameters not set to detect at least two isotope peaks Adjust parameters See Peak Processing parameters spectrum data only on page 3 26 Peaks are more than 1 Da apart No action Normal occurrence Peaks are not from the same isotope species No action Normal occurrence Filter width is set too high to detect other isotope peaks Adjust Filter Width See Advanced Settings spectrum data only on page 3 28 Maximum Charge State for charge state determination is set lower than the charge state of the peak Adjust the Maximum Charge State See Peak Processing parameters spectrum data only on page 3 26 9 20 Applied Biosystems Peak Detection and Labeling Troubleshooting Table 9 12 Charge State and Isotope Determination Troubleshooting Mariner Only Continued
213. ing 3 From the Process menu select Multiple Charge then select Mass Deconvolution Data Explorer Software User s Guide 5 37 Chapter 5 Examining Spectrum Data The Multiply Charged Deconvolution dialog box Figure 5 11 is displayed 4 Inthe Spectrum window right click drag one multiply charged peak 5 Right click drag a second multiply charged peak adjacent to the first selected peak and in the same envelope of charged peaks Mass Charge values are entered in the list box Nil im bam iscia a han I paa alxizlel Hanns F dade O hiia Lead F im Hoo Che ipen Aga Fims i To Fea aa hijai lan plein Figure 5 11 Multiply Charged Deconvolution Dialog Box 5 38 Applied Biosystems Mass Deconvolution Mariner Data Only 6 Select the method to use for calculation e Automatic Selects additional multiply charged peaks based on the selected peaks and performs the calculation NOTE If the trace is noisy the software may not accurately select additional multiply charged peaks e Manual Performs the calculation using only selected peaks Does not select additional multiply charged peaks 7 Select Apex or Centroid mass to use for the calculation 8 Enter the mass charge range to include in the calculation This range is used for two purposes e If you selected Automatic mode the software searches this range for other peaks that are in the same charge series as the peaks s
214. ing 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 Zoom in NOTE Display data cursors then click the point you want to zoom on before clicking this button See Setting data cursors on page 1 27 e Zoom out to the previous zoom Full Unzoom 2 14 Applied Biosystems Manipulating Traces Centering a peak To center a peak in the trace window in the trace 4 Display the trace containing the peak of interest 2 Click the Spec Peak List or Chro Peak List tab in the Output window 3 Double click the peak to center The peak is centered in the active trace Customizing a See Section 1 5 Setting Graphic Options for information on trace customizing trace parameters such as line widths and data compression 2 4 2 Duplicating a Trace To make a duplicate of a trace 1 Click the trace 2 From the Display menu select Duplicate Active Trace The active trace is duplicated and displayed in another trace position 2 4 3 Dividing the Active Trace You can divide the active trace into equal segments with one command This is useful to expand the x axis range to better see spectral and chromatographic features To divide a trace 1 Click the trace 2 Fromthe Display menu select Divide Active Trace then select the number of segments to divide the trace into The active trace is automatical
215. ing to the To return to the original trace see Returning to the original original trace trace on page 4 4 Data Explorer Software User s Guide 4 29 Chapter 4 Examining Chromatogram Data 4 8 Using UV Trace Offset Mariner Data Only 4 30 Applied Biosystems To align a UV trace with a chromatogram trace 1 2 Display the chromatogram and UV traces of interest Display the x axis in retention time for each trace by selecting Traces from the Display menu selecting X Axis In then selecting Time Set the Replace Mode to Add a New Trace For information see Setting the Replace mode on page 2 17 From the Process menu select Realign UV Trace The UV Trace Offset dialog box is displayed Figure 4 13 UY Trace Offset x Original UY Peak Value min Aligned UY Peak To min Figure 4 13 UV Trace Offset Dialog Box Enter the retention times as follows Click the Original UV Peak Value min text box then right click drag over the peak to offset in the UV trace Select the Aligned UV Peak To min text box then right click drag over the peak to align with in the chromatogram trace Hint You can type retention times in the UV Trace Offset dialog box Use an X data cursor to read the retention time of the peak If you enter spectrum numbers by mistake the peak does not align properly Using UV Trace Offset Mariner Data Only Click OK The UV trace peak is shifted t
216. intains scaling of individual traces 6 Set trace colors as needed e If you selected Use same settings for all traces click Autocolor to allow the software to automatically assign trace colors NOTE The active trace color stays at its original setting Other trace colors are set based on the active trace color For example if the active trace is yellow other traces are assigned the colors pale blue pale green and medium gray which are the colors listed after yellow in the Trace color list excluding white White is not used in Autocolor because white may not print on certain printers e If you did not select Use same settings for all traces click Graph Setup then select trace colors See Setting colors on page 1 25 for information 7 Click OK Data Explorer Software User s Guide 2 27 Chapter 2 Using Chromatogram and Spectrum Windows 2 4 9 Annotating Traces You can add text annotation to traces by Copying a line of results from the Output window or copying any ASCII text from any source then pasting the copied information on the trace e Typing text on the trace Copying results To copy results 1 Generate results as desired by selecting commands from the Tools or Applications menu For more information see e Section 5 3 Manual Calibration or Section 5 4 Automatic Calibration e Section 5 6 Mass Deconvolution Mariner Data Only Section 6 2 Using the Isotope Calculat
217. io Figure 7 1 Improving Signal To Noise Ratio with an Extracted lon Chromatogram Data Explorer Software User s Guide 7 3 Chapter 7 Data Explorer Examples 7 1 2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks 7 4 Overview Creating a combined spectrum You can use the Data Explorer software to deconvolute chromatographic peaks and obtain masses for each component by e Creating a combined spectrum to identify the coeluting species by mass e Creating extracted ion chromatograms for the masses and comparing the extracted ion chromatograms to the original TIC containing the unresolved peaks You can then create a combined spectrum from each extracted ion chromatogram to evaluate the spectral data The following example illustrates how to deconvolute two unresolved chromatographic peaks in a tryptic digest of cytochrome c 1 Display the data file 2 Inthe Chromatogram window zoom in on the unresolved peaks Figure 7 2 S pikttiuin Mitten Figure 7 2 Unresolved Peaks in Cytochrome C Applied Biosystems 3 Inthe Chromatogram window right click drag over the unresolved peak pair aa h PA eiii ane ni ag a aa a mu Mariner Data Examples The combined spectrum is displayed Figure 7 3 with two intense peaks at 410 Da and 723 Da Generate extracted ion chromatograms as described below to determine if these peaks are the coeluting components NOTE If these peak
218. ion User s Guide Use this document to learn detailed information about the Mariner Workstation Voyager DE Biospectrometry Workstation User s Guide Use this document to learn detailed information about the Voyager DE Workstation Printer documentation depends on the printer you purchase Use this documentation to set up and service your printer Microsoft Windows NT User s Guide and related documents Use this guide to learn detailed information about the Microsoft Windows NT user interface We welcome your comments and suggestions for improving our manuals You can send us your comments in two ways On the web at www appliedbiosystems com about contact html By e mail at TechPubs appliedbiosystems com 1 Data Explorer Basics This chapter contains the following sections tet OVENICW Ss cut Astee icine niiteeeneel idee 1 2 1 2 File Formats and Types eeeeeeeeeeees 1 5 1 3 Parts of the Data Explorer Window 1 11 1 4 Customizing the Data Explorer Window 1 17 1 5 Setting Graphic Options 0 ee 1 23 1 6 Managing Files aenn 1 30 Data Explorer Software User s Guide 1 1 Chapter 1 Data Explorer Basics 7 1 1 Overview Description The Data Explorer Version 4 0 processing software is graphical software that you use to analyze calibrate and report data You can use the Data Explorer software to analyze data collected on e Mariner Workstations
219. ith the Chromatogram window displayed then selecting Select To display TIC Total lon Chromatogram which includes the entire mass range saved in the data file Each data point represents the sum of all ion intensities in the corresponding spectrum BPI Base Peak Intensity trace which includes only the base peak in each spectrum Each data point represents the single most intense ion in the corresponding spectrum Analog Trace of the input from an outside source representing any signal that changes over time for example the UV signal from an LC system or the Analyzer Temperature Air Temperature Spray Tip Potential or Nozzle Temperature from the Mariner mass spectrometer DAD TAC Diode Array Detector DAD Total Absorbance Chromatogram TAC which displays data acquired from LC samples Hint To display spectra for DAD data double click the TAC or Ch Absorbance trace DAD Diode Array Detector Channel chromatograms which display Channel N specific wavelength ranges where N is the channel number set during acquisition The data file can include up to five channels Hint To display spectra for DAD data double click the TAC or Ch Absorbance trace 4 2 Applied Biosystems Overview You can display extracted chromatograms from Mariner data files by selecting the Process menu with a Chromatogram window displayed then selecting Select To display Extracted lon Center Window o
220. ize the Chromatogram and Spectrum windows after hiding the Output window click E or in the toolbar Customizing the Data Explorer Window 1 4 Customizing the Data Explorer Window This section includes e Setting default values e Customizing Graphic and Processing settings e Customizing toolbars 1 4 1 Setting Default Values You can set defaults for most dialog boxes in the Data Explorer software by setting a value or selecting a button closing the dialog box then closing the data file you are viewing The next time you open the dialog box the last settings specified are displayed 1 4 2 Customizing Processing and Graphic Settings SET This section includes e Overview of processing and graphic settings e What settings contain Default processing and graphic settings Default graphic settings e Customizing settings saved in a data file e Making a copy of default SET files before customizing e Opening customizing and saving SET files e Applying a SET file Data Explorer Software User s Guide 1 17 Chapter 1 Overview of processing and graphic settings What settings contain Default processing and graphic settings 1 18 Applied Biosystems Data Explorer Basics Processing and graphic settings control how data is processed and displayed in the Data Explorer software The last settings used are automatically saved in the data file when you close it The next time y
221. izing the Data Explorer Window Additional SET files that have been developed for detection of different types of data are included in the C VVOYAGER PROGRAMSET FILES directory The names of the SET files indicate the type of data the files can be used for The appropriate default settings for the type of data you open are automatically applied to a data file the first time you open it in Data Explorer You can also manually apply these settings if desired For information see Applying a SET file on page 1 20 Two additional default settings files stand alone SET files that contain graphic settings only are also provided on your system in the C MARINER PROGRAM directory or the C VOYAGER PROGRAM directory e DEFAULTWHITE SET e DEFAULTBLACK SET These SET files contain the default graphic settings applied when you select White Background or Dark Background after selecting Default from the Display menu For information see Section 1 5 1 Changing Background Color You can customize settings saved in a data file by adjusting graphic or processing settings in the Data Explorer window Settings are saved with a data file when you close the data file and are automatically applied the next time you open the data file if specified For more information see Section 2 1 Opening and Closing Data Files You can also save the settings in a SET file for use with other data files as described in Saving SET files
222. just peak detection ranges and thresholds to screen out noise in low mass regions and to detect peaks in high mass regions 3 Setthe Set the Base Peak Intensity or Max Peak Area in peak detection until the unwanted noise peaks are removed from the Spec Peak list C Running To run the MS Fit MS Tag macro MS Fit MS tag 4 in the Toolbox Palette dialog box click MS Fit MS Tag HS FtM Tag Toolbos a mert Me t Mereigate To Parent lon Hi imiz Copy Paska Chae HS FivWS Tag Toolbox 5 MFE ms Ta terme Cony Peski cka Figure 3 15 MS Fit Dialog BoxMS Tag Dialog Box C 18 Applied Biosystems Using MS Fit MS Tag Toolbox Click e MS Fit tab lf you are examining peptide data from a protein digest e MS Tag tab lf you are examining PSD data Navigate to the web site containing the database to search Adjust settings on the web site as needed Minimize the web page Do not exit In the MS Tag dialog box enter Parent lon mass if you are using MS Tag Click Copy Peaks The Spec Peak list for the data file is copied to the web page Start the search from the web site The database search is performed Results of the search are displayed on the web site Data Explorer Software User s Guide _C 19 Appendix C Data Explorer Toolbox Visual Basic Macros C 20 Applied Biosystems Index Numerics and Symbols in spectrum header 2 31 Base Peak Intensity definiti
223. k Processing tab in Peak Detection change the default Integration Baseline Setting from Valley to Valley to Valley to Baseline Peaks represent partially On the Basic Settings tab in Peak Detection decrease resolved isotopes and the Mass Resolution setting until the isotopic envelope is you want to label and detected detect the average mass Figure 7 9 shows a spectrum with partially resolved peaks that represent two compounds Data Explorer Software User s Guide 7 11 Chapter 7 Data Explorer Examples Spec 1 gt NF1 0 BP 6147 5 42723 100 4117 663 49 757 4430 658 90 1577 8 Intensity 0 4068 0 4174 4 4280 8 4387 2 4493 6 Mace im 7 0 4600 0 Figure 7 9 Partially Resolved Peaks That Represent Two Compounds Minor Component Not Detected Adjusting peak To adjust peak detection detection 4 Click in the toolbar or select Peak Detection from the Peaks menu The Spectrum Peak Detection Setup dialog box is displayed with the Basic Settings tab Figure 7 10 displayed Bane etings Pek Frocenara advanced Saing Ginta Perens limfetiorap Oooo T Erabi SEP jairai T hecsteadd Tiata Par Feti E Ue Feie Pepan Ganinge r imidd irim Pasi Namikan ET aH Fem ee eg el ha P vee ER Canoe Figure 7 10 Spectrum Peak Detection Setup Basic Settings Tab 7 12 Applied Biosystems Voyager Data Examples 2 Do either of the following Chan
224. l Type peak apex or peak centroid to use for calibration Click OK 2 From the Process menu select Mass Calibration and then select Manual Calibration The Manual Calibration dialog box is displayed Figure 7 16 eget cahan melee be Refs paps rai hg ord palin rene Merits fE ER a e j ipe Ban pirea fiz O H Fd j Fmi pmjirg lir iras poy Calta re Epa i idei Figure 7 16 Manual Calibration Dialog Box 3 Click J then select the VOYAGER REF calibration reference file 4 Enter Reference Matching and Calibration Criteria NOTE For descriptions of calibration parameters see Section 5 3 Manual Calibration 7 16 Applied Biosystems Voyager Data Examples Matching peaks 5 Click Match Peaks and Solve The software compares observed masses in the spectrum to reference masses in the selected reference file lists the matches in the Peak Matched list calibrates the spectrum and displays the calibration statistics in the Output window NOTE If you set Mass Tolerance too low no peaks will match You can also add peaks to the Peak Matched list by right click dragging on a peak in the spectrum then selecting the mass from the reference Peak Information dialog displayed Click Solve and Plot after manually adding masses The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Output window Applying 6 Ifthe calibration statistics a
225. le from the list of most recently used files NOTE When you apply a SET file from the Recent Processing Settings menu only the process settings are applied e Select an option from the Settings command on the File menu 1 20 Applied Biosystems Customizing the Data Explorer Window To use the Settings option 1 Select Settings from the File menu 2 Select one of the following e Restore Processing Settings e Restore Graphic Settings e Restore Graphic Processing Settings Revert to the Last Saved Graphic Processing Settings 3 If you select a Restore Settings option select or type the name of the SET file in the Restore dialog box then click OK Hint You can also restore default processing and graphic settings when you open a file or files See Section 2 1 Opening and Closing Data Files 1 4 3 Customizing Toolbars Customizing toolbars To customize the toolbar 1 Select Customize Toolbar from the Tools menu to display the Customize dialog box 2 To display or hide a toolbar section click the Toolbars tab then select or deselect a toolbar 3 To add a button to a toolbar click the Commands tab select the appropriate category then click drag the button to any toolbar in the main toolbar Hint To display a button description click the button within the Customize dialog box You can add buttons from any category to any toolbar For example you can add buttons from the File cate
226. lick OK to accept the reference mass for matching The Manual Mass Calibration dialog box is displayed again see Figure 5 2 on page 5 9 with the observed mass and the reference mass you selected displayed in the Peaks Matched list Hint You can sort the list of matches by clicking the column header buttons You can display complete information about a reference mass by double clicking the mass Data Explorer Software User s Guide 5 11 Chapter 5 Examining Spectrum Data 5 12 Eliminating data 10 points Applied Biosystems Repeat step 7 and step 8 until all desired peaks are in the matched list To eliminate unacceptable data points from the calibration do either of the following e Select the data point mass in the Peaks matched list then click Delete Selected Match e Click Eliminate Fit Outlier The software deletes from the calibration the data point with the largest calibrated Fit Error difference between the calibrated mass and the reference mass as reported in the Output window Figure 5 4 NOTE The point deleted may not be the point with the largest Initial Error difference between the pre calibration observed mass and the reference mass listed in the Peaks Matched List in the Manual Calibration dialog box Manual Calibration Eliminate Fit Outlier removes the mass associated with the largest fit error in the Output window not the mass associated with the largest initial e
227. ly split into the selected number of traces with the range evenly divided among the traces Data Explorer Software User s Guide 2 15 Chapter 2 Using Chromatogram and Spectrum Windows For example if you select Divide Active Trace to Four when the active trace has a range of 0 0 to 20 0 the active trace is divided into four traces e First trace represents the range from 0 to 5 e Second trace represents the range from 4 to 10 e Third trace represents the range from 9 to 16 e Fourth trace represents the range from 15 to 20 NOTE To restore the display to a single trace select Remove Inactive Traces from the Display menu 2 4 4 Adding Traces from the Same Data File to a Window This section describes e Overview Setting the Replace mode e Adding a trace Overview By default the Data Explorer software displays Data Type Window Mariner Chromatogram and Spectrum Voyager single Spectrum only spectrum Voyager Spectrum only Can also optionally multispectrum display chromatogram Chromatogram and Spectrum windows each contain one trace see Figure 1 3 on page 1 11 2 16 Applied Biosystems Manipulating Traces When you perform certain functions for example smoothing a new trace is created You can set the Replace mode to add to or replace the active trace You can add up to seven new traces to a window to allow you to keep original data displayed when you generate
228. m the Processing History list from the history 4 From the Display menu select Processing History list 2 Select Remove History 3 Select the traces to remove from the list then click OK 2 22 Applied Biosystems Manipulating Traces Setting To set Processing History options Processing 1 From the Tools menu select Processing History History options Options The Processing History Options dialog box Figure 2 10 is displayed Pyeng Hier Chel ere i um preg aior oO T on lahan ciang daia iis Figure 2 10 Processing History Options Dialog Box 2 Turn Processing History on or off If you turn on specify Purge processing history Records all processing functions performed Stores the history log in a CTS file and purges the history log when you close the data file Purging a history file does not affect the data contained in the data file It clears the contents of the CTS file that contains the processing history Save processing history Records all processing functions performed Stores the history log in a CTS file and maintains the history log when you close the data file If you save processing history CTS files can become very large You can periodically delete older CTS files to clear disk space 3 If you want a reminder dialog box to appear when you close a data file that prompts you to selectively save or purge the history file select Show Save History dialog 4 Click O
229. mining Chromatogram Data Describes processing and analyzing chromatographic data Data Explorer Software User s Guide xi How to Use This Guide Chapter Appendix Content Chapter 5 Examining Spectrum Data Describes processing and analyzing mass spectral data Chapter 6 Using Tools and Applications Describes how to generate results using several tools and applications the Centroid calculator Elemental Composition calculator Isotope calculator Mass Resolution calculator lon Fragmentation calculator and Signal to Noise calculator Also describes using the Macro Recorder and the Elemental Targeting Application Chapter 7 Data Explorer Examples Includes specific examples for Mariner data and Voyager data Examples include how to improve the signal to noise ratio for reserpine deconvolute unresolved peaks in cyctochrome c Mariner data and label partially resolved peaks Voyager data Chapter 8 Viewing Voyager PSD Data Describes how to view label and calibrate PSD data Chapter 9 Troubleshooting Includes symptoms and possible causes of and corrective actions for potential system problems Appendix A Warranty Provides warranty and service information Appendix B Overview of Isotopes Includes background information you need for understanding isotopes Appendix C Data Explorer Toolbox Visual Basic Macros Describes loading Visual Basic
230. mining if mass difference 4 9 trace label 4 9 4 10 Extracted ion chromatogram XIC creating 4 5 definition 4 3 4 4 example 7 2 7 5 7 10 improving signal to noise ratio 4 6 including only mass of interest 4 6 trace label 2 31 4 7 4 8 4 16 Extraction mode setting 4 7 4 11 F Failed to create empty document message 9 3 Failed to open chromatogram data message 9 4 File format converting SPC to DAT 1 30 File management 1 30 File name does not print 9 13 full name not displayed 1 14 File properties searching 1 32 viewing 1 32 File size reducing 1 33 File types 1 6 to 1 8 Files automatically running macros when opening or closing 6 45 cannot find 9 3 closing 2 10 error message displayed when opening PSD 8 2 9 3 moving between open 2 8 opening manually 2 2 opening PSD 8 2 read only 2 7 Filter Width in peak detection algorithm 3 3 3 67 PSD segments 8 11 setting automatically 3 3 setting manually chromatogram 3 20 3 21 setting manually spectrum 3 31 too high peaks not labeled 3 20 3 21 3 31 9 19 value used when resolution based detection enabled 3 3 Data Explorer Software User s Guide Index 11 Filter Width Increment setting manually spectrum 3 31 suggested value spectrum 3 31 value used when resolution based peak detection enabled 3 31 Filtered traces viewing see Filtering Filtering chromatogram traces 4 23 event tags Mariner data only 4 24 monoisotopic peaks 3 43 noise chromatogram
231. mula 3 49 procedure 3 48 requirements 3 47 Deleting peak labels from the trace 3 44 3 59 peaks from the peak list 3 44 3 59 text annotation 2 29 Detection Ranges adding chromatogram 3 19 adding spectrum 3 29 calculating automatically 3 4 combining chromatogram 3 20 combining spectrum 3 29 deleting chromatogram 3 20 deleting spectrum 3 29 example 3 4 overlapping regions 3 5 PSD data 8 11 Data Explorer Software User s Guide Index 9 Detection Ranges continued setting manually chromatogram 3 19 3 20 setting manually spectrum 3 28 setting parameters globally spectrum 3 22 setting parameters locally spectrum 3 30 Detection see Peak detection DI in spectrum header 2 32 3 49 Diode array detector data see DAD Diode array detector data see DAD data Disk space conserving by converting profile data to centroid 1 33 Display range scaling 2 11 X range expanding 2 11 y range expanding 2 11 Display Trace dialog box 2 18 Displaying peak labels 3 54 3 56 Voyager chromatogram window 2 7 DNA residues labeling C 5 Double bond equivalents DBE definition 6 6 determining 6 6 Duplicating a trace 2 15 E EF in chromatogram header 2 30 4 25 redisplaying original data 4 26 Elemental composition calculating 6 2 description 6 2 elements adding 6 9 fragment ions electron state for accurate results 6 5 fragment ions identifying 6 2 Index 10 Applied Biosystems Elemental composition continued Isotope
232. mulating 4 22 adding subtracting from different data files 5 64 adding subtracting within a data file 4 20 averaging 4 22 combining Mariner data only 5 2 5 4 7 4 saturated correcting 5 62 single charge conversion 5 59 subtracting 4 20 Spectra continued summing non contiguous 4 21 troubleshooting 9 17 truncating 5 56 types of 5 2 Spectrum noise threshold setting locally 3 30 Spectrum window see also Peak labels see also Spectrum window traces see also Traces adding spectra from different data files 5 64 adding spectra from same data file 5 4 baseline correction 5 47 baseline correction advanced 5 48 baseline offset 5 45 centroid creating 5 36 centroiding 3 69 5 36 charge state labels incorrect 3 60 9 20 charge state labels not displayed 9 19 charge state labeling peaks with 3 53 combined spectrum creating 5 4 context sensitive menus 1 14 DAD diode array detector displaying Mariner data 4 2 description 1 13 display range adjusting 2 11 extracted absorbance chromatogram XAC creating from 4 13 extracted ion chromatogram XAC creating from 4 15 extracted ion chromatogram XIC creating from 4 5 4 8 linking in different data files 2 13 Mariner DAD data displaying 1 13 mass deconvolution 5 37 noise filtering 5 42 Spectrum window continued organizing 2 13 peak list 3 38 peak centering 2 15 resolution mass calculating 6 20 results opening 2 40 signal to noise ratio calculating 6 23 smoo
233. n see Section 2 1 Opening and Closing Data Files Data Explorer Software User s Guide 1 29 Chapter 1 Data Explorer Basics 4 1 6 Managing Files This section describes Converting SPC file format to DAT file format Mariner only Converting data from profile to centroid Mariner only Converting to and exporting ASCII data Importing a trace in ASCII format Extracting and saving information from DAT RSD and RCD files Copying from data files 1 6 1 Converting SPC File Format to DAT File Format Mariner Data Only When to convert 1 30 This section includes When to convert Before you begin Converting Viewing file properties Searching file properties NOTE You cannot convert Voyager MS files to DAT format You are not required to convert Mariner SPC file format for data acquired in software versions earlier than 3 0 to DAT file format However the DAT format allows you to store all information associated with the file such as data results settings in one file simplifying file management Applied Biosystems Before you begin Converting Confirm that the SPC and CGM files are located in the same directory Use Windows NT Explorer to display the directory contents and to move the SPC and CGM files as necessary If the SPC and CGM files are not in the same directory when you open the SPC file a Failed to open chromatogram data message is display
234. n generate the complete sequence ladder by appending new results to existing results NOTE When you search using reference peaks selected from the Sequence Results list new results retain the label of the original reference peak and new labels are appended For example if you select 433 08 Thr as the reference results are listed as 362 13 Thr Ala 331 98 Thr Thr and 334 07 Thr Val The Thr immediately following the mass corresponds to the selected reference the result of the original search The residue following Thr is the residue that corresponds to the mass difference between the mass and the reference mass Using the Peptide Fragmentation Toolbox 5 Click Copy to Output Window Result Tab to copy results to the Result tab You can then copy from the Result tab to another application such as Notepad or print as needed Correlation To list ion pair correlations 1 Click the Correlation tab 2 Select a peak in the Spec Peak List 3 Select the correlation you want to determine for the selected peak that is click a and b if you want to determine if the selected peak is a member of an ab fragment ion pair Peptide Fragmentation Toodkor Ei Spec Peak Lit Setup Pais Sequence Correlation Comelsteores Pound for Selected Pask aby lt 1 Pare related bo pritina j H22 Bales A Ladder Coresma it Ligt 44 Hi 11 75 2S Phat Figure 3 13 Peptide Fragment
235. n to determine the optimum settings for your data Refer to General guidelines for setting parameters on page 5 54 for more information Parameter Description Specifies Peak Width at half height Value the software uses to estimate the baseline amplitude at regularly spaced points in the spectrum The number of regularly spaced points used is derived using the Peak Width parameter and the Flexibility parameter The illustration below shows how using a higher number of points estimate the baseline accomplished by using higher Peak Width and Flexibility settings can affect the shape and slope of the calculated baseline Lower number of points eee s x Higher number of points iO aa oe continued 5 50 Applied Biosystems Adjusting the Baseline Parameter Description Specifies Peak Width Set Peak Width according to the data you are correcting at half height e For best results set to the peak width at half height continued of the narrowest peak However smaller peak width values increase processing time e If peak width varies across the spectrum set to the average peak width e If peaks are narrow relative to the baseline region you are correcting set a larger value for example 10 times the width of widest peak to increase processing speed Ifthe baseline changes sharply across the spectrum set a smaller value closer to the narrowest peak width
236. nal spectrum was an unprocessed spectrum select Spectrum from the Display menu The number of the original spectrum is displayed in the Select Spectrum dialog box Click OK e Ifthe original trace was a processed spectrum select Processing History from the Display menu then select the original trace Data Explorer Software User s Guide 5 3 Chapter 5 Examining Spectrum Data 5 2 Creating a Combined Spectrum NOTE Before creating a combined spectrum for Voyager multispectrum data files calibrate the data See Section 5 3 Manual Calibration To create a combined spectrum 1 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 Inthe Chromatogram window right click drag across the region of the chromatogram that contains the spectra to combine The combined spectrum Figure 5 1 is displayed Combined spectrum label Spec 8 13 BD 558 3 799 100 5 3 798 7 B0 70 60 e 450 40 E 10 0 r 0 552 0 554 8 557 6 560 4 563 2 566 0 Mass im 7 Figure 5 1 Combined Spectrum 3 To return to the original trace see Returning to the original spectrum on page 5 3 5 4 Applied Biosystems Manual Calibration 5 3 Manual Calibration This section describes e Overview of manual calibration e Manually calibrating e Creating or modifying a calibration reference file e Reverting to instrument calibration e Hi
237. nd lists all masses in the selected reference file The selected entry is the nearest match in the calibration reference file to the selected peak mE leeterernt m Pesi jiranmrnn E Chii i a er a e o Howe toca S a a E roie haope Mate bmi o din irie diris amp F tt Po 1 i ts z na hego P Farhad Pign a Feseia Bhan Za Pd hog F Femted hogh HH Pepa Mhairi z Faad Hewi w Fanad Pogon 13 Lol khanrkten i j Figure 5 3 Select or Create Reference Peak Information Dialog Box 5 10 Applied Biosystems Manual Calibration 8 Do any of the following Click OK to accept the selected reference mass for matching then add it to the Peaks Matched list Select a different reference mass then click OK Type new reference mass information in the Name Theoretical m z Charge and Elemental Composition text boxes select the mass type then click OK to accept the reference mass for matching NOTE You must type a minus sign preceding the charge in the Charge text box for negative charge states Type new reference mass information in the appropriate text boxes click Save or Save As to add the information to the reference file then click OK to accept the reference mass for matching Type new reference mass information in the appropriate text boxes click Update to replace an entry in the reference file click Save or Save As to add the information to the reference file then c
238. ng viii 9 1 9 2 9 3 9 4 9 5 9 6 OVOIVIOW stectcecvens sie tec ieee vaelawneuna an enara sie eid sien ineade eeu teeta ae 9 2 General Troubleshooting ceceeceee cece cence cence eee ee eee eeeeeeeeeaeee 9 3 Processing Tools and Applications Troubleshooting eseeeeee 9 6 Calibration Troubleshooting ceeeeeeee cece eee eeee eee e neta eeeeeaeeeeees 9 10 Printing TroubDleShOoting ecceee eee eee e ee eee ANAKENT SEEPS ENKAY 9 14 Peak Detection and Labeling Troubleshooting ecseeeeeeeeeeeees 9 15 Applied Biosystems Table of Contents Appendix A Warranty Information 00000 ee A 1 Appendix B Overview of Isotopes 00 eeee B 1 Appendix C Data Explorer Toolbox Visual Basic Macros ccccceeeeeeeeeeeeeeeeeeeeeees C 1 Gall 1OVENVIOW fsa l one ies rnanan taraa i r i Seaton eaa aaa e Guanes C 2 C 2 Preparing Data Before Accessing Macros 2 sceeeeeeeeeeeeeeeeeeeees C 3 C 3 Accessing the MaCrOS ccceeeee cece cece eee ia nao na C 4 C 4 Using the Ladder Sequencing Toolbox cceeeeeeee eee eeeeeeeeeeeeees C 5 C 5 Using the Peptide Fragmentation ToolboX 2 eceeeeeeeeeeeeeeeeeeeees C 9 C 6 Using the Polymer Analysis TOOIDOX 2 e eceeeeeeeeeeeeeeeeeeeeeeeee C 15 C 7 Using MS Fit MS Tag Toolbox ceeeeeee eee e eee ee eee ee eee ee eeeeeeeeeee C 18 Index Data Explorer Software U
239. ng macros 5 2 to combine processing functions Applied Biosystems e Single spectrum Double click any point in the TIC to display the corresponding spectrum e Combined spectrum Click drag across a region of the chromatogram to sum the intensity at each mass for all selected spectra Combining spectra can improve peak shape signal intensity and signal to noise ratio NOTE You cannot display a combined spectrum for Voyager single spectrum data files e DAD spectrum Mariner DAD data only Click the Chromatogram window select Traces from the Display menu select the DAD TAC or channel of interest then double click any point in the TAC or channel data trace to display the corresponding DAD spectrum Voyager PSD data files contain the precursor spectrum and fragment ion spectra You can view up to eight spectra at a time and can click and to display other fragment ions You can create macros that perform multiple functions for example smooth and baseline correct and then start the macro with one mouse click For information see Section 6 7 Using the Macro Recorder Overview Returning to the Many processing functions generate a new trace If you have original spectrum Trace Replace mode set to Replace the new trace replaces the original trace For information on Replace mode see Section 2 4 4 Adding Traces from the Same Data File to a Window To return to the original spectrum e If the origi
240. ng mode to Display Relative as described in Section 2 2 Adjusting the Display Range 3 Link the data files by selecting the Spectrum window for each data file then selecting Link View from the View menu You must select Link View for each data file NOTE Clicking in the toolbar links traces not views 4 Organize the Spectrum windows for the data files by clicking and in the toolbar 5 Zoom and manipulate traces as needed 6 To print all data files select Print All Views from the File menu 2 36 Applied Biosystems Working with Multiple Data Files NOTE If you select Print All Views when more than two data files are open certain printers may not print the data file name To ensure data file names are printed print views individually or only open two data files before you select Print All Views 2 5 2 Copying Traces from Multiple Data Files to a Window You can copy up to seven traces from open data files to a different trace window 1 Select the trace to copy 2 From the Edit menu select Copy then Trace Data or right click the trace then select Copy Trace Data 3 Activate the window in which to paste the trace NOTE The Add Replace Trace state as set in the Display Trace dialog box determines whether the copied trace replaces or is added below the active trace See Setting the Replace mode on page 2 17 4 From the Edit menu select Paste then Trace Data or
241. nium ions labeling C 9 IMMONIUM_IONS REF 5 18 8 19 Import Calibration error displayed 9 11 9 12 procedure 5 16 PSD 8 20 Importing macros into DATA EXPLORER VB6 6 43 PKT files to Excel 3 41 trace from ASCII format 1 35 Improving signal to noise ratio 5 2 7 2 Initial Error calibration 5 12 Inserting peaks in peak list 3 39 In source CID data labeling C 9 Instrument calibration reapplying 5 22 reverting to 5 22 Instrument settings extracting from DAT file 1 36 tab Output window 1 16 viewing 1 16 Integration description 3 70 setting chromatogram 3 21 setting spectrum 3 26 Valley to Baseline chromatogram 3 21 3 70 Valley to Baseline spectrum 3 26 3 70 Valley to Valley chromatogram 3 21 3 70 Valley to Valley spectrum 3 26 3 70 Internal mass calibration 5 5 5 26 Internal standard calibration see Calibrating mass lon Fragmentation Calculator ISO in spectrum header 2 32 6 6 description 6 25 procedure 6 25 PSD segment spectra labeling 8 8 REF file PSD creating 8 21 results 6 29 sequence codes acceptable 6 26 6 16 6 18 6 33 Isotope average mass labeling 3 10 7 11 description B 1 displaying theoretical 6 33 isotopic envelope B 5 list of common B 8 monoisotopic and average masses B 6 monoisotopic labeling 3 43 overview B 1 parameters controlling charge state determination 3 27 partially resolved labeling 7 11 resolution limits of system 3 53 resolution overview B 4
242. njection Number fi OK Cancel Figure 4 10 Event Tag Dialog Box 4 24 Applied Biosystems Displaying MS Method Data Mariner Data Only NOTE Only tags present in the data file are available 6 Select the event tags to display then click OK The filtered trace is displayed with an EF trace label Figure 4 11 1 9E 5 2 a S r r 1 0 850 890 930 970 1010 1050 TIC gt EF CID 1005 CID 1 9E 5 904 804 704 604 amp 504 404 304 204 104 0 r r 7 r 0 850 890 930 970 1010 1050 Spectrum Number Figure 4 11 Event Filtered Trace NOTE The spectra in an event filtered trace are numbered contiguously 1 2 3 regardless of their relation to the overall acquisition However because the axes of the trace reflect the numbering of the overall experiment you may see spectra with numbers that do not correspond to the x axis Hint Line mode is useful when displaying filtered LC TIC traces Vertical bar mode may be more useful when filtering direct infusion TIC traces If you have the Replace mode set to Add in the Display Trace dialog box a new trace is added Data Explorer Software User s Guide 4 25 Chapter 4 Examining Chromatogram Data Hint Add mode is useful when you are filtering the same trace for different event tags The original trace remains displayed and accessible Each filtered trace up to four total tr
243. nown standard analyzed in PSD mode You can then export the PSD CAL file and include the PSD CAL file in a PSD instrument settings BIC file you use to acquire unknown PSD samples Internally calibrate an unknown PSD data file by specifying known monoamino acid fragment ions as the peaks to match during calibration To create a PSD CAL file Acquire a peptide standard with a known sequence in PSD mode Check peak detection as described in Section 8 3 1 Checking Peak Detection Create a calibration reference REF file as described in Section 8 3 4 Creating PSD Calibration Reference REF Files Internally calibrate the PSD DAT file as described in Section 8 3 2 Calibrating Specify the calibration reference REF file created in Section 8 3 4 Creating PSD Calibration Reference REF Files when calibrating Export the calibration CAL file from the PSD DAT file to use when acquiring unknown samples in PSD mode Calibrating a PSD Spectrum 8 3 1 Checking Peak Detection Checking Before calibrating check that peaks in all segment traces of interest are properly peak detected and that noise is not detected as peaks Note the following when setting peak detection parameters Peak detection settings are applied to the currently displayed composite spectrum or segment traces The default peak detection settings in VOYAGERPSD SET have different detection ranges for different mass ranges You can fine tune ran
244. ns are saved with the data file 1 5 1 Changing Background Color White or dark You can switch background color by selecting Default from background the Display menu then selecting White Background Displays blue traces and red labels by default Default settings are contained in DEFAULTWHITE SET Dark Background Displays yellow traces and green labels by default Default settings are contained in DEFAULTBLACK SET NOTE These SET files contain graphic settings only They do not contain processing settings You can customize the graphic settings associated with default settings if desired For information see Section 1 4 2 Customizing Processing and Graphic Settings SET Data Explorer Software User s Guide 1 23 Chapter 1 Data Explorer Basics 1 1 5 2 Customizing Graphic Options 1 24 This section includes Accessing graphic options Setting colors Setting line widths Setting data cursors Setting traces in line or bar mode Setting graphic compression Accessing To access graphic options graphic options Applied Biosystems 1 2 3 Display the trace of interest From the Display menu select Graphic Options To use the graphic options settings for all traces click Use same settings for all traces in the View Setup tab Click a Graph Setup tab in the Graph and Plot Options dialog box Figure 1 4 Set colors line widths data cursors and graphic compression as
245. nt calibration e Mass Calibration Edit Create Reference File e Dual Spectral Trace Arithmetic e Multiple Charge Mass Deconvolution Peaks e Peak Detection Peak Label e Insert Peak chromatogram Tools e Processing History Options e Customize Toolbar e Customize ToolMenu e Macro commands including Automatic Macros Applications e Elemental Targeting e lon Fragmentation Calculator Window No commands supported You can apply specific Graphic Options or Peak Detection settings by creating a macro that loads a SET file containing the desired settings You can apply specific peak labels by creating a macro that loads a USR user label file containing the desired labels Functions you perform in the Output window for example sorting or copying the peak list are not supported by the Macro Recorder 6 36 Applied Biosystems 6 7 2 Recording a Macro To record a macro 1 2 Using the Macro Recorder Open a data file From the Tools menu select Record New Macro The Record Macro dialog box Figure 6 23 is displayed Record Macro xj Macro Name Macro2 Description ps Macro Recorded 02 22 99 OK Cancel Figure 6 23 Record Macro Dialog Box Type a name and a description if desired Click OK Select the commands you want to automate with the macro For example select Noise Filter Smooth from the Process menu select the smoothing method specify the associated pa
246. nts are saved with the spectrum Apply to All All spectra in the data file are calibrated using NOTE This the currently displayed calibration and are button is displayed with an MC trace label The calibration displayed only if constants are saved with the data file Each you are spectrum in the data file is calibrated when calibrating a displayed Voyager NOTE If you select Apply to All you overwrite multispectrum any calibrations previously applied to individual data file spectra by clicking Apply Calibration Data Explorer Software User s Guide 5 15 Chapter 5 Examining Spectrum Data Exporting The calculated calibration constants can be exported to a calibration CAL file for use with other data files You have two options for constants amp xporting CAL file Export the current calibration Click Export in the Manual Mass Calibration dialog box to calculate and export the current calibration constants from the masses displayed in the Peaks Matched list in the Manual Mass Calibration dialog box NOTE The calibration constants saved in the CAL file are not calculated from the active trace on the screen or taken from the calibration in the data file They are calculated from the listed masses Export a calibration from a previously calibrated data file Open an existing data file then export the calibration constants in a CAL file See Exporting BIC MSM and CAL files on page 1 3
247. nts for calibrating Mariner data e Hints for calibrating Voyager data NOTE Manual calibration is not supported for Mariner DAD data 5 3 1 Overview of Manual Calibration Overview During manual calibration e You specify a calibration reference file REF that contains reference masses The software matches peaks by comparing observed masses in the spectrum to reference masses e The software lists masses that match within the specified peak matching criteria and calculates calibration constants e You can apply the calibration constants to all spectra in a Mariner or Voyager data file e You can apply individual calibration constants to each spectrum in a Voyager data file Data Explorer Software User s Guide 5 5 Chapter 5 Examining Spectrum Data The manual calibration feature provides two modes for peak matching e Automatic The software automatically compares reference masses to observed masses and lists peaks that are within the specified peak matching criteria e Manual You manually select an observed mass then select the reference mass for comparison Hint It is often useful to automatically perform a match and fit first then manually adjust the fit as needed Calibration Default calibration reference files are provided with the references files software You can use these files as a starting point and add REF provided delete reference masses as needed For information
248. o align with the chromatogram trace peak NOTE To restore the original UV trace open the UV Trace Offset dialog box see step 4 then click Reset To return to the original trace see Returning to the original trace on page 4 4 Data Explorer Software User s Guide 4 31 Chapter 4 Examining Chromatogram Data 4 32 Applied Biosystems 5 Examining Spectrum Data This chapter contains the following sections 5 1 OVEIWVIOW iet 5 2 5 2 Creating a Combined Spectrum s s s 5 4 5 3 Manual Calibration c cccccecsseeeeeeeeseeeeeeeaes 5 5 5 4 Automatic Calibration 5 26 5 5 CONTOMMING scrieti Pevbaeesi aiaia 5 36 5 6 Mass Deconvolution Mariner Data Only 5 37 5 7 Noise Filtering Smoothing ceeeeeeees 5 42 5 8 Adjusting the Baseline eeen 5 45 5 9 Truncating a SpeCtruM c eeseeeeeeeeeeeeeees 5 56 5 10 Converting to a Singly Charged Spectrum Mariner Data Only sseeeeeeeeeeeeeeeeeees 5 59 5 11 AutoSaturation Correction Mariner Data Only c seeeeeeeeeeeeeeeeeeees 5 62 5 12 Adding and Subtracting Raw or Processed Spectra From the Same or Different Data Files Dual Spectral Trace Arithmetic 5 64 Data Explorer Software User s Guide 5 1 Chapter 5 Examining Spectrum Data 5 1 Overview Types of spectra You can display the following types of spectrum data you can display Voyager PSD spectra Creati
249. o one of the following e Select an existing range then click This creates a new range with boundaries ranging from the end of the existing range to the end of the trace If the existing range ends at the end of the trace the region of the existing range is split in half between the existing range and the new range e Double click an existing range to manually enter lower and upper boundaries e Select a range in the dialog box then click drag the X data cursors labels in the trace to set the lower and upper boundaries To delete a range select the range then click To combine all ranges in the list into one range click Em The peak detection settings displayed in the dialog box correspond to the selected range To view peak detection settings for another range select the range of interest Continued Data Explorer Software User s Guide 3 29 Chapter 3 Peak Detection and Labeling Table 3 4 Advanced Settings Spectrum Data Only Continued Parameter Description Active Range Thresholds NOTE These settings apply to the Detection Range selected and override the Global Thresholds specified on the Basic Settings tab described on page 3 19 BP Intensity See Base Peak Intensity on page 3 20 Max Peak Area See Max Peak Area on page 3 20 Minimum Intensity Specifies the absolute peak intensity below which peaks are not detected Calculated relative to zero NOTE
250. oceco eo sz a OF Coemi Figure 6 3 Element Limits Dialog Box Data Explorer Software User s Guide 6 7 Chapter 6 Using Tools and Applications 2 To change the limits for an element double click an element to display the Isotope dialog box Figure 6 4 Isotope Mass Abund Minimum Maximum 0 500 12C 12 00000 0 99 0 0 13C 13 00335 0 01 0 0 14C 14 00324 0 00 0 0 OK Cancel Figure 6 4 Isotope Dialog Box NOTE Ignore the column of check boxes to the left of the Isotope column if it is displayed 3 Change the Minimum and Maximum number of occurrences for the element as needed NOTE The software ignores changes you make to the individual isotope minimum and maximum values Click OK Change limits for other elements as needed then click OK to return to the Elemental Composition Calculator 6 8 Applied Biosystems Using the Elemental Composition Calculator Adding new To add new elements and set limits elements and 1 To add new elements and set limits for Elemental setting limits results click Element Limits in the Elemental Composition Calculator dialog box The Limits dialog box is displayed Figure 6 5 SS A Senn Pal pirani hiia Hoe Fi J a a 1h H i ify r o D 5 u 5 F D Ha fi i oOo oO br L F D l o D OF Coemi Figure 6 5 Element Limits Dialog Box 2 Click 3 The Periodic Table Figure 6 6 is displayed
251. of peaks that must meet the matching criteria for this calibration to be successful e Maximum Outlier Error m z or ppm Tolerance within which all matched peaks must fall for this calibration to be successful NOTE If the software finds fewer than three matches the maximum Outlier Error is not significant Masses are labeled with the specified reference masses 12 Click Save Settings to save the automatic calibration settings reference masses matching criteria and fit rejection parameters as part of processing settings in the DAT file Saving the SET if you are preparing Automatic Calibration settings for use by file the Voyager Sequence Control Panel save the SET file by selecting Settings from the File menu then select Save Graphic Processing Settings As Data Explorer Software User s Guide 5 33 Chapter 5 Examining Spectrum Data 5 4 3 Automatically Calibrating Mariner Data Only This section includes e Automatically calibrating e Applying new constants to the data file e Calibration results e Applying auto calibration settings to other files Automatically To automatically calibrate calibrating 1 Open the data file to calibrate 2 Click the Spectrum window 3 From the Process menu select Mass Calibration select Auto Calibrate State then select On NOTE The Auto Calibrate State command setting is stored in a DAT file That is if you turn on this command then close the D
252. og box Figure 6 20 is displayed Enter the Mass Tolerance for the evaluation If the displayed Mass Peak Resolution is not appropriate for this calculation change the setting in the Basic Settings tab of the Spectrum Peak Detection Setup dialog box For more information see Section 3 2 4 Peak Detection Parameter Descriptions Enter formulas in the Element list by doing either of the following e Click 3 type a formula select a charge state then click OK e Click Import then select a tab delimited TXT file that contains formulas and charge states Data Explorer Software User s Guide 6 31 Chapter 6 Using Tools and Applications Note the following when entering formulas e Spaces do not matter for formula The first letter of two letter elemental symbols must be capitalized for example Na e To ensure a better match between theoretical and observed isotopes include the appropriate number of protons in the formula you enter for multiply charged ions Elemental Target x Mass Tolerance Mass Peak Resolution Dalton joo m PPM fo Element List Import Saves Cancel Figure 6 20 Elemental Targeting Dialog Box 7 Click Calculate 6 32 Applied Biosystems Displaying results CHATS mre edb th 7 eats SOM mall Si it 1 DIH CHH Using the Elemental Targeting Application The results of the calculation are displayed in the Elemental Ta
253. old e Scanning from the valley regions toward the apex region using the number of data points defined by the Filter Width If the difference between two consecutive filtered regions is greater than the Noise Threshold the midpoint of the filter region closest to the apex is used as the peak bound e Compares the area of the new peak to the Max Peak Area and the Minimum Area A Minimum Area of 2 is used for chromatogram data If the new peak is within the specified area settings the peak is added to the peak list 3 68 Applied Biosystems Process that Occurs During Peak Detection Centroiding and Integration After peak After peaks are detected detection _ Gentroid mass is calculated for spectral data then modified by Gaussian peak fitting if it is selected e Chromatographic and spectral peaks are integrated e Peak lists are generated for chromatograms and spectra Centroiding The Data Explorer software calculates the peak centroid for spectral data by e Drawing a projected centroid baseline at the percentage of the peak height specified by the Centroiding entered Centroiding is measured between the top of the peak and 0 For example a centroiding of 10 uses the top ten percent of the peak where peak height is determined from 0 e Searching to the left and right of the apex for data points that bound the projected centroid baseline then interpolating the centroid baseline e Calculating the intensity weighte
254. ols menu select Automatic Macros The Automatic Macro Setup dialog box Figure 6 26 is displayed Aeon Sco iat acu Dacie P Fi 3 Figure 6 27 Automatic Macro Setup Dialog Box 2 Select File Open Macro or File Close Macro as desired then select the macro to run when you open or close a data file 3 Click OK Data Explorer Software User s Guide 6 45 Chapter 6 Using Tools and Applications 6 46 Applied Biosystems 7 Data Explorer Examples This chapter contains the following sections 7 1 Mariner Data Examples 7 1 1 Improving Signal To Noise Ratio 7 2 7 1 2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks 7 4 7 1 3 Determining if a Peak is Background Noise secese 7 8 7 2 Voyager Data Examples 7 11 7 2 1 Detecting and Labeling Partially Resolved Peaks 7 11 7 2 2 Processing Before Calibrating to Optimize Mass Accuracy 0000 7 14 7 2 3 Detecting Peaks from Complex Digests cceeceeeseeeeeeees 7 18 Data Explorer Software User s Guide 7 1 7 Chapter 7 Data Explorer Examples 7 1 Mariner Data Examples This section includes e Improving signal to noise ratio e Deconvoluting and evaluating unresolved chromatographic peaks e Determining if a peak is background noise 7 1 1 Improving Signal To Noise Ratio 7 2 Overview Creating an extracted ion chromatogram Applied Biosystems You can impr
255. omplete file name will be Cal_PSD CAL You can apply calibration constants from a CAL file to any data file To apply the new constants from a mass calibration file to a different file 1 2 3 Display the spectrum to calibrate From the Process menu select Mass Calibration Select Import Calibration Select the CAL file to use Click Open The software displays the calibrated spectrum with an MC trace label To save the calibration to the data file select Mass Calibration from the Process menu then select Apply Calibration Calibrating a PSD Spectrum 8 3 4 Creating PSD Calibration Reference REF Files Overview Using the lon Fragmentation calculator to create a calibration reference REF file You can manually create a calibration reference file by typing masses in a text file as described in Section 5 3 3 Creating or Modifying a Calibration Reference File REF You can also use the lon Fragmentation calculator to generate theoretical fragments and masses from the sequence for a standard compound then automatically save the masses and associated information in a calibration reference file For detailed information on using the lon Fragmentation calculator see Section 6 5 Using the lon Fragmentation Calculator To create a PSD calibration reference file using the lon Fragmentation calculator 1 From the Applications menu select lon Fragmentation Calculator The lon Fragmentation Calculato
256. omponent To evaluate further create an extracted ion chromatogram for 391 Da as described below and evaluate the signal To create the extracted ion chromatogram right click drag over the peak at 391 Da in the spectrum trace NOTE You can also create an extracted ion chromatogram by selecting Trace from the Display menu selecting Extracted lon then entering a mass Figure 7 8 illustrates the extracted ion chromatogram for mass 391 The signal is relatively consistent indicating that it is background instead of a component peak OTS rinrin i Ft Dei ap KIC UEIS 5 peliuin Himbar Figure 7 8 Extracted lon Chromatogram for Mass 391 Da Applied Biosystems Voyager Data Examples 7 2 Voyager Data Examples This section includes e Detecting and labeling partially resolved peaks e Processing before calibrating to optimize mass accuracy e Detecting peaks from complex digests 7 2 1 Detecting and Labeling Partially Resolved Peaks If peaks are not if peaks are partially resolved and the peaks of interest are not labeled _ labeled you can adjust the following peak detection parameters If Adjust the following Peaks represent two In the Peak Label dialog box select Allow Overlapping compounds and you Peak Labels want both peaks labeled oR On the Basic Settings tab in Peak Detection set Max Peak Area to 0 then adjust the Base Peak Intensity until peaks are detected OR On the Pea
257. ompound is different for example charge state or name Each mass in the list is considered during calibration Do not include multiple entries with the same m z value in the Calibration Reference file See Section 5 3 3 Creating or Modifying a Calibration Reference File REF REF file created with Windows Notepad not listed when you select Reference file Reference file does not include REF extension Some applications automatically append a TXT extension to file names To name the file with a REF extension include the file name and extension in double quotes in the Save File dialog box for example CAL REF Data Explorer Software User s Guide 9 11 Chapter 9 Troubleshooting Table 9 7 Calibration Troubleshooting Mariner Only Symptom Possible Cause Action Mass Calibration commands are dimmed Chromatogram window is selected Select Spectrum window Apply Calibration command is dimmed when calibrating MS Method data For MS Method data calibration is valid for an individual spectrum You cannot apply the calibration from one spectrum to the entire data file No action Normal occurrence Error displayed when you import a calibration CAL file corrupted Create new CAL file See Exporting BIC MSM and CAL files on page 1 36 Importing a CAL file generated from a Voyager data file Import a M
258. on chromatogram 3 20 definition spectrum 3 22 in peak detection algorithm 3 67 Mariner data 3 7 setting for active detection range spectrum 3 30 setting global spectrum 3 22 setting with data cursor chromatogram 3 11 3 16 setting with data cursor spectrum 3 23 setting chromatogram 3 20 Voyager data 3 10 Max Peak Area definition chromatogram 3 20 definition spectrum 3 23 Mariner data 3 7 setting for active detection range spectrum 3 30 setting global spectrum 3 23 setting chromatogram 3 20 Voyager data 3 9 in spectrum header 2 31 in spectrum header 2 31 A a and b ion pairs labeling C 10 a b y 17 ion pairs labeling C 10 Absolute Threshold replaced by Minimum Intensity 3 30 AC in spectrum header 2 31 5 34 Accelerating Voltage changes compensated for by system 5 25 effect on calibration 5 25 Accumulate spectra 4 22 Acquiring data comment displaying 1 15 comment displaying for open data file 1 15 comment displaying when opening a data file 2 3 version of software used for 1 15 Acquisition comment displaying for open data file 1 15 displaying when opening a data file 2 3 Activate file 2 8 Add mode setting for added traces 2 18 Adding and subtracting spectra from different data files 5 64 from same data file 5 4 raw data only 4 20 5 4 raw or processed data 5 64 within a data file 4 20 Adding peaks to peak list 3 39 Adding text to traces 2 29 Adding traces maximum number 2 18 procedure 2
259. on constants CAL from a DAT or results file 1 Open or activate the data DAT or results RSD or RCD file 2 From the File menu select Export then select e Calibration To export the last applied calibration constants in a CAL file Saving SET files 3 Managing Files NOTE To export calibration constants used to acquire the data select Mass Calibration from the Process menu then select the Revert to Instrument Calibration before exporting For more information see Section 5 3 4 Reverting to Instrument Calibration e Configuration To export BIC or MSM files MSM Mariner data only NOTE To access the instrument settings for each spectrum in a data file acquired using a Mariner MS Method you must first extract the MSM file from the data file as described above Then export the BIC files from the MSM file using the Export button in the MS Method editor For more information on exporting a BIC file from MSM see the Mariner Workstation User s Guide In the Save As dialog box type a name for the exported file then click Save To save processing and graphic settings SET from a DAT or results file 1 2 Open or activate the DAT RSD or RCD file From the File menu select Settings then select one of the following e Save Processing Settings As e Save Graphic Settings As e Save Graphic Processing Settings As In the Save As dialog box type a name for th
260. ond equivalent of 0 5 with an actual number of double bonds of 1 Formula Elemental composition for each mass Isotope Match Score Number between 0 and 1 0 that reflects how well the observed peak matches the theoretical formula based on mass and isotope pattern A higher number represents a better match NOTE An Isotope Match Score of 0 00000 is always reported for fragment ion calculations If no results are if no results are displayed it may indicate that the specified displayed Tolerance is too limiting and no matches were found 6 6 Applied Biosystems Using the Elemental Composition Calculator Displaying the To display the theoretical isotope distribution for a calculated theoretical formula double click the corresponding line in the Elemental isotope Analysis tab of the Output window distribution The theoretical isotope trace is displayed in the Spectrum window with an ISO trace label and the elemental formula To return to the original trace see Returning to the original spectrum on page 5 3 6 1 2 Setting Limits This section includes Setting limits for existing elements e Adding new elements and setting limits Setting limits for other result types Setting limits for To set limits for existing elements existing elements 1 To set limits for Elemental results click Element Limits in the Elemental Composition Calculator dialog box The Limits dialog box is displayed Figure 6 3 BS
261. ontinued Symptom Possible Cause Action Results not saved for all Only results for the active Display individual traces traces in an overlaid trace trace are saved then save results for each trace For more information see e Section 2 4 8 Overlaying Traces e Section 2 6 Saving Opening and Deleting DAT Results e Section 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only Link View command does You did not select Link You must select Link View not have an effect on View for each window or for each window and each windows or open data files data file data file you want to link See Linking views on page 2 13 Data Explorer Software User s Guide 9 7 Chapter 9 Troubleshooting Table 9 4 Processing Tools and Applications Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action After Single charge Conversion of multiply charged peaks you see charge states other than 0 or 1 Peaks in the original spectrum are labeled with an incorrect charge state Set peak detection thresholds to disregard these peaks See Section 3 2 5 Charge State Determination and Examples Convert the spectrum again See Section 5 10 Converting to a Singly Charged Spectrum Mariner Data Only 9 8 Applied Biosystems Processing Tools and Applications Troubleshooting Table 9 5 Processing
262. ontinued instrument settings viewing 4 23 spectrum numbers in filtered trace 4 25 MSM files extracting from DAT file 1 36 overview 1 7 Multiple Charge command dimmed on menu 5 37 9 8 not displayed on menu 9 8 Multiple data files comparing 2 36 2 38 copying traces into a window 2 37 printing 2 36 working with separately 2 36 zooming 2 13 2 36 Multiple spectra in Voyager data files 2 7 Multiply charged peaks deconvoluting 5 37 single charge conversion 5 59 Multi point calibration see Calibrating mass N Negative ion z label 3 58 Neutral loss chromatogram see Extracted ion chromatogram CNL NF in chromatogram header 2 30 4 19 in spectrum header 2 32 5 44 Noise filtering chromatogram 4 17 spectrum 5 42 spectrum recommended setting 4 18 5 43 Noise Threshold chromatogram calculated automatically 3 21 3 68 in peak detection algorithm 3 68 setting locally spectrum 3 30 Noise screening out noise filtering command 4 17 5 42 subtracting spectra and creating extracted ion chromatogram Mariner data 7 8 truncate spectrum 5 56 Nozzle Temperature displaying trace 4 2 NR in chromatogram header 2 30 4 19 in spectrum header 2 32 5 44 Number average molecular weight determining C 15 Number of data points across a peak determining 3 21 3 31 5 51 O Offsetting baseline chromatogram 4 27 spectrum 5 45 Offsetting UV trace Mariner data only Mariner data 4 30 restoring original trace 4 31 Open file
263. oolbar If a numbered macro button is disabled gray no macro has been assigned to it If a numbered macro button is enabled green a macro has been assigned to it For a description of a toolbar button place the cursor on the toolbar button The name you assign to the macro or the default macro name is displayed below the button Functions not Most commands in the Data Explorer software can be supported included in a macro that you create with the Macro Recorder The following menu commands are not supported by the Macro Recorder Menu Commands Not Supported File Open Close Close All Result Spectrum Delete Convert All Spectra New Data Format Export Result Spectrum Settings Save Processing Settings Settings Restore Graphic Settings Settings Save Graphic Settings Settings Revert to last saved Graphic Processing Settings Print Setup Exit Continued Data Explorer Software User s Guide 6 35 Chapter 6 Using Tools and Applications Menu Commands Not Supported View No commands supported Display e Add Remove Traces NOTE The and buttons are supported by the Macro Editor e Processing History e Range e Graphic Options e Default Revert to Last Graphic Settings Process e PSD processing e Mass Calibration Manual Calibration e Mass Calibration PSD calibration e Mass Calibration Automatic Calibration e Mass Calibration Revert to instrume
264. or e Section 6 3 Using the Mass Resolution Calculator Section 6 4 Using the Signal to Noise Ratio Calculator 2 Click the Results tab in the Output window 3 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 2 28 Applied Biosystems Manipulating Traces Annotating the To annotate the trace trace 4 Click the trace at the location where you want to insert text 2 Right click then select e Paste text lf you copied results Add text annotation lf you want to type in text Type in text as needed The text is added to the trace and remains in the Spectrum window until you delete it NOTE The text is associated with the x coordinate If you display another spectrum the text remains in view 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 If you annotate overlaid traces only the text associated with the active trace is displayed NOTE To move the text click drag the text to the desired position 3 To customize the appearance of the annotated text see Section 1 5 Setting Graphic Options 4 To delete an annotation do either of the following e Select the text right click then select the appropriate delete or cut option e Right click the trace
265. or information 2 34 Applied Biosystems Dedicating a printer to landscape orientation Print Setup Manipulating Traces NOTE Line Widths of 0 or 1 or lines set to the color white may not print on certain printers If traces do print change the line width or color not To dedicate the printer to landscape orientation 1 From the Windows desktop click Start then select Settings Click Printers Select the printer name in the displayed list Click File then select Document Defaults a FF oO PN In the Page Setup tab select Landscape orientation NOTE If you cannot select Landscape orientation you do not have access permission See your administrator The Print Setup function allows you to select a printer and set printer options For more information on Print Setup and on connecting printers to your computer refer to the documentation provided with your computer Data Explorer Software User s Guide 2 35 Chapter 2 Using Chromatogram and Spectrum Windows 2 5 Working with Multiple Data Files When you have multiple data files open you can e Work with the data files separately to view zoom and print Copy traces from one data file to another to compare or combine data 2 5 1 Working with Separate Data Files To view zoom and print multiple data files 1 Open the data files as described in Section 2 1 Opening and Closing Data Files 2 Setthe Y Scali
266. or problems related to a rising global baseline and signal spikes Hint If you are analyzing digest data set Max Peak Area to 0 before deisotoping to ensure that all peaks of interest are detected For more information see Section 7 2 3 Detecting Peaks from Complex Digests Filter Width Specifies the number of data points used in smoothing for peak detection before integration The software uses a Filter Width Increment of 1 when applying smoothing NOTE If you set Filter Width too high narrow peaks may not be detected Continued 3 20 Applied Biosystems Peak Detection Table 3 1 Chromatogram Settings Continued Parameter Description Filter Width Hint Set Filter Width to a number equal to the number of points across the peak To determine the number of points across a peak you can change the trace display from Line to Vertical Bars Each vertical bar represents one data point For more information see Section 1 4 Customizing the Data Explorer Window continued Integration Baseline Settings NOTE You can set peak labels to display peak start peak end and baseline marks See Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels Valley to Baseline Drops a vertical line from all valleys to a horizontal baseline The level of the horizontal baseline is determined using the minimum peak valley point left or right for each peak See Figure 3 25 on page 3 70 Valley
267. orer Mariner data file open Figure 1 2 shows the Data Explorer main window with a Voyager data file open To exit the software select Exit from the File menu in the Data Explorer window The Data Explorer software closes l Davr Espiorer 091 4neuro_frag001 dat ioj xj File Edit View Display Process Peaks Tools Applications Window Help jemja i aise se rhe o Se Beeler els ao Ih CHRO 0914neuro_frag001 dat 12 4 Spectrum Number Spec 1 AS MC Intensity 4594 639 6 Mass m z SPEC 091 ln CHRO 091 For Help press F1 Figure 1 1 Data Explorer Window with Mariner Data Data Explorer Software User s Guide 1 3 Chapter 1 Data Explorer Basics Data Expherer ca _mix20001 dat File Edit View Display Process Peaks Tools Applications Window Help jenja twe egba sie lioe a4 Bite lee uy IMSPEC cal_mix20001 dat Spec 1 BP 1314 6 58462 1314 616 2 a 3 3 S 2117 574 2491 999 3691 346 1852 7242132 643 3473 874 2891 505 3199 147 3599 498 2199 4 2799 6 Mass m z l SPEC cal For Help press F1 Figure 1 2 Data Explorer Window with Voyager Data Default colors Tne default colors are different for Mariner and Voyager e Mariner Black background yellow traces and green labels e Voyager White background blue traces and red labels You can customize the default colors as needed See Section 1 5 1 Changing Backgroun
268. orting the Peak List Filtering Peak list filtering allows you to display only desired peaks in the spectrum the spectrum peak list Only peaks that are included in the peak list peak list are labeled on the trace To filter the peak list 1 From the Peaks menu select Filter Peak List Hint You can also display the Mass Peak List Filter dialog box by right clicking the Spec Peak List then selecting Peak Filter Applied Biosystems Peak List 2 Select Enable Peak List Filter then select Filter Type Description Monoisotopic Labels the peak of the lowest detected mass in an isotope envelope Before applying filtering set the values in Peak Processing detection parameters to accurately detect and label all peaks in the isotope envelope with the correct charge state If the parameters are not correctly set to yield correct charge states for all peaks the monoisotopic peak may not be correctly labeled See Section 3 2 5 Charge State Determination and Examples for more information on setting parameters Monoisotopic filtering is useful on isotope clusters where the approximate elemental composition and isotopic ratios are not known for example in PSD analysis where the precursor ion filtering enhances the precursor ion isotope intensity relative to the rest of the cluster or in a spectrum that contains molecules with very different elemental compositions For most applications especially peptide analysis the
269. ory 2 4 8 Overlaying Traces ccceceeeeeeeeeeeeeeeeeeeeeaeaaaeaeeeeeeeeeeeeeeees 2 4 9 ANMotating Trae svsrcisspevcedasteedece tninebenenvant ARS 2 4 10 Viewing Trace Labels 0 0 eceecceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaes 2411 Printing Traces srana ee ee 2 5 Working with Multiple Data Files 0c eceeee seen eee eee eee seen eens 2 5 1 Working with Separate Data Files ccceeeeeeeeeeeeeeeeeee 2 5 2 Copying Traces from Multiple Data Files to a Window 2 6 Saving Opening and Deleting DAT Results eeeeeeeeee es 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only espiri iiipin ciated seth cee cta ced divin stantial eta 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only cuiere ndienin ta euch aai ee AEE Aai iv Applied Biosystems Table of Contents Chapter 3 Peak Detection and Labeling 3 1 3 2 3 3 3 4 3 5 3 6 3 7 OVeMEW os tieb soda e a a E a a aaa aa 3 2 3 1 1 Default Peak Detection enerne ea a R EETA 3 2 3 1 2 The Resolution Based Peak Detection Routine 3 3 Peak DeteCtiOn lt ceneititie arets tasunen thats seat eie dete eee EIEEE OS ANKE SEP ARA 3 6 3 2 1 Strategy for Mariner Peak Detection ecese 3 6 3 2 2 Strategy for Voyager Peak Detection cceeeeeeeeeeeeeeeeeeees 3 8 3 2 3 Setting Peak Detection Parameters ccccssseeceeeeeenneeeeees 3 11 3 2 4 Peak Detection Pa
270. os 1 Access the Visual Basic Editor from Data Explorer as described in Section 6 7 6 Advanced Macro Editing 2 From the File menu select Import File 3 Select the BAS file Basic File module containing the macros or FRM file form containing the user interface for the macros if applicable then click Open Data Explorer Software User s Guide 6 43 Chapter 6 Using Tools and Applications 6 44 Exporting Applied Biosystems The selected macros are imported into the DATAEXPLORER VB6 project The BAS files are included in the Modules folder in the DataExplorerProject and the FRM files are included in the Forms folder in the DataExplorerProject All macros imported into the DataExplorerProject are displayed in the list of macros you can assign in the Data Explorer software See Section 6 7 3 Assigning Macros to Buttons To export macros J Access the VBA Editor from Data Explorer as described above From the File menu select Export File Select the BAS or FRM file to export then click Save NOTE You have to export modules or forms one at a time 6 7 8 Running Macros Automatically When Opening and Closing Files Using the Macro Recorder You can set the Data Explorer software to automatically run macros you previously created when you open or close a data file For information on creating macros see Section 6 7 2 Recording a Macro To set up automatic macros 1 From the To
271. otopic Peak List Filtering Turned On 3 34 Applied Biosystems Peak Detection Max Isotope If the Max Isotope is set too low the software incorrectly set too low groups peaks into different isotope clusters In this example with the Max Isotope set to 2 and Max Charge State set to 4 the software labels all peaks as charge state 3 Figure 3 10 Spec 1 BP 558 3 824 558 3 23 80 558 6 23 558 9 23 Intensity 559 3 23 T os 554 556 558 560 562 564 Mass m z Figure 3 10 Max Isotope Set Too Low Max Charge State Set Correctly The effect of setting Max Isotope too low is apparent when you turn on Monoisotopic peak list filtering Figure 3 11 shows that the software has grouped the peaks into two clusters and has identified two monoisotopic peaks Spec 1 BP 558 3 824 100 558 3 z3 824 90 80 2 7 a 60 S 50 40 558 9 23 30 i 20 10 0 r T meg z x en tf 554 556 558 560 562 564 Mass m z Figure 3 11 Max Isotope Set Too Low Monoisotopic Peak List Filtering Turned On Data Explorer Software User s Guide 3 35 Chapter 3 Peak Detection and Labeling Effect of Minimum The Minimum Intensity setting on the Advanced Settings tab Intensity can also determine how charge states are determined for a peak because it determines if the software can find a match for a peak Figure 3 12 illustrates two peaks correctly labeled with ch
272. ou open the data file you can select any of the following to apply Settings from the data file Described in Section 2 1 Opening and Closing Data Files Default settings Described below e Settings from a selected set file You can also apply processing and graphic settings that have been exported as stand alone SET files from other data files See Applying a SET file on page 1 20 Graphic settings include the attributes you set in Graphic Options described in Section 1 5 Setting Graphic Options Processing settings include e Peak detection parameters described in Section 3 2 4 Peak Detection Parameter Descriptions e Smoothing points described in Section 5 7 Noise Filtering Smoothing e Automatic calibration settings described in Section 5 4 Automatic Calibration NOTE You can open a SET file in Microsoft Notepad to view the complete file contents The following default settings files stand alone SET files that contain both processing and graphic settings are provided on your system in the C MARINER PROGRAM Set Files directory or the C VOYAGER PROGRAM Set Files directory e MARINER SET e VOYAGERLINEAR SET e VOYAGERREFLECTOR SET e VOYAGERPSD SET For more information on Peak Detection settings see Section 3 7 Default Peak Detection Settings Default graphic settings Customizing settings saved in a data file Making a copy of default SET files before customizing Custom
273. ough room for the label to be displayed the label may be suppressed Zooming the region of interest expands the trace and may allow the labels to be displayed To display labels when peaks are close together select Allow overlapping peak labels or change Orientation to Vertical or 45 degrees in the Peak Label dialog box e Peak filtering is enabled and only peaks that meet the peak list filtering criteria are labeled e Filter Width is set too high to detect the peak e If you have Match Charge State enabled for spectrum user labels the peak of interest does not have the charge state specified in the user label 3 66 Applied Biosystems Process that Occurs During Peak Detection Centroiding and Integration 3 6 Process that Occurs During Peak Detection Centroiding and Integration This section gives an overview of the process that occurs during peak detection centroiding and integration Parameters are defined in detail in Section 3 2 4 Peak Detection Parameter Descriptions Peak detection During peak detection the Data Explorer software e Divides the trace into detection ranges based on the number of data points per peak described in Section 3 1 2 The Resolution Based Peak Detection Routine e Starts scanning the chromatogram or spectrum at the origin of the x axis e Applies a Gaussian sliding filter to the number of raw data points defined by the Filter Width setting The filter moves across the raw data
274. ove the signal to noise ratio for low level components in the total ion chromatogram TIC by creating an extracted ion chromatogram for the mass of interest Each data point in the TIC represents the sum of all ion intensities for all masses in the corresponding spectrum By creating an extracted ion chromatogram for a specific mass you can screen out ion contribution from masses that you are not interested in The following example illustrates how you can create an extracted ion chromatogram to improve the signal to noise ratio for reserpine 609 Da 1 Display the data file 2 Inthe Chromatogram window click in the toolbar or set the Trace Replace mode to Add if you want to adda new trace See Section 2 4 4 Adding Traces from the Same Data File to a Window for information 3 From the Display menu select Trace then select Extracted lon 4 Inthe Extracted lon Chromatogram window select Center Window then type the mass of interest 609 Da and the window for the mass for example 0 5 Da 5 Click OK Mariner Data Examples Figure 7 1 illustrates the improved signal to noise ratio in the extracted ion chromatogram for three replicate loop injections Midian oa 11 ibai Be isi Higi EFI dit 4 nic w a 2 g ete M g ee mpi E yn Ope PS ees a ae ee ee ae m Fi s 1 nja rs pectin Hira Original TIC Extracted ion containing all chromatogram for 609 Da masses with improved signal to noise rat
275. page 8 7 e Applies baseline correction and two standard deviation noise removal to the composite region of each segment e Stitches the composite regions together and displays the composite spectrum PSD calibration The equation that the Voyager software uses to calculate the equation mass for fragment ions is 2 aR B my a Rit B i t ht SE mp where ms Fragment ion mass a 8 y Calibration constants R Mirror Ratio t Fragment ion flight time tp Precursor ion flight time at R 1 calculated during acquisition m Precursor ion mass entered in PSD Acquisition settings before acquisition or changed in PSD Processing 8 6 Applied Biosystems Displaying PSD Data If you are performing an internal standard calibration the software determines the constants as listed below PSD Internal Standard Calibration constant valle SPE One point a Calculated from standard mass Bandy 10 Two point or aand Calculated from standard masses three point Yy 0 More than three point B y Calculated from standard masses Region of The composite spectrum is generated from portions of the segments included segment traces The upper mass limit of the composite region in composite in each segment is determined by the PSD Mirror Ratio Rp with which the segment was acquired and the mass of the spectrum p precursor ion mp Figure 8 3
276. pe for highly charged narrow peaks Manual Calibration 3 To modify an entry click the entry to select it modify the entry as needed then click Update 4 To delete an entry click the entry to select it then click Delete 5 To add an entry type the Name and Theoretical m z for a reference compound then select the charge state Optionally enter the Elemental Composition for the compound 6 Specify the mass type Resolved Isotope or Average 7 Click Insert CAUTION The software allows you to add multiple items with the same m z value to the calibration list box if any other attribute of the reference compound is different for example charge state or name Each mass in the list is considered during calibration If the mass list contains duplicate entries the calibration may return an invalid number of matches 8 Click Save or Save As 9 Inthe Save As dialog box select a location and type a name for the file then click Save When specifying highly charged non isotopically resolved species with peaks less than 1 Da wide for example myoglobin 20 as reference masses in a calibration reference file set the peak type as a Resolved Isotope Mass even though it is not a resolved isotope The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the software mis
277. pend new labels to existing user label list For more information see Section 3 5 3 Setting Custom Peak Labels Click OK Data Explorer Software User s Guide 6 27 Chapter 6 Using Tools and Applications Setting 13 Click User Defined Amino Acids user defined amino The User Defined Amino Acids dialog box Figure 6 17 acids is displayed Defined amino acids Symbol one char E Monoisotopic mass Average mass A formula Glutamic Acid N Glutamine Sidechain formula Glycine Histidine Sidechain a formula Isoleucine cee Sidechain b formula Methionine Phenylalanine Pe Proline Immonium ion mass fe Validity of neutral loss sequence ions calculations Ul il Phosphorylated Ser H20 f NH3 T PO4 f SOCH4 Phosphorylated Thr Delete Close Phosphorylated Tyr Met Sulfoxide hi Bel Update Figure 6 17 User Defined Amino Acids Dialog Box 14 Add amino acid definitions and codes as needed NOTE You cannot modify pre defined amino acids User defined amino acids are not saved when you close the Data Explorer software 15 Click Close Calculating the 16 Click Induce Fragmentation fragment ions Results are displayed in the lon Fragmentation Calculator dialog box Figure 6 18 6 28 Applied Biosystems Using the lon Fragmentation Calculator Himma ia Cjan Hrein pme Free Sind reer Poeteery Tiera ma eawn br erame z Du
278. playing 1 12 DAD spectrum displaying 5 2 Mariner data continued examples 7 2 in source CID labeling C 9 isotope resolution limits 3 53 mass accuracy affected by peak shape 5 7 5 47 peak detection strategy 3 6 Mariner Sequence Control Panel automatic calibration settings reference masses for 5 27 MARINER REF viewing masses in 5 10 MARINER_NEG REF provided 5 18 MARINER_POS REF provided 5 18 Mass apex copying from peak list 1 41 apex labeling 3 57 average B 6 centroid see Mass centroid in chromatogram header 2 30 in chromatogram header replaced by XIC 2 31 labels 3 56 monoisotopic see Monoisotopic peak Mass accuracy affected by peak shape 5 7 5 47 multi point calibration versus single point calibration 5 8 8 10 Mass accuracy optimizing by AutoSaturation Correction Mariner data only 5 62 by baseline correcting and deisotoping before calibrating 7 14 by baseline correcting and noise filtering before calibrating 5 7 Data Explorer Software User s Guide Index 15 Mass Calibration commands dimmed on menu 9 11 not displayed on menu 9 8 Mass calibration see Calibrating mass Mass centroid calculating 3 39 copying from peak list 1 41 definition B 6 displaying peaks as histograms 5 36 labeling 3 57 Mass deconvolution command dimmed on Process menu 5 37 9 8 example 7 4 performing 5 37 result failed to calculate 9 8 results 5 40 Mass Difference peak label from adjacent peak regular labels 3
279. ples 3A011 dat 148 59 0 71 6 107 4 179 0 Spectrum Number 143 2 Spec 1 ASC BP 121 1 59 58 6 303 0391 3479 3 590 5 683 6772 4 429 4 619 6 809 8 Mass m z Intensity 239 2 1000 0 IMSPEC plate with 7 different samples 3A010 dat Spec 1 ASC BP 121 1 60 100 BaP Zi 30123913 503 6590 6 683 5771 5 239 2 429 4 619 6 809 8 Intensity 0 0 49 0 1000 0 LATERE 5 1 4 06 30 36 72 108 Spectrum Number Intensity 144 INSPEC plate with 7 different samples 3A009 dat Spec 1 ASC BP 121 1 58 211 1 307 3391 3 507 4 649 4 749 6 429 4 619 6 809 8 Mass m z 121 1 8310 0 49 0 100 Intensity 239 2 1000 0 59 8 Figure 2 2 Data Explorer Window with Four Mariner Data Files Open Each DAT File Displays a Chromatogram and a Spectrum Trace IMsPec Intensity 0 3999 0 GSTest1_1P100002 dat Spec 1 BP 6640 4 4421 6640 5 8070 6 5999 8 7000 2 8000 6 Mass m z 4999 4 Spec 1 BP 6642 8 22119 6643 7 Intensity 5999 8 7000 2 Mass m z 04 T 3999 0 4999 4 Spec 1 BP 6644 2 2996 2 2 g 7107 8 7667 0 8405 9 0 5999 8 7000 2 8000 6 9001 0 Mass m z Spec 1 BP 6640 8 8713 6642 4 Intensity 7021 4 7610 2 9342 0 5999 8 7000 2 8000 6 Mass m z 4999 4 0 9001 0 Figure 2 3 Data Explorer Window with Four Voyager Data Files Open Spectrum Traces Only Displaye
280. pplied Biosystems Peak Detection NOTE Resolution dependent settings do not apply to Mariner chromatogram data or Voyager PSD data For more information see Section 3 1 2 The Resolution Based Peak Detection Routine To apply settings to all traces select Use same settings for all traces in view To set parameters independently for all traces ina window deselect Use same settings for all traces in view Type the Mass Resolution value to use for peak detection The default value for the type of data displayed is acceptable for most applications Defaults are listed in Basic Settings spectrum data only on page 3 22 NOTE The Mass Resolution you set here is also used by the Elemental Composition Calculator the Elemental Targeting Application and the Default Smoothing function For more information see Section 6 1 Using the Elemental Composition Calculator Section 6 6 Using the Elemental Targeting Application and Section 5 7 Noise Filtering Smoothing Click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box Data Explorer Software User s Guide 3 15 Chapter 3 Peak Detection and Labeling Setting Peak To set Peak Processing parameters Processing 4 Click the Peak Processing tab in the Spectrum Peak parameters Detection Setup dialog box rum spect a ens The Peak Processing tab is displayed Figure 3 5 Bewe
281. r Range Extracted lon Chromatogram XIC which includes only the signal response from a mass window or range For more information see Section 4 2 1 Creating an Extracted lon Chromatogram XIC Extracted lon Neutral Loss Constant Neutral Loss chromatogram which extracts only the response from peaks that are separated by a selected mass difference For more information see Section 4 2 2 Creating a Constant Neutral Loss CNL Chromatogram Extracted Absorbance XAC Extracted Absorbance Chromatogram which includes only the response from a specified wavelength window or range For more information see Section 4 3 Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only Types of Voyager chromatograms can optionally be displayed for Voyager data multispectrum DAT files To display a Voyager chromatogram select Restore Chromatogram from the View menu For more information see Section 2 1 3 Displaying Voyager Chromatograms If you have a chromatogram displayed you can display the following type of Voyager data by selecting Traces from the View menu then selecting Select To display TIC Total lon Chromatogram which includes the entire mass range saved in the data file Each data point represents the sum of all ion intensities in the corresponding spectrum Data Explorer Software User s Guide 4 3 Chapter 4 Examining Chromatogram Data Yo
282. r dialog box is displayed Figure 8 4 on page 8 8 2 Inthe Sequence text box type the amino acid sequence of the standard compound Use single letter codes Set other parameters as needed For parameter descriptions see Section 6 5 Using the lon Fragmentation Calculator 3 Select Monoisotopic from the Calculate fragment ion masses as drop down list Data Explorer Software User s Guide 8 21 Chapter 8 Viewing Voyager PSD Data 8 22 Applied Biosystems NOTE This selection determines the mass type specified for the reference masses in the calibration reference file Use a calibration reference REF file that specifies the peak type for reference masses as Resolved Isotope Mass even if they are not resolved isotopes The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the software mistakes these narrow peaks as isotopically resolved and ignores the reference mass For more information on creating calibration reference files see Section 5 3 3 Creating or Modifying a Calibration Reference File REF Click Induce Fragmentation Results are listed in the ions table and fragment ions are labeled if they are present in the spectrum Click Create Reference File A Save As dialog box is displayed Name and save the calibration reference file Hint PSD
283. r more than one peak the mass of the highest peak is used 6 Click OK Data Explorer Software User s Guide 6 21 Chapter 6 Using Tools and Applications The result is displayed in the Output window Figure 6 13 Data Explorer SPEC 091 4neuro_frag001 dat iol x laj x l Eile Edit View Display Process Peaks Tools Applications Window Help jenar as eins stew emo st 2 Ele Spec 1 ASC BP 558 3 938 558 3 558 6 a 3 2 Resolution calculator results For Help press F1 Figure 6 13 Resolution Calculator Results 6 22 Applied Biosystems Using the Signal to Noise Ratio Calculator 6 4 Using the Signal to Noise Ratio Calculator Description A signal to noise ratio is typically used to describe how well a peak of interest in a spectrum or chromatogram is distinguished from background noise The Data Explorer software provides two signal to noise ratio methods that calculate signal to RMS noise electronic and chemical e Auto You specify the peak for calculation and the software automatically calculates the average noise across the spectrum Manual You specify the peak and the baseline region for calculation For accurate results this method requires a flat non rising baseline that does not include peaks The manual method is useful when evaluating a narrow region around a peak to determine the relative significance of a peak Calculating
284. r 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 Limited Product Warranty Limited warranty Applied Biosystems warrants that for a period of ninety 90 days from the date of installation the Data Explorer software designated for use with Mariner API TOF Workstations or Voyager Biospectrometry Workstations will perform substantially in accordance with the function and features described in its accompanying documentation when properly installed on the instrument system Applied Biosystems does Data Explorer Software User s Guide A 1 Appendix A Warranty Warranty period effective date Warranty claims A 2 Warranty exceptions Applied Biosystems 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 but no later than three 3 months from the date of shipment for 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
285. race 4 30 Reapplying instrument calibration 5 22 Recording a macro 6 37 REF files see Calibration reference file REF Reference masses calibration see also Calibration reference file REF adding to REF file 5 18 displaying list of 5 10 8 16 for Voyager Sequence Control Panel 5 27 5 28 selecting 5 10 5 14 7 17 8 15 8 16 References required for Data Explorer Toolbox Visual Basic macros provided C 3 Related documents xiv Removing traces active 2 21 inactive 2 21 Replace mode setting for added traces 2 18 Resolution Calculator not displayed 9 8 Resolution mass calculating 6 20 command not on menu 9 8 default peak height used 6 20 defaults used in peak detection 3 24 isotope B 4 isotope Mariner data 3 53 isotope Voyager data 3 53 isotope limited B 5 PSD segment optimum observed near Max Stitch Mass 8 4 results 6 22 trend within PSD segments 8 11 Resolution based peak detection see Peak detection resolution based Result tab Output window 1 15 Results see also RSD and RCD annotating traces with 2 28 copying 2 28 displaying in Output window 1 15 elemental composition 6 5 elemental targeting 6 33 exporting 2 39 extracting information from 1 36 ion fragmentation 6 29 isotope 6 19 mass deconvolution 5 40 name of raw data file result is derived from 1 15 peak list 3 38 RCD and RSD files 2 39 resolution mass 6 22 signal to noise ratio 6 24 Results DAT copying 2 28 deleting results 2 38 opening results
286. ram XIC eee 4 5 4 2 2 Creating a Constant Neutral Loss CNL Chromatogram 4 9 Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only wicsoies sisig boas talk Hace seine ade acne ete 4 13 Noise Filtering SMoothing s eeceee cece cece eee ee eee teat este eeeeeeee eee 4 17 Adding and Subtracting Raw Spectra Within a Data File 4 20 Displaying MS Method Data Mariner Data Only eeeeee ee 4 23 Adjusting the Baseline ssrrsrserersrereiis randha seceded KEARSKE ENES KEENE KAENA 4 27 4 7 1 Using Baseline Offset nesseeseesesserrrersrerrrrsssssrrsrnnrnrrrerns 4 27 4 7 2 Using Baseline Correction 0 cccccceeceeeeeeaeeaeeeeeeeeeeeeeeeeeeeeees 4 29 Using UV Trace Offset Mariner Data Only s eeeeee eens eee eee ee 4 30 Chapter 5 Examining Spectrum Data vi 5 1 5 2 5 3 5 4 OVOIVIOW E ees E be de Aecig cael ab cadice edendicn eae cenewanenecd seseed 5 2 Creating a Combined Spectrum ccceeceeeeee eee cece eee e eee eeeeeeeeeaeee 5 4 Manual Calibration cccccccce ene eee eee eee e eae eeaeeaeeeaeenaenaeenaeeaeenaes 5 5 5 3 1 Overview of Manual Calibration cccccceccseseeeeeeeeeeeeeeeeees 5 5 5 3 2 Manually Calibrating c ccceeeeeeeeeeeeeeeeaeeaeeeeeeeeeeeeseeeeeeeees 5 7 5 3 3 Creating or Modifying a Calibration Reference File REF 5 17 5 3 4 Reverting to Instrument Calibration sessen 5 22
287. rameter then click OK Not all functions in Data Explorer are supported See Functions not supported on page 6 35 From the Tools menu select Stop Macro Recording The macro is saved and added to the DATAEXPLORER VBS6 file Data Explorer Software User s Guide 6 37 Chapter 6 Using Tools and Applications 6 7 3 Assigning Macros to Buttons Only macros present in the DATAEXPLORER VB6 file can be assigned to buttons and run in the Data Explorer software NOTE If you have installed a new version of Data Explorer software new macros may be provided New macros are not available for use until you import them into the Data Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 Assigning a You can assign macros to 10 buttons To assign a macro macro toa button 4 From the Tools menu select Assign Macro The Assign Macro dialog box Figure 6 24 is displayed Assign Macros xi Macro Button Macro to run Macro 1 Emooth5pt a Macrol Macro 2 Macro4 Macro 3 Macro5 Macro 4 Macro 5 xl Assign De assign Cancel Figure 6 24 Assign Macro Dialog Box 2 Select the button to assign from the list on the left and the macro to assign from the list on the right 3 Click Assign The macro button in the toolbar to which you assigned the macro turns from gray to green if it did not previously have a macro assigned to it 6 38 Applied Biosystems
288. rameter Descriptions acnee 3 19 3 2 5 Charge State Determination and Examples ceecccseeeeeeeeeeeeeeeeeeaaeeeeeeees 3 32 Pak LISt E E tadeae en Genvendie reaediee ie edeiies Genactaiins 3 37 3 3 1 Displaying the Peak List 1 0 0 eeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeaeeeaees 3 37 3 3 2 Inserting Peaks in the Peak List ccc eeeeeeesnseeeeeeeeaeaeeeees 3 39 3 3 3 Saving the Peak List 0cccccceeeeeeeeeceeeeaeeeeeeeeeeeeeeeeeeeeeeeaaes 3 40 3 3 4 Sorting Filtering and Printing the Peak List e 3 42 Deisotoping a Sp ctrUM icin esnan ae a doves Aaaa Saeed 3 45 Pak Labelng eiere rine aaea ean EEA cee nce EARE RE RA EEN EAEAN TE KAAN ERA ASE 3 52 3 5 1 Charge State Labels argian iniaa n a as 3 53 3 5 2 Setting Chromatogram and Spectrum Peak Labels 3 54 3 5 3 Setting Custom Peak Labels ccccceccseeeeeeeeeeeeeeeeeeeeeeeees 3 61 Process that Occurs During Peak Detection Centroiding and Integration cceceee eee cece eee eee eee ee tees teenies 3 67 Default Peak Detection Settings ceeeeeeee eee e eee e teen eee eeeeeeeee 3 71 Data Explorer Software User s Guide v Table of Contents Chapter 4 Examining Chromatogram Data 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 OVEWICW esaea E E edie deseohveee edie see betdeneven de ANKARA 4 2 Creating an Extracted lon Chromatogram 2 cceeeeeeeeeeeeeeee teenies 4 5 4 2 1 Creating an Extracted lon Chromatog
289. re acceptable click Apply Calibration Figure 7 17 shows the deisotoped spectrum after calibration with improved mass accuracy Intensity Spec 1 gt BC gt DI gt MC BP 754 3 981364 199 5 4E 5 80 3657 9294 70 60 i 2093 0867 30 1296 6853 2465 1989 20 i __ 967 0 1707 2 2447 4 3187 6 3927 8 4668 0 Mace im 7 Figure 7 17 Deisotoped Spectrum After Calibration Data Explorer Software User s Guide 7 17 Chapter 7 Data Explorer Examples 7 2 3 Detecting Peaks from Complex Digests Overview Complex digests often contain hundreds of peaks which may have relatively low signal to noise ratios To quickly screen out noise and detect peaks of interest e Noise filter smooth to remove initial noise e Set initial peak detection thresholds low enough to detect all peaks to ensure that monoisotopic peaks are detected for proper deisotoping e Deisotope to identify isotope peak clusters The deisotope function amplifies peak intensity based on the total area of all peak areas in the isotope cluster It does not amplify noise peaks e Increase peak detection thresholds to eliminate noise peaks Figure 7 18 represents a spectrum trace from a digest Overlapping Peak Labels are enabled to illustrate the large number of peaks detected in the spectrum over 300 peaks contained in the peak list displayed in the Output window Spec 1 BP 1012 4 46590 1012 380 4 7E 4 Intensity
290. re version used for acquisition e Acquisition time and sample comments entered when data was acquired For result files RCT RST RCD RSD displays any processing functions that have been performed and saved in the result file Data Explorer Software User s Guide 1 15 Chapter 1 1 16 Displaying clearing and closing Applied Biosystems Data Explorer Basics Instrument Setting Displays a list of instrument settings used to obtain the data The settings are taken from instrument settings pages in the Instrument Control Panel Also displays segments event numbers and event tags from Mariner MS Method acquisitions and LC information if LCMS was acquired using Mariner e Elemental Analysis Displays results for the Elemental Composition calculator For information see Section 6 1 Using the Elemental Composition Calculator Elemental Targeting Displays results for the Elemental Targeting application For information see Section 6 6 Using the Elemental Targeting Application The Output window is automatically displayed when you generate results for example when you calculate resolution To display the Output window manually select Output Window from the View menu To clear the Output window right click the Output window then select Clear Window To close the Output window deselect Output Window from the View menu or right click the Output window then select Hide Hint To maxim
291. reating a Constant Neutral Loss CNL Chromatogram You can create an extracted ion chromatogram e To improve the signal to noise ratio for a mass or mass range of interest To determine if mass differences in a data file correspond to loss of specific fragments by generating a Neutral Loss Chromatogram 4 2 1 Creating an Extracted lon Chromatogram XIC This section describes how to create an XIC e From the Chromatogram window From the Spectrum window From the To create an extracted ion chromatogram XIC from the Chromatogram Chromatogram window window 1 Click the Chromatogram window to activate it 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Extracted lon or click in the toolbar Data Explorer Software User s Guide 4 5 Chapter 4 Examining Chromatogram Data 4 Inthe Extracted lon Chromatogram dialog box Figure 4 1 on page 4 7 select one of the following from the Mass Range Difference Type drop down list e Center Window then type the mass of interest and the mass window for masses to include NOTE When analyzing multiple components with similar masses set a Window of less than 0 5 to include only the mass of interest Range then type the From and To values for masses to include NOTE To improve the signal to noise ratio in the extracted ion chromatogram set the starting range
292. rection does not completely correct for saturation of the detection system even if it is enabled AutoSaturation Correction Mariner Data Only Effect on Mariner The AutoSaturation Correction feature is not applied to RST RST files files saved from the Mariner Instrument Control Panel even when Saturation Correction is turned on Saturation requires information about the pulser frequency used to acquire the data and this information is not stored in RST files saved from the Instrument Control Panel Trace label When AutoSaturation Correction is turned on spectra in the data file are displayed with an ASC AutoSaturation Correction trace label Data Explorer Software User s Guide 5 63 Chapter 5 Examining Spectrum Data 5 12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files Dual Spectral Trace Arithmetic The Dual Spectral Trace Arithmetic function lets you add two spectra together or subtract one spectrum from another Spectra can be raw or processed and can be from the same or different data files To use the Dual Spectral Trace Arithmetic function 1 To perform this function on spectra from different data files copy a spectrum trace from one data file to another See Section 2 5 2 Copying Traces from Multiple Data Files to a Window for information on copying traces 2 Activate the Spectrum window that contains the two traces of interest 3 Process the traces as ne
293. resholds to a setting between 0 1 and 1 until the peaks of interest are detected and noise is screened out Figure 7 23 shows the trace after deisotoping Note that many fewer peaks are detected By further fine tuning Max Peak Area you can screen out additional noise peaks Spec 1 gt NFO0 7 gt DI BP 859 4 1269981 100 1 3E 6 2 80 60 40 1012 381 alli 1p34 352 _ 755500 1561 604 1723 656 1987760 Ee a a a a PORNP EPPS L n 1 a pare POPON 0 860 0 1113 2 1366 4 1619 6 1872 8 2126 0 Mass im 7 Figure 7 23 Digest After Noise Filtering Deisotoping and Adjusting Detection Thresholds 7 22 Applied Biosystems 8 Viewing Voyager PSD Data This chapter contains the following sections 8 1 Displaying PSD Data i e 8 2 8 2 Applying Fragment Labels eee 8 8 8 3 Calibrating a PSD Spectrum 8 10 8 3 1 Checking Peak Detection 8 11 8 3 2 Calibrating sissioni taai 8 12 8 3 3 Creating PSD CAL Files and Applying to Other Data Files 8 20 8 3 4 Creating PSD Calibration Reference REF Files essc 8 21 8 3 5 Changing the Precursor Mass 8 23 Data Explorer Software User s Guide 8 1 Chapter 8 Viewing Voyager PSD Data 8 1 Displaying PSD Data This section includes e Displaying the composite spectrum e Advancing through segment traces e Displaying multiple segment traces e Redisplaying the composite spectrum How the composite spectrum is generat
294. rge state field To calculate a theoretical isotope distribution for doubly sodiated B cyclodextrin with a 2 charge state m z 545 The software performs With these parameters specified this calculation Formula C42H60030 C42H60030 2Na 2 Group type Na Group Count Charge 2 Add 6 16 Applied Biosystems Using the Isotope Calculator To calculate a theoretical isotope distribution for doubly sodiated and deprotonated cyclodextrin with 1 charge state m z 1089 The software performs this calculation With these parameters specified Formula C42H60030Na2 C42H60030Na2 H Group type H Group Count Charge 1 Subtract To calculate a theoretical isotope distribution for B cyclodextrin with 2 charge state m z 522 The software performs With these parameters specified this calculation Formula C42H60030 C42H60030 2 Group type deselected Charge 2 Add Formula entry is case sensitive Data Explorer Software User s Guide 6 17 Chapter 6 Using Tools and Applications Evaluating traces The theoretical isotope distribution is displayed in the Spectrum window with an ISO trace label Figure 6 10 Intensity Ge C100 H202 BP 1404 6 100 100 oii 1403 6 80 70 60 50 A 6 40 30 20 1406 6 10 A 6 0 gt 0 1404 2 A 4 i 1406 6 4407 8 j 1409 0 Returning to the original spectrum 6 18
295. rget tab of the Output window Figure 6 21 Figure 6 21 Elemental Targeting Results in the Output Window Displaying the theoretical isotope distribution Results include e Index Sequential number assigned to each result Formula Elemental composition you entered e m z Mass charge of an observed peak that compared to the theoretical mass of the formula specified is within the Mass Tolerance and Resolution you specified e Charge Charge of the observed peak Isotope Match Score Number between 0 0 and 1 0 that reflects how well the observed peak matches the theoretical formula based on mass and isotope pattern A higher number represents a better match e Isotope Match Intensity Peak area counts that overlaps between the observed isotope pattern and the theoretical isotope pattern A higher number represents a better match To display the theoretical isotope distribution for a formula double click the line in the Elemental Target tab of the Output window The theoretical isotope trace is displayed in the Spectrum window with an ISO trace label and the elemental formula Data Explorer Software User s Guide 6 33 Chapter 6 Using Tools and Applications 6 7 Using the Macro Recorder Description Macros provided In this section The Macro Recorder feature in Data Explorer allows you to set up multi step tasks to execute automatically when you click a macro button The Macro Recorder
296. right click the window then select Paste Trace Data The copied trace is added to the active window The original trace label is preceded by the name of the file from which you copied the trace You can repeat step 1 through step 4 until a maximum of eight traces are displayed in a trace window If eight traces are displayed and you copy a new trace to the window the active trace is replaced Data Explorer Software User s Guide 2 37 Chapter 2 Using Chromatogram and Spectrum Windows Comparing After you copy a trace to another trace window you can copied traces compare traces by overlaying see Section 2 4 8 Overlaying Traces or by using trace arithmetic see Section 5 12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files Dual Spectral Trace Arithmetic 2 6 Saving Opening and 2 Deleting DAT Results Saving results for To save results for DAT files DAT files 4 Process a data file to generate results as needed 2 From the File menu select Result Spectrum or Result Chromatogram then select Save As 3 Use the default title or enter a name up to 31 characters for the results in the Title text box then click OK Only the results for the active trace are saved NOTE Results are stored within the DAT file not as separate files Title is an identifier you can use to recall the results It is not a file name Opening results To open results for DAT files for
297. rmination Troubleshooting Mariner Only Symptom Possible Cause Action Known isotope labeled with incorrect charge state too low Max Charge State parameter set too low See example in Max Charge State set too low on page 3 33 Set Max Charge State correctly See Peak Processing parameters spectrum data only on page 3 26 9 18 Applied Biosystems Peak Detection and Labeling Troubleshooting Table 9 12 Charge State and Isotope Determination Troubleshooting Mariner Only Continued Symptom Possible Cause Action Known isotope not labeled with charge state Charge State peak labels disabled Turn on Charge State peak labels See Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels Max Charge State parameter set too low See example in Max Charge State set too low on page 3 33 Set Max Charge State correctly See Peak Processing parameters spectrum data only on page 3 26 Peak List Filtering is enabled with Charge State filter enabled and set too low Disable Peak List Filtering See Filtering the spectrum peak list on page 3 42 Minimum Intensity set too high to detect other isotope peaks See example in Effect of Minimum Intensity on page 3 36 Set Minimum Intensity correctly See Advanced Settings spectrum data only on page 3 28 Filter width set too high to determ
298. rror in the Peaks Matched list gt Peaks matched Initial Error m2 17814380 178 12246 00213 The 267 Da mass is 267 18560 267 15452 0 0311 g removed when you 495 16970 495 10982 0 0599 eaaa click Eliminate Fit Outliers because it generated the TETEPI largest fit error below ality Delete Entire List Delete Selected Metct Eliminate F itOutlier zion cons J LON cons 3 178 14380 Calibrated Peak Mass 178 14343 Error 0 00037 m z i 07 1950 cnasorotea pear naso 20720029 teeor 0 00069 we 3 495 16970 Calibrated Peak Mass 495 16938 Error 0 00032 m z Chro Peak List A Spec Peak List A Sample Info Instument Setting Elemental Analysis Elemental Target Figure 5 4 Eliminate Fit Outlier Deletes the Match with the Largest Fit Error from the Output Window To clear the entire list click Delete Entire List Plotting 11 To apply the calibration constants to the displayed spectrum click Plot The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Result tab of the Output window Figure 5 5 Data Explorer Software User s Guide 5 13 Chapter 5 Examining Spectrum Data L Bate Eee PCE ete eet ee ee bib TTT bei u L Fe Edi Wea ie Pomm Pa i Joib Ajokoe aiie Ha aol Bl eet he a wie soe ieee So Fe Bernd Nar eearem n a CO E ce oh HHEEBGAES E i 4 wet Th E i anai THN D
299. s 3 If multiply charged peaks are present convert to a singly charged spectrum See Section 5 10 Converting to a Singly Charged Spectrum Mariner Data Only 4 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 5 From the Peaks menu select Peak Deisotoping The Deisotoping dialog box Figure 3 18 is displayed Repeat Formula Adduct JH Formula CSH 5NO OK Cancel Figure 3 18 Deisotoping Dialog Box 6 Inthe Adduct text box type the adduct that is the charge carrying species in the spectrum you are examining 3 48 Applied Biosystems Troubleshooting Deisotoping a Spectrum CAUTION If you enter an invalid value in the Adduct field for example numbers the spectrum is still converted to a deisotoped spectrum and the peak height is proportional to the original peak area 7 Inthe Formula text box type the generic formula using any elements in the Periodic table that approximates the isotopic pattern for the compound class you are examining Generic formulas include e Peptides C6H5NO e DNA C38H49N15024P4 e RNA C38H47N15028P4 e Carbohydrate C6H1206 8 Click OK The deisotoped spectrum is displayed with a DI trace label The mass of the vertical bar corresponds to the mass of the monoisotopic peak The height of the vertical bar is proportional to the peak areas of all peaks in the isotope cluster If you see s
300. s at Tolerance Da f eee Get Spec Peak List Clear Spec Pesk Let Adducts to Remove Fe ia acdeluets F E adducts F cridation Snnitabe Spebctrurri D Reime Mass T Macs Diference Double chick the pesk Peak Mass to zoom in Figure 3 9 Ladder Sequencing Toolbox Using the Ladder Sequencing Toolbox 7 Under Annotate Spectrum select the types of labels you want displayed Reference Mass Mass of the reference peak against which the current peak is compared Mass Difference Difference between the current peak and the reference preceded by a minus sign Peak Mass Mass of the current peak preceded by an equal sign 8 Click Label Peaks If Peptide is selected the software Examines the spectrum in 55 Da increments and selects the most intense ion in the range The 55 Da increment is used because it is less than the smallest mass difference related to a residue Labels mass differences plus or minus the specified Tolerance that correspond to amino acids Labels the reference peak from which the mass difference was derived with an asterisk Applies the additional labels you selected under Annotate Spectrum Data Explorer Software User s Guide C 7 Appendix C Data Explorer Toolbox Visual Basic Macros If DNA or RNA is selected the software e Examines the spectrum in 270 Da increments and selects the most intense ion in the range The 270 Da increment
301. s moving between 2 8 Opening data files 2 2 data files and automatically running a macro 6 45 PSD data files 8 2 result files 2 40 Data Explorer Software User s Guide Index 17 Output window acquisition comment displaying 1 15 calibration statistics displaying 5 13 8 18 Chro Peak list tab 1 15 clearing 1 16 closing 1 16 copying results from 2 28 instrument settings 1 16 Instrument Settings tab 1 15 peak list chro 1 15 peak list displaying 1 15 peak list importing and saving in Excel 3 41 peak list saving as a file 3 40 peak list spec 1 15 Results tab 1 15 results displaying 1 15 sample info 1 15 Sample Info tab 1 15 Spec Peak list tab 1 15 Overlapping peak labels 3 55 3 58 Overlaying traces active trace color 2 26 advancing 2 26 autocolor 2 27 colors setting 2 27 procedure 2 24 scaling options 2 27 troubleshooting 9 6 P Partially resolved peaks labeling 7 11 Peak area see Area peak centroid see Centroid mass charge state see Charge state peak deisotoping see Deisotoping inserting 3 39 Index 18 Applied Biosystems Peak bounds color changing 1 25 in peak labels 3 55 3 58 Peak detection see also Detection Ranges see also Peak detection parameters see also Peak detection Mariner data see also Peak detection resolution based see also Peak detection Voyager data accessing 3 11 3 13 Advanced Settings tab not available 3 17 3 28 Advanced Settings spectrum 3 17 3 2
302. s present 3 62 determining ion polarity 3 58 examples 3 32 filtering peak list by 3 42 labeled incorrectly 3 60 9 20 labels 3 53 3 58 3 62 labels not displayed 3 60 9 19 parameters setting 3 27 Data Explorer Software User s Guide Index 5 Charge state peak continued requirements for labeling 3 53 single 5 59 tolerance calculation 3 32 troubleshooting 9 17 z labels 3 58 zero 3 40 3 43 Chro peak list tab Output window 1 15 CHRO window see Chromatogram window Chromatogram noise threshold calculated automatically 3 21 3 68 in peak detection algorithm 3 68 Chromatogram window see also Chromatogram window traces see also Peak Labels see also Traces adding and subtracting spectra 4 20 baseline correction 4 29 baseline offset 4 27 composite PSD data displaying 1 13 constant neutral loss CNL chromatogram creating from 4 9 DAD diode array detector displaying Mariner data 4 2 description 1 12 display range adjusting 2 11 event tags Mariner data only filtering 4 24 extracted absorbance chromatogram XAC from Mariner data creating from 4 13 extracted ion chromatogram XIC chromatogram creating from 4 5 improving signal to noise ratio 7 2 Mariner data displaying 1 12 2 2 Index 6 Applied Biosystems Chromatogram window continued multispectrum Voyager data displaying 1 13 noise filtering 4 17 peak labels 3 52 peak list 3 38 results opening 2 40 reten
303. s Horizontal 45 degree or Vertical labels 8 Select label content to be displayed in addition to m z Area lIntegrated area of peak displayed with an A label e Charge State Charge state of peak displayed with a z label where the z represents a positive or negative charge NOTE A z is displayed for positive or negative ions To determine the actual charge of the ions display the Instrument Setting tab in the Data Explorer Output window See Output window on page 1 15 for information Check the listed polarity For spectra acquired in positive ion mode positive ions are produced For spectra acquired in negative ion mode negative ions are produced 9 To create custom labels select User Labels then click User Label Setup See Section 3 5 3 Setting Custom Peak Labels 10 Click OK The trace is displayed 3 58 Applied Biosystems Deleting labels Labels not displayed Peak Labeling If peak list filtering is enabled only the detected peaks that meet the peak filtering criteria are labeled according to the peak label settings Otherwise all detected peaks are labeled according to the peak label settings See Section 3 2 4 Peak Detection Parameter Descriptions and Filtering the spectrum peak list on page 3 42 To delete a peak label from the trace click the Spec Peak List tab in the Output window select the item right click the spectrum peak list then
304. s do not yield chromatograms with profiles that correspond to the unresolved peaks try another spectral peak Spec PES StOPH 1971 Figure 7 3 Combined Spectrum for Unresolved Peaks in Cytochrome C Creating To create extracted ion chromatograms extracted ion chromatograms 1 With the Chromatogram window activated click in the toolbar two times to add two traces Click an added trace to activate it In the Spectrum window right click drag over the first peak The extracted ion chromatogram is displayed In the Chromatogram window click the second added trace to activate it In the Spectrum window right click drag over the second peak The extracted ion chromatogram is displayed Figure 7 4 illustrates the peaks deconvoluted from the original unresolved peaks Data Explorer Software User s Guide 7 5 Chapter 7 Data Explorer Examples EDDM a hia bite Spectum Humber Original TIC Extracted ion containing chromatograms with unresolved peaks deconvoluted peaks Figure 7 4 Deconvoluting Unresolved Chromatographic Peaks Creating Create a combined spectrum for each extracted ion combined spectra chromatogram 1 Activate the Spectrum window then click in the toolbar two times to add two traces 2 Right click drag over the first half of the mass range in the 410 extracted ion chromatogram 3 Activate the second trace in the Spectrum window 4 Right click drag over the
305. s from the File menu then set the Replace Mode by selecting one of the following e Replace the Active Trace Add a New Trace 3 From the File menu select Import then select ASCII Chromatogram or ASCII Spectrum the menu item is context sensitive to the selected window An Open dialog box appears 4 Select the ASCII file to import 5 Click OK The imported trace is displayed in the active window Data Explorer Software User s Guide 1 35 Chapter 1 Data Explorer Basics CAUTION An imported ASCII format trace contains only the data points for the trace The Sample Info and Instrument settings tabs in the Output window display data from the data file you opened in step 1 These tabs do not include information about the imported trace 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files Overview Exporting BIC MSM and CAL 1 36 files Applied Biosystems You can extract the following information from a DAT data file RSD spectrum results file and RCD chromatogram results file then save the information as a stand alone file for use with other files e Instrument settings BIC e MS Method MSM Mariner data only e Calibration constants CAL e Processing graphic settings SET e Spectrum or chromatogram peak labels LBS or LBC e ASCII Spectrum and ASCII Chromatogram TXT To export Instrument settings BIC MS Method settings MSM or calibrati
306. s nc a Ea E aa suletievendsind esd a naaa 1 2 File Formats and Typos Seire aa E A AAN 1 5 1 2 1 Software Applications Compatibility eeeeeeeeeeeeeeeeeeeeeeee 1 5 1 2 2 Data DAT File Format ssssssssesrsssserrrressrrrsrsesrrrrresrrrrinnernnns 1 5 Parts of the Data Explorer WINdOW ussssssssssnnssnnnnnnnnnnnnnnnnnnnn 1 11 Customizing the Data Explorer WINdOW sssssesssssssssssnrenrrnrersrennes 1 17 1 4 1 Setting Default Values 00 0 0 cecceceeeeeeeeeeeeeeaeeaeeeeeeeeeeeeeeeeeeeeeees 1 17 1 4 2 Customizing Processing and Graphic Settings SET 1 17 1 4 3 Customizing Toolbars cccccceeeeeeeeeeeeeeaeeeeeeeeeeeesteeeeeeeeeeees 1 21 Setting Graphic Options eor eyr ieis cece cece EAEEREN A 1 23 1 5 1 Changing Background Color ccccceeeeeeeeeeeeeeeeeeeeeeeeeaeaaes 1 23 1 5 2 Customizing Graphic Options ccseeeeeeeeeeeeeeeeeeeeeeeaeaaes 1 24 1 5 3 Reverting to Previous Graphic Options 1 29 Managing Files tsccissteciigsi aioe EA EEA EA AAE NAT 1 30 1 6 1 Converting SPC File Format to DAT File Format Mariner Data Only cceceeeeeeeeeee eee aeeaeeeeeeeeeeeeeeeeeeeeaeeanaaaes 1 30 1 6 2 Converting Data from Profile to Centroid Mariner Data ONIY srira eiai aa a aaaea aa adeta iaa 1 33 1 6 3 Converting to and Exporting ASCII Data oo eee 1 34 1 6 4 Importing a Trace in ASCII Format seese 1 35 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files ess
307. se the method appropriate for your data to remove noise spikes For more information see Section 5 7 Noise Filtering Smoothing It is critical to perform both these functions which affect the peak centroid before calibration It is also good practice to perform the same processing functions on calibrants and unknowns NOTE If you are calibrating Mariner data baseline correction and noise filtering or smoothing are not recommended before calibrating Due to the shorter flight times and fewer data points associated with Mariner data these functions may affect peak shape which in turn affect mass accuracy Data Explorer Software User s Guide 5 7 Chapter 5 Examining Spectrum Data 5 8 Manually calibrating a single spectrum Applied Biosystems NOTE Multi point calibration yields higher mass accuracy than one point calibration Selecting calibrant peaks that bracket the mass of interest also yields higher mass accuracy To manually calibrate a single spectrum J Click the Spectrum window to activate it then select the spectrum trace of interest From the Peaks menu select Peak Label set the Mass Label Type to Centroid then click OK NOTE For spectra containing broad peaks that have unresolved adducts or impurities such as proteins you may obtain better results if you use apex instead of centroid settings From the Process menu select Mass Calibration then select Manual Calibr
308. second half of the mass range in the 723 extracted ion chromatogram 7 6 Applied Biosystems Mariner Data Examples Figure 7 5 illustrates the combined spectra for the deconvoluted peaks Note that both spectra contain a peak at 391 Da which requires investigation to determine if it is a low level component or background noise See Section 7 1 3 Determining if a Peak is Background Noise T intensity LG oan Spec a MAPl 75 Pat Spec Pik TIGJBPM 137 naar 137 1 Maa nwaj Both combined spectra contain a peak at 391 Da Figure 7 5 Combined Spectra for Deconvoluted Peaks Data Explorer Software User s Guide 7 7 Chapter 7 Data Explorer Examples 7 1 3 Determining if a Peak is Background Noise 7 8 Overview Subtracting spectral peaks Applied Biosystems To determine if spectral peaks represent low level components or if they are due to solvent contribution you can e Subtract the spectral peaks from the chromatogram e Create an extracted ion chromatogram for the spectral peaks The following example uses the chromatograms and spectra from Section 7 1 2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks It illustrates how to subtract baseline and determine if the spectral peak observed at 391 Da Figure 7 5 on page 7 7 is eliminated To subtract spectra 1 Activate the first extracted ion chromatogram in Chromatogram window see Figure 7 4 on page 7 6
309. sed Spectra from the Same or Different Data Files Dual Spectral Trace Arithmetic 2 5 64 Chapter 6 Using Tools and Applications 6 1 6 2 6 3 6 4 6 5 6 6 6 7 Using the Elemental Composition Calculator eeeeeeeeeeeee ees 6 2 6 1 1 Determining Elemental Composition ccceceeeeeeeeeeeeeeeeeeees 6 2 6 1 2 Setting Limits sis e a ert Aiea Ae ce cea east 6 7 Using the Isotope Calculator 2 ceeceeeeee cece eee e eee ee teens eeeeeeeeeee 6 13 Using the Mass Resolution Calculator cccceeeeeeeeeeeeeeeeeeeaeees 6 20 Using the Signal to Noise Ratio Calculator ccceceeeeeeeeeeeeeeeeee 6 23 Using the lon Fragmentation Calculator ccceeeeeeeeeeeeeeeeeeeeeees 6 25 Using the Elemental Targeting Application eeeeeeeeee eee renee 6 31 Using the Macro Recorder 2ccccceee aaaea iana aa 6 34 6 7 1 Before Using the Macro Recorder cs eeeeeeeeeneneeeeeeeeaaeeeeees 6 34 6 7 2 Recording amp Macro nerean E EET 6 37 6 7 3 Assigning Macros to Buttons cecceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 6 38 6 7 4 RUAMING a Macro sinit reene ere de neqeepars Soe R T 6 39 6 7 5 Deleting A MACO aasa erakar eee eeeeeeeeeeeaeeeeeeseeaeeeeeeeeaaeeeeesaeeaees 6 41 6 7 6 Advanced Macro Editing cceceeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaaes 6 42 6 7 7 Importing or Exporting Macros in DATAEXPLORER VBB 6 43 6 7 8 Running Macros Automatically Wh
310. sed as the window for isotope peak comparison because related isotope peaks will usually have lower mass accuracy than monoisotopic peak Setting Tolerance too low can result in a falsely low Isotope Match Score see page 6 6 6 Specify the Mass Type to calculate 7 Ifthe displayed Mass Peak Resolution is not appropriate for this calculation change the setting in Peak Detection For more information see Section 3 2 4 Peak Detection Parameter Descriptions 8 Specify the Result Type Elemental Amino Acid Carbohydrate DNA or RNA 9 Specify the Max Number of Results for each m z entered in step 4 NOTE Max Number of Results is per m z entered not total number of results generated for the calculation 10 Set limits as described in e Setting limits for existing elements on page 6 7 6 4 Applied Biosystems Using the Elemental Composition Calculator e Adding new elements and setting limits on page 6 9 e Setting limits for other result types on page 6 12 11 Click More Parameters then enter the Minimum and Maximum Double Bond Equivalents to include in the calculation Electron state for 12 Set the Electron State to calculate If you are intact molecules or calculating composition for fragment ions Intact molecules Use the default of Even Only e Fragment ions Set to Both Odd and Even if you are analyzing mass differences or absolute masses NOTE You may be able
311. see Section 5 3 3 Creating or Modifying a Calibration Reference File REF When to use Use manual calibration when you manual e Calibrate Voyager data calibration l l e Calibrate Mariner data and any of the following occur e You have one or only a few spectra to calibrate You do not know in advance which reference masses to use e You do not know in advance if the quality of the reference mass signals is acceptable You want to fine tune the results of an automatic calibration For information on automatic calibration see Section 5 4 Automatic Calibration Accurate mass Before performing an internal calibration refer to the measurements Mariner Workstation User s Guide e Voyager Biospectrometry Workstation User s Guide 5 6 Applied Biosystems Manual Calibration 5 3 2 Manually Calibrating This section describes Before calibrating Voyager data e Manually calibrating a single spectrum e Applying new constants to the data file e Exporting calibration constants CAL file e Applying new constants to additional files Before calibrating Before you calibrate Voyager data do the following to improve Voyager data mass accuracy e Baseline correct The Centroid peak detection value is derived from a percentage of the peak height which is measured from 0 not from the local baseline For information see Section 5 8 2 Using Baseline Correction e Noise filter or use default smoothing U
312. ser s Guide ix Table of Contents X Applied Biosystems How to Use This Guide How to Use This Guide Purpose of this guide Audience Structure of this guide The Applied Biosystems Data Explorer Software User s Guide describes processing and analyzing data with the Data Explorer software You can use the Data Explorer software to analyze data collected on e Mariner Workstations with Version 3 0 and later software e Voyager DE Biospectrometry Workstations with Version 5 0 and later software This guide is intended for novice and experienced Mariner or Voyager workstation users who are analyzing biomolecules The Applied Biosystems Data Explorer Software User s Guide is organized into chapters and appendixes Each chapter page is marked with a tab and a header to help you find information The table below describes the material covered in each chapter and appendix Chapter Appendix Content Chapter 1 Data Explorer Basics Describes file formats file management the parts of the Data Explorer window and how to customize the Data Explorer software Chapter 2 Using Chromatogram Describes window and trace handling Also and Spectrum Windows describes saving results Chapter 3 Peak Detection and Labeling Provides background information on peak detection centroiding and integration Describes peak detection peak labeling and peak deisotoping Chapter 4 Exa
313. ses the maximum data point within the range of data points being compressed range of 10 in the example above Average Uses the average of the data points within the range of data points being compressed range of 10 in the example above e Sum Binning Uses the sum of the data points within the range of data points being compressed range of 10 in the example above NOTE Changing the graphic compression mode may alter the displayed intensities of peaks in a trace 1 28 Applied Biosystems Setting Graphic Options 1 5 3 Reverting to Previous Graphic Options You have two options to revert to previously used graphic options e Revert to Last Saved Graphic Setting Reverts to the last graphic settings saved in the data file Does not affect processing settings To access select Default from the Display menu then select Revert to Last Saved Graphic Setting e Revert to Last Saved Graphic Processing Settings Reverts to the last graphic and processing settings saved in the data file To access select Settings from the File menu then select Revert to Last Saved Graphic Processing Settings Hint Instead of applying the settings saved with a data file you can apply the default settings stored in the default SET file for your system see page 1 18 To apply the default settings close the data file open the data file again then select Use Default Settings in the Open dialog box For more informatio
314. sful or failed calibration message To apply auto calibration settings to other files 1 Extract the SET information from the data file containing the auto calibration settings by selecting Settings from the File menu then selecting Save Processing Settings As For more information see Section 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files 2 Copy the settings to the new file by selecting Settings from the File menu then selecting Restore Processing Settings For more information see Section 1 4 2 Customizing Processing and Graphic Settings SET Data Explorer Software User s Guide 5 35 Chapter 5 Examining Spectrum Data 5 5 Centroiding 5 36 NOTE Centroiding is not supported for Mariner DAD data To display peaks as centroid traces 1 Click the Spectrum window to activate it 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing From the Process menu select Centroiding The centroid spectrum is displayed with a CT trace label see Figure 5 10 The height of each vertical bar corresponds to the original peak area Intensity a Spec 1 gt CT BP 558 3 2539 558 3 2539 0 556 6 558 9 aa 1 557 8 7 558 6 550 4 7 560 2 56 Mass im 7 Applied Biosystems Figure 5 10 Centroid Spectrum To return to the original trace see Returning to the original spectrum on page 5 3
315. ssseresieerrsrrseresrrerns 1 36 1 6 6 Copying from Data Files ceeeeeeeeeee eee aeeeeeeeeeeeeeeeeeeeeeeeaeaaee 1 38 Data Explorer Software User s Guide iii Table of Contents Chapter 2 Using Chromatogram and Spectrum Windows 2 1 Opening and Closing Data Files cceeceeeee cess eee eeeeeeeeeeees 2 1 1 Opening Data Files ccceceeceeeeeeeeeeeaeeaeeeeeeeeteeeeeeeeeeeees 2 1 2 Displaying Mariner DAD Traces ccce 2 1 3 Displaying Voyager Chromatograms ccceeeeeeeeeeeeeees 2 1 4 Viewing Read Only Files ccccecececseeeeeeeeeeeeeeeeeeeeeeeeees 2 1 5 Moving Between Open Files cceeeeeeeeeeeeeeeeeeeeeeeeeeeeees 2 1 6 Closing Data Files ccccceeceeeeeeeeeee eee eee eeeteeeeeeeeeeeeeeees 2 2 Adjusting the Display Range cceeeeeee eee eee tees eee eeeeeeeeeeees 2 3 Organizing WiINdOWS ece eee ee eee eee eee eee eens eee aaar aaa 2 4 Manipulating TaCeS ci ic cteescecieedteat ens teectedies sie ebpeniennieaiens tetees 2 4 1 Zooming Centering and Customizing a Trace 5 2 4 2 Duplicating A Traco sensasi aa A 2 4 3 Dividing the Active Trace asssssssssrrreessrrriressrrrrrsssrrnns 2 4 4 Adding Traces from the Same Data File to a Window sseseeseeeeeeseresrrrerresrnes 2 4 5 REMOVING Tacas see sexta ek A ee A Ae 2 4 6 Expanding and Linking Traces cceeeeeeeesseeeeeeeeaaaeeeeeees 2 4 7 Recalling and Rearranging Traces Processing Hist
316. stems File Formats and Types Table 1 2 Additional File Types Continued Category File Type File Content Reference REF List of masses to select from during calibration See Creating and saving a calibration reference file on page 5 18 Process RCT Results file saved from Mariner e Mariner SPC format data file versions earlier only than 3 0 in the Data Explorer software after a chromatogram is manually processed See Section 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only e Mariner DAT format data file version 3 0 and later in the Mariner Instrument Control Panel for a snapshot of chromatogram data NOTE Results for a DAT file are stored within the DAT file not as a separate file RST Results file saved from e Mariner SPC format data file versions earlier than 3 0 in the Data Explorer software after a spectrum is manually processed See Section 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only e Voyager MS format data file in the Data Explorer software after a spectrum is manually processed e Mariner DAT format data file version 3 0 and later in the Mariner Instrument Control Panel for a snapshot of spectrum data NOTE Results for a DAT file are stored within the DAT file not as a separate file Data Explorer Software User s Guide 1 9 Chapter 1 Data Explorer Basics Table 1 2 Additional File Typ
317. t spectrum then return to the current spectrum For more information see Section 5 4 Automatic Calibration During calibration the software is not matching spectrum masses to some reference masses in the calibration reference file The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the software mistakes these narrow peaks as isotopically resolved and ignores the reference mass When specifying highly charged non isotopically resolved species with peaks less than 1 Da wide for example myoglobin 20 as reference masses in a calibration reference file set the peak type asa Resolved Isotope Mass even though it is nota resolved isotope See Section 5 3 3 Creating or Modifying a Calibration Reference File REF Mass tolerance or Minimum Intensity set too high Adjust See Section 5 3 Manual Calibration 9 10 Applied Biosystems Calibration Troubleshooting Table 9 6 Calibration Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action Calibration returns an invalid number of matches When creating a reference mass list in Manual or Automatic calibration the software allows you to add multiple items with the same m z value to the calibration list box if any other attribute of the reference c
318. t box type the amino acid sequence of the compound Use single letter codes Set other parameters as needed For parameter descriptions see Section 6 5 Using the lon Fragmentation Calculator Click Options to specify the fragment peaks to label Click Induce Fragmentation Click Label Peaks The ion peaks specified in Options are labeled on the trace if they are present Hint To more selectively apply labels decrease the Mass Tolerance in the Options dialog box Hint This function creates User Labels in the data file To view select Peak Label from the Peaks menu then click User Label Setup Click Save As to save the labels in a LBS file for use with other data files For more information see Section 3 5 3 Setting Custom Peak Labels To display the original labels select Peak Label from the Peaks menu then deselect User Labels Data Explorer Software User s Guide 8 9 Chapter 8 Viewing Voyager PSD Data 8 3 Calibrating a PSD Spectrum When to use this 8 10 procedure Overview of creating a PSD CAL file Applied Biosystems NOTE Multi point calibration yields higher mass accuracy than one point calibration This section includes Checking peak detection Calibrating Creating PSD CAL files and applying to other data files Creating PSD calibration reference REF files Changing the precursor mass Use this procedure to Generate a PSD calibration CAL file from a k
319. ta files or between Chromatogram and Spectrum windows for a Mariner data file See Moving Between Open Files on page 2 8 Data file names are displayed in the Title bar of each window e Tab for each window NOTE If the full name of the data file does not fit in the tab part of the name is displayed followed by To display the full name of the data file place the cursor on the tab The full name of the data file is displayed below the tab Parts of the Data Explorer Window Output window The Output window see Figure 1 3 on page 1 11 displays tabs at the bottom that you can click to switch between the types of information displayed Output window e Result Displays results generated using commands on tabs the Process Tools and Applications menus For more information on results see Section 5 3 Manual Calibration or Section 5 4 Automatic Calibration Section 5 6 Mass Deconvolution Mariner Data Only Section 6 2 Using the Isotope Calculator Section 6 3 Using the Mass Resolution Calculator Section 6 4 Using the Signal to Noise Ratio Calculator e Chro Peak List Displays results of chromatogram peak detection and integration For more information see Section 3 3 Peak List e Spectrum Peak List Displays results of spectrum peak detection integration and centroiding For more information see Section 3 3 Peak List e Sample Info For data files displays the e Softwa
320. takes these narrow peaks as isotopically resolved and ignores the reference mass Data Explorer Software User s Guide 5 21 Chapter 5 Examining Spectrum Data 5 3 4 Reverting to Instrument Calibration The Revert to Instrument Calibration function does the following Mariner data Reapplies the original calibration constants used to acquire the data Voyager data Applies default calibration to the data regardless of whether default or external calibration is used to acquire the data To revert the calibration s From the Process menu select Mass Calibration then select Revert to Instrument Calibration The spectrum is recalibrated with the original calibration constants used during acquisition Mariner data or default calibration Voyager data and displayed with an MC trace label The original and new constants are displayed in the Output window 2 To save the reverted calibration with the data file select Mass Calibration from the Process menu then If r yon z Select The following occurs calibrating Mariner Apply Calibration All spectra in the data file are calibrated and data displayed with an MC trace label The including calibration constants are saved with the data MS Method file Each spectrum in the data file is data calibrated when displayed 5 22 Applied Biosystems Manual Calibration vee le Select The following occurs calibrating Voyager Apply Cal
321. ted in the same directory For information see Before you begin on page 1 31 If the SPC and CGM files are not in the same directory when you open the SPC file a Failed to open chromatogram data message is displayed To convert from profile to centroid format 1 Open or activate the DAT or SPC file to convert 2 Select Convert from the File menu then select Centroid The Save As dialog box is displayed The software appends a CT suffix to the file name This is the default file name You can enter a new file name for the centroid file 3 Click Save NOTE The m z range in a data file that is converted from profile to centroid is determined by the peak detection range set in Data Explorer not the m z range in the original data file For more information see Section 3 2 Peak Detection Data Explorer Software User s Guide 1 33 Chapter 1 Data Explorer Basics 1 1 6 3 Converting to and Exporting ASCII Data This section describes e Converting a data file to ASCII format e Exporting a trace to ASCII format Converting a data You can convert a data file for use in a spreadsheet or another file to ASCII application and export and import single traces in ASCII format format To convert an entire active data file to an ASCII text file 1 Open the data file to convert 2 From the File menu select Convert then select ASCII text A Save As dialog box is displayed 3 Specify the
322. tering the spectrum peak list on page 3 42 NOTE If peak labeling is disabled no labels are displayed even for peaks in the peak list If you delete a peak from the peak list it is not labeled Labeling peaks If peak detection settings do not detect and label desired manually peaks you can also manually label peaks by inserting them into the peak list See Section 3 3 2 Inserting Peaks in the Peak List 3 52 Applied Biosystems Peak Labeling 3 5 1 Charge State Labels Charge state A unique feature of the Data Explorer software is the ability to labels label multiply charged isotope peaks with their charge state In general the observed spacing between isotopes is determined by the charge state of the detected ion Spacing between isotope peaks is narrower at higher charge states For more information on isotopes see Appendix B Overview of Isotopes To determine charge state the software uses the following charge state determination parameters e Max Charge State e Max Isotope e Min and Max Intensity For a description of these parameters see Advanced Settings Spectrum data only on page 3 28 and Charge State Determination and Examples on page 3 32 NOTE If Charge State Determination parameters are set incorrectly the charge state is determined incorrectly For more information see Charge state parameter examples on page 3 32 and Charge State and Isotope Determination Troubles
323. tes it is a by contaminant monoisotopic peak If first three peaks are part of va Contaminant the same isotope cluster but yA F Peak that is not contaminant is also present J Patt of cluster Increased amplitude of peaks indicate they are monoisotopic peaks If two isotope clusters are present Figure 3 17 Interpreting a Deisotoped Trace Requirements The Deisotope function requires a singly charged spectrum If the spectrum you are examining includes multiply charged peaks use the Single Charge Conversion function on the Process menu before using the Deisotope function For information see Section 5 10 Converting to a Singly Charged Spectrum Mariner Data Only CAUTION If you use the Deisotope function on multiply charged peaks invalid results are reported Data Explorer Software User s Guide 3 47 Chapter 3 Peak Detection and Labeling Using the To use the Deisotope function Deisotope 1 Display the spectrum trace of interest function l Make sure peak detection thresholds are set low enough to detect the monoisotopic peak before deisotoping If the detection thresholds are not set low enough adjust them For information see Section 3 2 3 Setting Peak Detection Parameters If you are analyzing digest data set Max Peak Area to 0 before deisotoping to ensure that all peaks of interest are detected For more information see Section 7 2 3 Detecting Peaks from Complex Digest
324. th Flexibility 0 5 Degree 0 5 Baseline is gently curving f Peak Width 10 to 20 times peak width Flexibility 0 Degree 0 5 decrease further to raise the baseline Baseline rise should be ignored and treated as signal Peak cluster gt Peak Width Half of the width of the cluster to correct in the illustration shown if the width of the cluster is 200 set Peak Width to 100 Flexibility O Degree 0 5 decrease further to raise the baseline 5 54 Applied Biosystems Adjusting the Baseline Troubleshooting f the baseline rises preceding and following a peak after the correction a hump under the peak adjust the following parameters in the order listed e Decrease Flexibility e Decrease Degree e Decrease Peak Width NOTE Lower Peak Width values increase the time needed for processing Returning to the To return to the original trace see Returning to the original Original spectrum spectrum on page 5 3 Data Explorer Software User s Guide 5 55 Chapter 5 Examining Spectrum Data 5 9 Truncating a Spectrum Description The Truncate function removes data points from a trace outside a selected region Truncating spectra is useful to Mariner data Remove noise at the low end of a spectrum before generating a result file e Voyager data Eliminate the Low Mass Gate spike and background in the low mass range Truncating To truncate spectr
325. the dialog box select Result Spec Files RS or Result Chro Files RC Saving Opening and Deleting SPC Results Files Mariner Data Only 3 Select the RST or RCT file to open then click OK NOTE Saturation Correction is not applied to Mariner RST files For more information see Section 5 11 AutoSaturation Correction Mariner Data Only 4 Click the Sample Info tab in the Output window to display the following information for the result file Name of the original raw data file from which the result file was generated e Processing functions that were performed and saved in the result file Deleting results Use Windows NT Explorer to delete RST and RCT result files for SPC files generated from SPC files Data Explorer Software User s Guide 2 41 Chapter 2 Using Chromatogram and Spectrum Windows 2 42 Applied Biosystems 3 Peak Detection and Labeling This chapter contains the following sections 3 1 OVEWIOW i eters tilciiiats Alita NST 3 2 3 2 Peak Detection cccececeeceeeeeeeeeeeeeseeeeeneees 3 6 3 3 Peaks hist ch T a ea a A aaia aea eee be 3 37 3 4 Deisotoping a SpectrUM esssssessseersrreree 3 45 3 5 Peak Labeling ccccecceeseeeeeeeeeeeeeeeeeeeeeees 3 52 3 6 Process that Occurs During Peak Detection Centroiding and Integration ceee 3 67 3 7 Default Peak Detection Settings 0 3 71 Data Explorer Software User s Guide 3
326. the mass difference and includes it in the chromatogram if any mass difference corresponds to a mass difference plus tolerance specified You can generate a CNL extracted chromatogram To determine if a fragment of known mass is present e To increase sensitivity of a trace by including signal from both the parent and one or more fragment ions For LCMS data a CNL extracted chromatogram is useful to identify eluting components with a given neutral loss For other types of data a CNL extracted chromatogram is useful to screen samples for a given mass difference For example create a CNL extracted chromatogram to survey a peptide digest for the presence of peaks separated by a glycosyl residue as you might see in a digest containing glycopeptides Data Explorer Software User s Guide 4 9 Chapter 4 Examining Chromatogram Data Labeling spectrum peaks 4 10 with mass difference optional Procedure Applied Biosystems You can label spectrum peaks with mass differences to assist you in determining the mass differences to specify in the CNL extracted chromatogram To label spectrum peaks with mass differences 1 Display a spectrum of interest from the data file for which you are creating the CNL extracted chromatogram Click the Spectrum window to activate it then select Peak Label from the Peaks menu In the Peak Mass Label Type select Mass difference from the selected peak In the Spectrum
327. the new file by selecting Settings from the File menu then selecting Restore Processing Settings For more information see Section 1 4 2 Customizing Processing and Graphic Settings SET NOTE All processing settings not just calibration settings are applied to the new file Specifying To specify automatic calibration settings 1 Click the Spectrum window to activate it then select the spectrum of interest Data Explorer Software User s Guide 5 29 Chapter 5 Examining Spectrum Data 2 From the Peaks menu select Peak Label then set the Mass Label Type to Centroid NOTE For spectra containing broad peaks that have unresolved adducts or impurities such as proteins you may obtain better results if you use apex instead of centroid settings 3 From the Process menu select Mass Calibration then select Automatic Calibration The Automatic Calibration Settings dialog box is displayed Figure 5 7 Auto Calibration Settings x M Select Calibration Reference File Reference Masses To Match Reference Mass Type Name Char Elemental Com Seleul New File Add Refeierwee Tu List gt AJJAI gt gt r Reference Matching Criteria Min Intensity fi Jz Relative Intensity x Mass Iolerance p wz Tit Nejection Parameters Min Peaks to Match 1 ae e s Peak Weighting Factor Max Outlier Error m2 0 5 inverse width 7 Save Settings Close Delete All References Delete Selected Reference
328. the region of the existing range is split in half between the existing range and the new range Double click an existing range to manually enter lower and upper boundaries Select a range in the dialog box then click drag the X data cursors labels in the trace to set the lower and upper boundaries Continued Data Explorer Software User s Guide 3 19 Chapter 3 Peak Detection and Labeling Table 3 1 Chromatogram Settings Continued Parameter Description Detection Ranges continued To delete a range select the range then click To combine all ranges in the list into one range click Eu The peak detection settings displayed in the dialog box correspond to the selected range To view peak detection settings for another range select the range of interest Base Peak Intensity NOTE This parameter was previously named Peak Threshold and Base Peak Relative Specifies a percentage of the base peak intensity as the threshold value To be detected peaks must be above this threshold and above the Max Peak Area value Hint For most Voyager applications leave this parameter set at 0 and adjust the Max Peak Area Max Peak Area Specifies a percentage of the peak with the largest area as the threshold value To be detected peaks must be above this threshold and above the Base Peak Intensity value Max Peak Area is calculated above the local baseline and can compensate f
329. the second cursor displays the appropriate value relative to the first cursor For example if you place the first X cursor in a spectrum at 100 m z and the second X cursor at 80 m z the cursors are labeled 100 and 20 Data Explorer Software User s Guide 1 27 Chapter 1 Data Explorer Basics Setting traces in You can change the trace display from Line to Vertical Bars Line or Vertical Each vertical bar represents one data point 1 Bar mode Vertical bar mode is useful when setting peak detection parameters to determine the number of points across a peak Setting graphic NOTE Graphic Compression mode is not saved as part of compression graphic settings When you close a data file it is automatically reset to the default Local Max setting By default all data is compressed when it is displayed on your computer screen The degree of compression is determined by the number of data points in the data file and the resolution setting of your computer monitor For example assume that a data file contains 10 000 data points and needs compression to 1 000 data points to fit on your computer screen Every 10 data points will be compressed into a single data point To set graphic compression settings 1 Select Graphic Options from the Display menu 2 Inthe Graph Setup tab of the Graph and Plot Options dialog box see Figure 1 4 on page 1 25 select a Graphic Compression mode e Local Max default U
330. thing 5 42 spectrum numbers on trace do not match axis 9 5 subtracting spectra from different data files 5 64 types of data 5 2 Spectrum window traces adding 2 18 displaying as vertical bars 5 36 do not print 2 34 overlaying 2 25 overlaying from different data files 2 24 previewing and printing 2 33 Spray Tip Potential displaying trace 4 2 Spreadsheet saving peak list in 3 41 Starting Data Explorer software 1 3 Stitched PSD in spectrum header 2 32 8 2 Stitched spectrum see Composite spectrum Stopping Data Explorer software 1 3 Subtracting background 4 20 raw spectra within a data file 4 20 spectra from different data files 5 64 spectra example 7 8 Summing data graphic compression 1 28 Summing spectra see Adding spectra see Combined spectrum Data Explorer Software User s Guide Index 25 T Tabs for open files 2 8 in Data Explorer window 2 8 TAC in chromatogram header 2 30 Mariner data optional 1 12 Tag see Event tag Target compounds determining if present in spectrum 6 31 Technical support contacting 9 2 for computers with altered configuration A 1 Temperature trace displaying 4 2 Text files saving peak list in PKT 3 40 Threshold peak detection chromatogram 3 20 global spectrum 3 22 local spectrum 3 30 TIC see also Chromatogram window description 4 2 4 3 Title when saving results 2 38 Toolbars buttons adding and removing 1 21 customizing 1 21 1 22 description 1 12 Macro displaying 6 3
331. tion time displaying 1 12 smoothing 4 17 spectrum number displaying 1 12 traces selecting type 4 2 vial displaying 3 55 Voyager data displaying multispectrum 2 7 Chromatogram window traces adding 2 18 do not print 2 34 overlaying 2 25 overlaying from different data files 2 24 previewing and printing 2 33 trace labels in chromatogram 2 30 CID data labeling C 9 CNL see also Extracted ion chromatogram CNL in chromatogram header 2 30 Colors background changing 1 23 changing to black before printing 2 33 customizing 1 25 default Mariner 1 4 default Voyager 1 4 overlaid traces 2 27 setting 1 25 Combined spectrum creating 5 4 definition 5 2 example 7 4 7 6 label 5 4 CombiSolv data displaying one injection 4 24 Event Tag Filtering command dimmed Mariner data only 4 24 Comment acquisition displaying 1 15 acquisition displaying for open data file 1 15 acquisition displaying when opening a data file 2 3 result file 1 15 2 40 Comparing data files 2 38 Composite spectrum PSD automatically processed when generated 8 6 displaying segment traces 8 3 generating 8 2 8 5 how it is generated 8 6 labeling fragment ions 8 8 Max Stitch Masses 8 4 precursor mass changing 8 23 Composition elemental 6 2 Compression data 1 28 Computer configuration requirement A 1 technical support for altered configuration A 1 Configuration exporting from DAT file 1 36 Constant neutral loss CNL se
332. tions and technical support please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com Applied Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 07 2001 Part Number 4317717 Rev C an Applera business
333. to refine results for fragment ions by setting Electron State to Odd Only 13 Click OK Calculating 14 Click Calculate To cancel a lengthy calculation click Cancel Calc Results The results of the calculation are displayed in the Elemental Analysis tab of the Output window Figure 6 2 z sini Haih iig CHRE HUTE OI PID 02961 Get WIR Ra DEL r med iL Tra UEH amans TEE r Aani gi Hi a P T it ii T irsi Figure 6 2 Elemental Composition Calculation Results in Output Window Data Explorer Software User s Guide 6 5 Chapter 6 Using Tools and Applications Hint You can sort the results in a column by clicking the column header Results include Index Sequential number assigned to each result Input m z Entered m z for each composition calculation Calculated Mass Calculated mass for the entered m z and charge state for each theoretical composition mDa Error and ppm Error Error for each calculation Double Bond Equivalents DBE Value that corresponds to the number of double bonds or rings that the valence bond theory requires to construct a molecule from each calculated composition If the target mass is a protonated molecular ion M H the DBE value includes 0 5 and corresponds to an even electron state To convert the reported DBE value to the actual number of double bonds or rings add 0 5 For example H C NH has an M H total mass of 30 0344 and a reported double b
334. to the original trace see Returning to the original trace on page 4 4 and apply the Noise Filter again with a higher Correlation Factor setting Applying the filter more than one time with the same Correlation Factor setting does not improve noise removal Higher molecular Gaussian Specify a Filter Width in data points odd weight data Smooth integers only The maximum number of SM smoothing points is 2001 Points less than 1 Filter Width from the edge of the spectrum are May affect not smoothed peak resolution 4 18 Applied Biosystems Noise Filtering Smoothing Type of Data Suggested Method Description High resolution data Noise Removal NR Does not affect peak resolution Specify the number of standard deviations of noise to remove The software automatically calculates the average white noise for all frequencies across the spectrum then removes the specified number of standard deviations of noise This method slightly affects peak intensity and removes peaks with a signal to noise ratio less than the specified standard deviation In general If you set White Noise Std Dev to removed is 1 68 2 95 3 99 If applying Noise Removal with a certain Std Dev does not yield the necessary noise removal return to the original trace see Returning to the original trace on page 4 4 and apply the Noise Removal again with a higher Standard Deviation setting Applying the
335. to the peak list to be included in the calculation by typing a mass in the Add Mass to Peak List field then clicking Add Peak Remove unwanted peaks from the list that you do not want included in the calculation by clicking the peak in the list then clicking Delete Selected Peaks 4 If you will be identifying y and b pairs select the precursor peak then click Use Selected Peak Pairs To list ion pairs 1 Click the Pairs tab Poptds Fragmentation Toolbox E Spec Peak Lit Setup Pars Sequence Correlation i T i a bu v 17 pars jean b pars Lowa of HAO Remoa Mass From Seleched Par Left Fiaht Chnwablie click he peak bo mom in Recut Tab Clos Figure 3 11 Peptide Fragmentation Pairs 2 Click the button that corresponds to the ion masses you want identified e aand b pairs Lists peak pairs with a 28 Da mass difference a b y 17 pairs Lists peak pairs with a 17 Da mass difference which corresponds to the loss of NH3 C 10 Applied Biosystems Using the Peptide Fragmentation Toolbox e y and b pairs Lists peak pairs whose combined masses plus 1 Da add up to the Precursor lon Mass you specified on the Setup tab e Loss of H2O Lists peak pairs with a 18 Da mass difference 3 To remove all pairs results click Clear List 4 To remove amass from the Spec Peak List to simplify the sequence interpretation select an entry in the pairs results then click Left or Right
336. tomatic Calibration function in Data Explorer to prepare Automatic Calibration settings contain reference masses and other matching information You then save a SET file that contains the settings then specify the SET file in the Sequence Control Panel The Sequence Control Panel automatically calibrates spectra using the calibration settings contained in the SET file Use automatic calibration for Voyager data to prepare Automatic Calibration settings The Sequence Control Panel uses Automatic Calibration settings to calibrate data as it is acquired For more information on the Sequence Control Panel and on specifying automatic calibration see the Voyager Biospectrometry Workstation User s Guide Automatic Calibration 5 4 2 Importing and Specifying Automatic Calibration Settings Importing Tee Eeo Hint Importing automatic calibration settings is useful when you calibrate batches of related samples Automatic calibration settings are saved as part of processing settings in a DAT file To use auto calibration settings from another DAT file 1 Open the data file containing the desired auto calibration settings files 2 Save the SET information from this file by selecting Settings from the File menu then selecting Save Processing Settings As For more information see Section 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files 3 Open or activate the new file 4 Copy the settings to
337. ttings tab and the Advanced Settings tab is displayed when you select Peak Detection See Setting Advanced Settings spectrum data only on page 3 17 4 Set Global Thresholds as needed These thresholds are applied to all detection ranges unless you override them for a detection range on the Advanced Settings tab For a description of the parameters see Basic Settings spectrum data only on page 3 22 Data Explorer Software User s Guide 3 13 Chapter 3 Peak Detection and Labeling a Paeh riartar bation AA ieee Biome Feteye Peak Piicemergy ddirt Serie Ginta Precint Time Pastira OO 3 T Hias Paak disa Eoo T Erabi SE haero T hened Tiaia Pit Detection E Ue Feine Pepan anir r imidd irtam Pasi Hamids Bhim Meade mm Fen ee eg ee ll ee P vere ER Canoe Figure 3 4 Spectrum Peak Detection Setup Basic Settings Tab 5 If you are detecting PSD data or want to override the Global Thresholds select Use Advanced Settings and skip to step 7 6 If you are detecting mass spectral data select Use Resolution Dependent Settings if you want the software to e Automatically determine the number of data points across a peak e Divide the trace into different detection ranges based on the resolution Apply a Filter Width and Increment appropriate for each detection range Apply a Minimum Area of 0 and a Minimum Intensity of 0 to all detection ranges 3 14 A
338. tup Click Save As to save the labels in a LBS file for use with other data files For more information see Section 3 5 3 Setting Custom Peak Labels To display the original labels select Peak Label from the Peaks menu then deselect User Labels Creating a To create a PSD calibration reference file click Create calibration Reference File For more information see Creating PSD reference file Calibration Reference REF Files on page 8 21 REF 6 30 Applied Biosystems Using the Elemental Targeting Application 6 6 Using the Elemental Targeting Application Description Using the Elemental Targeting application The Elemental Targeting application determines if observed masses in a spectrum correspond to chemical formulas you enter This application generates a theoretical isotope pattern for the mass you enter using the Mass Resolution specified in Basic Peak Detection settings It then compares each observed mass and isotope pattern to the theoretical mass and isotope pattern for each composition you entered and reports an isotope match score that reflects how closely they match Use the Elemental Targeting application to screen a spectrum for the presence of specific chemical compounds To use the Elemental Targeting application 1 2 3 Display the spectrum of interest Click the Spectrum window to activate it From the Applications menu select Elemental Targeting The Elemental Targeting dial
339. u can display the following types of Voyager data by selecting Extracted lon from the Process menu with a Chromatogram window displayed then selecting Select To display Center Window Extracted lon chromatogram which includes only the response or Range from a mass window or range For more information see XIC Section 4 2 1 Creating an Extracted lon Chromatogram XIC Neutral Loss Constant Neutral Loss Chromatogram which extracts only the CNL response from a mass difference from a selected peak For more information see Section 4 2 2 Creating a Constant Neutral Loss CNL Chromatogram Creating macros You can create macros that perform multiple functions for to combine example smooth and baseline correct then start the macro processing with one mouse click functions For information see Section 6 7 Using the Macro Recorder Returning to the Many processing functions generate a new trace If you have original trace Trace Replace mode set to Replace the new trace replaces the original trace For information on Replace mode see Section 2 4 4 Adding Traces from the Same Data File to a Window To redisplay the original trace select the trace type from the Display menu or click the corresponding toolbar button 4 4 Applied Biosystems Creating an Extracted lon Chromatogram 4 2 Creating an Extracted lon Chromatogram This section includes e Creating an Extracted lon Chromatogram XIC e C
340. uct fH b Polarity Positive C Negative OK Cancel Figure 5 20 Single Charge Spectrum Conversion Dialog Box In the Adduct text box type or select the adduct that is the charge carrying species in the spectrum you are examining Select the polarity for the converted spectrum You must select the same charge as the spectrum you are evaluating CAUTION If you do not select the same charge as the spectrum you are evaluating an incorrect mass is reported Click OK The converted spectrum is displayed with an SC trace label The height of each vertical bar corresponds to the original peak areas To return to the original trace see Returning to the original spectrum on page 5 3 Converting to a Singly Charged Spectrum Mariner Data Only Example Figure 5 21 and Figure 5 22 illustrate the effects of single charge conversion Before conversion Figure 5 21 the spectrum includes 2 and 3 charged species of neurotensin Intensity Spec 6 19 BP 558 3918 ja 918 0 A Neurotensin 70 multiply charged a species 50 40 30 149 0313 20 104 119 0891 200 0323 z1 338 3345 0 ee ee R E N E 100 280 460 1000 Mass m z Figure 5 21 Spectrum Before Single Charge Conversion After conversion Figure 5 22 the 2 and 3 charged species are converted to the 1 species of neurotensin Spec 6 19 gt SC BP 1672 8 3012 400 672 6007 z1 3012 2 90 Neurotensin a singly ch
341. um window of the selected file Data Explorer Software User s Guide 2 9 Chapter 2 Using Chromatogram and Spectrum Windows 2 1 6 Closing Data Files 2 10 Applied Biosystems You can close files in the following ways Select Close from the File menu to close the active file Select Close All Files from the File menu to close all files In the Open File dialog box select any open files from the Files Selected list click Remove then click Finish to close the selected files Select Exit from the File menu to close all files and exit the software If you enabled Processing History and selected the Show Save History option a dialog box is displayed Click Save or Purge For more information see Setting Processing History options on page 2 23 Adjusting the Display Range 2 2 Adjusting the Display Range To set the display range 1 Click the Chromatogram or Spectrum window to activate it From the Display menu select Range Select X Range to set the x axis range The scaling units depend on the window you are scaling e Chromatogram Scales in the same units currently displayed in the Chromatogram window Spectrum Number or Time Mariner data only e Spectrum Scales in m z units Select Y Range to scale the y axis range The Y Axis Setup dialog box Figure 2 6 is displayed Scaling Mode Display Relative gt Minimum Absolute Max Y T Use Limit fe Y Display Range From p 2
342. ure the correct reference masses and peak masses are selected Manual Calibration 5 3 6 Hints for Calibrating Voyager Data Importing a if you import a calibration you must import a calibration calibration generated from a data file that was acquired on the same instrument using identical settings for the following instrument setting parameters e Polarity Instrument mode If you import a calibration that was generated using settings that are different from the current Polarity and Instrument Mode settings an error message is displayed NOTE The calibration of the mass scale changes when you change the Accelerating Voltage Grid Voltage or Delay Time Default calibration adjusts for these changes However you observe more accurate calibration if you use an external calibration CAL file generated with the same Accelerating Voltage Grid Voltage or Delay Time Data Explorer Software User s Guide 5 25 Chapter 5 Examining Spectrum Data 5 4 Automatic Calibration This section includes e Overview of automatic calibration e Importing and specifying automatic calibration settings e Automatically calibrating Mariner data only NOTE Automatic calibration is not supported for Mariner DAD data 5 4 1 Overview of Automatic Calibration This section includes e During automatic calibration e Automatic calibration for Mariner data e Automatic calibration for Voyager data During automatic During
343. vanced Settings m Peak Detection Settings Detection Rangefs Full full XK X Range s Lower Bound Upper Bound Range 1 Start End Active Local Range Thresholds BP Intensity g 8933 Hm in Intensity fo a Max Peak Ateal2 n Min Area fo 4 Noise Threshold E sj m Filter Settings Width E Increment fi OK Cancel Figure 3 6 Spectrum Peak Detection Setup Advanced Settings Tab Data Explorer Software User s Guide 3 17 Chapter 3 Peak Detection and Labeling Resetting Basic 3 18 Applied Biosystems 3 4 Select a detection range then set parameters as needed Click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box For a description of the parameters and the data cursor see Advanced Settings spectrum data only on page 3 28 If you override Basic Settings by entering parameters on the Settings Advanced Settings tab you can reset Basic Settings by doing the following 1 2 3 Click the Basic Settings tab Select Use Resolution Dependent Settings Enter the desired Global Thresholds Click Apply or OK The following occurs e Detection ranges Filter Width and Filter Increment previously set on the Advanced Settings tab are reset to defaults e Minimum Area and Minimum Intensity are set to 0 e Global Threshold settings from the Basic Settings
344. viation In general If you set White Noise Std Dev to removed is 1 68 2 95 3 99 If applying Noise Removal with a certain Std Dev does not yield the necessary noise removal return to the original trace see Returning to the original spectrum on page 5 3 and apply Noise Removal again with a higher Std Dev setting Applying Noise Removal more than one time with the same Std Dev setting does not improve noise removal 5 44 4 Click OK The trace is displayed with an RSM NF NR or SM trace label 5 To return to the original trace see Returning to the original spectrum on page 5 3 Applied Biosystems Adjusting the Baseline 5 8 Adjusting the Baseline This section includes e Using Baseline Offset e Using Baseline Correction e Using Advanced Baseline Correction 5 8 1 Using Baseline Offset Use the Baseline Offset command to offset the y axis in a spectrum or to correct a sloping baseline 1 Activate the window in which you want to perform the offset NOTE You can select a Chromatogram or a Spectrum window If you do not activate the correct type of window before performing the next step the software does not select values when you click drag on the trace For example you must select a Spectrum window before starting baseline offset on a spectrum 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the
345. vidual Creates one trace for each specified wavelength Click OK The extracted absorbance chromatograms are displayed in the Chromatogram window with an XAC trace label Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only From the To create an extracted absorbance chromatogram for a Spectrum window wavelength range from the Spectrum window 1 Click the Chromatogram window to activate it 2 From the Display menu select Traces then select a DAD TAC or DAD Channel chromatogram The TAC or Sig Absorbance trace appears in the Chromatogram window 3 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 4 Double click the left mouse button or right click drag over an area of interest in the Chromatogram window A DAD Spectrum appears in the Spectrum window 5 Click the Spectrum window to activate it 6 Inthe Spectrum window right click drag over the region of interest The width of the box you draw defines the precise wavelength range used in the extracted absorbance chromatogram The extracted absorbance chromatogram is displayed in the Chromatogram window Figure 4 7 with an XAC trace label and the center mass and window or the mass range indicated in the trace label Data Explorer Software User s Guide 4 15 Chapter 4 Examining Chromatogram Data XAC 201 6 622 2 400 294 8 6E 6 90 80 70 60 50 162 Intensity
346. without opening the file in the Data Explorer software To view file properties 1 In Data Explorer close the file of interest 2 In Windows NT Explorer select the file then right click 3 Select Properties from the menu 4 Click the Summary tab NOTE If the Summary tab is not available the file may be open in Data Explorer Close the file and repeat the steps above Searching file To search for a file based on file properties properties 4 in Windows NT Explorer select Find from the Tools menu then select Files or Folders The Find All Files dialog box is displayed 2 Inthe Name amp Location tab type or select a directory in the Look in text box Select the Advanced tab Select Data Explore Document from the Of type drop down list 5 Type the information you are searching for for example a title or Keyword in the Containing text box then click Find Now 1 32 Applied Biosystems 1 6 2 Converting Data from Profile to Centroid Mariner Data Only Overview Before you begin Converting to centroid Managing Files You can convert an entire data file from profile to centroid format Centroid format files are smaller than profile format files NOTE Profile data is not automatically deleted when you convert to centroid data You can delete the profile data file using Windows NT Explorer If the file to convert is in SPC format confirm that the SPC and CGM files are loca
347. you are analyzing higher masses or want to label average isotope masses e Voyager reflector data 10 000 which is optimized for masses below 20 000 m z Decrease this value if you are analyzing higher masses or want to label average isotope masses If you set a resolution value of e 1 000 or greater The software uses the setting until it reaches the mass at which isotopic resolution is no longer possible then switches to a resolution of 1 000 the resolution that corresponds to isotopic clusters e Less than 1 000 The software uses the setting for all masses NOTE The Mass Resolution you set here is also used by the Elemental Composition Calculator the Elemental Targeting Application and the Default Smoothing function For more information see Section 6 1 Using the Elemental Composition Calculator Section 6 6 Using the Elemental Targeting Application and Section 5 7 Noise Filtering Smoothing 3 24 Continued Applied Biosystems Peak Detection Table 3 2 Basic Settings Tab Parameters Spectrum Data Only Continued Parameter Description Trace Settings Use same settings Applies settings to all traces in the active window for all traces in view NOTE In previous versions of Data Explorer software peak detection allowed you to specify Peak Width The software now automatically uses a minimum peak width that is equal to the Filter Width and a maximum peak width of 10 000 d
348. you override Global Thresholds by selecting Use Advanced Settings described on page 3 23 the software ignores Global Thresholds and uses the Base Peak Intensity Max Peak Area Minimum Area and Minimum Intensity thresholds set on the Advanced tab described on page 3 28 to detect peaks for the selected detection range NOTE If you are examining Voyager data that contains a Low Mass Gate spike the software may identify the Low Mass Gate spike as the Base Peak Truncate the data as described in Section 5 9 Truncating a Spectrum to eliminate the Low Mass Gate spike and correctly identify the Base Peak Hint For most Voyager applications leave this parameter set at 0 and adjust the Max Peak Area Continued 3 22 Applied Biosystems Peak Detection Table 3 2 Basic Settings Tab Parameters Spectrum Data Only Continued Parameter Description Max Peak Area Specifies a percentage of the peak with the largest area as the threshold value To be detected peaks must be above this threshold and above the Base Peak Intensity value Max Peak Area is calculated above the local baseline and can compensate for problems related to a rising global baseline If you override Global Thresholds by selecting Use Advanced Settings described on page 3 23 the software ignores Global Thresholds and uses the Base Peak Intensity Max Peak Area Minimum Area and Minimum Intensity thresholds set on the Advanced tab d

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