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Analyst® 1.6.1 Software Scripts User Guide

1. Analyst 1 6 1 Software Scripts User Guide AL Release Date March 2012 This document is provided to customers who have purchased AB Sciex equipment to use in the operation of such AB Sciex equipment This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited except as AB Sciex may authorize in writing Software that may be described in this document is furnished under a license agreement It is against the law to copy modify or distribute the software on any medium except as specifically allowed in the license agreement Furthermore the license agreement may prohibit the software from being disassembled reverse engineered or decompiled for any purpose Portions of this document may make reference to other manufacturers and or their products which may contain parts whose names are registered as trademarks and or function as trademarks of their respective owners Any such use is intended only to designate those manufacturers products as supplied by AB Sciex for incorporation into its equipment and does not imply any right and or license to use or permit others to use such manufacturers and or their product names as trademarks AB Sciex makes no warranties or representations as to the fitness of this equipment for any particular purpose and assumes no responsibility or contingent liability including indirect or consequential damages for any use to w
2. Item Description 1 Scan type 2 Buttons to cycle through different MS scans 3 Links Optimization Complete When the quantitative optimization for MS is completed or stopped a Results txt file is generated This file is automatically opened in Microsoft Notepad You can also view the file by clicking View Results from the MRM3 Optimization window The various parts of the Results txt file are described as follows Time and Duration Displays the date and time duration of optimization User Starting Conditions Displays the settings and Advanced Settings in this section e Optimization Conditions Found Displays the optimal conditions found during the ER and Q1MI scans MS3 Fragments Found and Associated Losses Displays the fragments and optimal conditions collision energy and excitation energy as well as associated losses found for the EPI scan and MS Scripts User Guide Analyst 1 6 1 Software 61 of 88 Analyst Software Scripts P Results txt Notepad Eje Edt Format View Help Quatitative Optimization for MS3 Thursday July 15 2004 Start 10 12 49 AM End 10 24 37 AM starting Parameters Analyst Project opt MS3 starting Method starter Method dam Compound Name Reserpine Resolution Unit Expected m z 609 281 amu Polarity Positive ER Scan Rate 250 amu s QIMI DP Ramp Ov to 200v with Sv step EPI Scan Rate 1000 amu s EPI 2nd Prec 5 MS3 Scan Rate 1000 amu s
3. A Blexempte y A EN PEARL TN IDA First Level Criteria Include Exclude Isotope Patter Select r e i m mostinensepesks F fe Dynamic Background J Subtraction of Survey scan Acquisition Method 3 Y Mass Spec 30 000 min 3 6 Period 30 000 min MRM B IDA Criteria Survey gt IDA Experiment Exclude former target ions C Always ju nv NM After occurrence s jz 1 m 2 C For sec 7 Wh nathan ae ier e ea AH UEG Scripts User Guide Analyst 1 6 1 Software 21 of 88 Analyst Software Scripts 2 Although the DBS feature is activated at the method level the DBS options are set using the script From the Analyst software menu click Script gt DBS Settings ig DBS Settings Al x Subtract 4 y previous spectra from current spectrum Smooth 5 v Data Points OK Cancel Subtract the _ previous spectra from current spectrum Use this field to select the number of spectra averaged to represent the background signal These spectra are taken immediately before the current survey spectrum Typical values used are between 2 and 5 Smooth Before Subtract Select to make sure that the current survey spectrum is smoothed using a Savitzky Golay smooth before the subtraction step Select the number of points to consider in the process A typical value is 5 Survey Scans Supported The DBS feature can be used with any IDA survey scans currently supported EMS EMC Precursor
4. To use the script 1 When creating a batch in the Batch Script field navigate to the script After submitting the batch to the queue the following dialog appears in which you can attach the additional acquisition scripts ia Multiple Batch Scripts Script Ea Batch Scripts Add Script Remove Selected Selected Remove all All Only show this dialog again if the control key is down Cancel i Figure 1 34 Multiple Batch Scripts Script dialog 2 To attach an additional script to this batch click Add Script to navigate to the acquisition script 3 Toremove a script click the script and then click Remove Selected Select the Only show this dialog again if the control key is down check box if you want the dialog to appear when submitting the batch 5 Click Run to attach all of the acquisition scripts to the batch Analyst 1 6 1 Software Scripts User Guide 66 of 88 Analyst Software Scripts Open In Workspace Use this script to open a sample in a previously created workspace This script loads the previous workspace and displays the sample in the same layout as specified in the saved workspace It is not possible to create a workspace that specifies pane arrangements so that any sample can be loaded into it For more information on workspaces refer to the online Help To use the script 1 Click Script gt OpeninWorkspace The Open in Workspace dialog appears with the current working project d
5. A given number of initial columns corresponds to the first spectrum a given number of following ones to the second spectrum and so forth Each sparse spectrum is essentially stored end to end A separate vector is written which you can use to determine the starting and ending position of any given spectrum in this matrix This vector contains the one based index of the start of a given spectrum The end for a given spectrum can be determined by subtracting one from the start of the following spectrum except for the very last spectrum which is determined by the size of the above mentioned matrix The first format is the default format for quadrupole spectra and is always used for diode array data The second sparse format is always used for TOF time of flight data and can optionally be used for quadrupole data The actual names of the various matrices are specified using the Options dialog described later in this document To use the script 1 With a chromatogram in an active pane click Script gt Wiff to MatLab Alternatively this script can be attached to a batch select this script using the Batch Editor If the chromatogram is associated with mass spectral data TIC XIC then MS data is exported to the MatLab file if the chromatogram is associated with diode array data TWC XWC then DAD data is exported 2 To change the conversion options open the Wiff to MatLab Conversion Options dialog by pressing the Ctrl key whi
6. Description 1 Checklist 2 Message In the spectral status window the previously generated spectrum or chromatogram is displayed When one of the checklist items is selected the corresponding graph is displayed The scan type name indicates which scan is currently being displayed For each completed step it is possible to open the acquisition method dam or data file wiff associated with the graph displayed If an MS MS MS scan is displayed you can use the buttons to cycle through the different MS scans Analyst 1 6 1 Software Scripts User Guide 60 of 88 MRM3 Optimization Intensity cps m z amu 5 49 43 PM No peaks found parent not below 10 total ion count 55203 PM Optimized AF2 96 552 03 PM No peaks found parent not below 10 total ion count MK 55203 PM gt MS MS MS scan of 2nd precursor 346 021 amu 9 67E 06 cps VeewResuts _ Senos Lose em i 2012 lt gt darn siti 54943 PM gt MS MS MS scan of 2nd precursor 207 065 amu 9 98E 06 cps wv Analyst Software Scripts Enhanced Resolution V Determine actual 1st precursor Q1 Multiple lon 7 Optimize DP Optimize EP EY Y Enhanced Product lon v Get 2nd precursors Multiple Reaction Monitoring v Optimiza CEs for 2nd precursors MS MS MS Generate Final Methods C Generate final MS MS MS Expected batch ime 155 511 sec aun Figure 1 31 MRM3 Optimization window during MS scan
7. E aj lon Source E CUR EIS m TEM E 6S1 GS2 m ihe CAD Figure 1 3 Convert Methods dialog 2 Click Open navigate to the method that you want to convert and then click Open The Method Converter dialog displays the instrument name of the original method 3 Click Save type a name for the converted method and then click Save Analyst 1 6 1 Software Scripts User Guide 14 of 88 Analyst Software Scripts Creating Quantitation Methods and Text Files The Create Text File From Quan Method script exports a quantitation method to a tab delimited text file The Create Quan Method From Text Files script imports the information contained in a tab delimited text file to a Quantitation Method File qmf Currently the Quantitation Method component in the Analyst software does not support this functionality The Create Text File from Quan Method script creates a text file representation of a quantitation method file A column in the text file is created only for all of the required fields The optional fields will be created if the field value is not the same for all peaks in the quantitation method The Create Quan Method From Text Files script specifies the default values for any of the non required fields in the text file such as integration algorithm or regression parameters For more information see Text File Format on page 17 To use the Create Quan Methods From Text Files script 1 Click Script Create Quan Methods Fro
8. Figure 1 37 Regression Calculator dialog Type data in pairs of x and y co ordinates 3 After entering two or more pairs to get the Slope Intercept and R2 value click Calc 4 To delete the values for x and y co ordinates click Clear 5 To exit the application click OK Scripts User Guide Analyst 1 6 1 Software 69 of 88 Analyst Software Scripts Remove Graph Selections Use this script to clear a selected area in the graph For example you can clear the graph selection line from the graph To use the script To remove selections from the graph click Script gt RemoveGraphSelections Analyst 1 6 1 Software Scripts User Guide 70 of 88 Analyst Software Scripts Repeat IDA Method Use this script on an acquisition workstation It opens the acquisition method for the data file and updates the exclusion list with the masses and times that have been acquired The method is saved under the same name To use the script 1 In Explore mode open an IDA data file 2 Click Script gt RepeatIDAMethod 3 To keep the previous exclusion list press the Ctrl key while clicking the script Scripts User Guide Analyst 1 6 1 Software 71 of 88 Analyst Software Scripts Savitzky Golay Smooth Using the Analyst software you can smooth a graph in Explore mode using two different algorithms When you are using the IntelliQuan integration algorithm in Quantitate mode a third smoothing algorithm the Savitzky Golay s
9. Final Methods Save all final methods Optimization Results Actual m z 609 172 amu 7 23E 07 cps aq 3 Optimized oP 90 ae initial value Optimized EP 10 10 initial value optimized CEP 24 24 774 initial value MS MS Fragment 1 195 117 amu Loss of 414 9 98E 06 cps optimized CE 47 10 initial value optimized AF2 70 100 initial value MS3 Peak Centrotd Mass amu 2nd Loss Centroid Intensity cps 5 00E 04 1 67E 04 Final M53 Method Reserpine_FinalMs3_195 117 dam MS MS Fragment 2 174 149 amu Loss of 435 8 60E 06 cps optimized CE 55 10 initial value optimized AF2 70 100 initial value MS3 Peak centroid Mass amu 2nd Loss centroid Intensity cps 1 00E 05 5 00E 04 Final MS3 Method Reserpine FinalMS3 174 149 dam Figure 1 32 Optimization Report Item Description 1 Time and Duration 2 User Starting Conditions 3 Optimization Conditions Found 4 MS3 Fragments Found and Associated Losses All of the generated acquisition methods have a descriptive file name in the format supplied compound name scan type m z dam These methods are saved in the same folder as the starter acquisition method All of the data Log txt and Results txt files are saved into a Data sub folder that is created in the same project as the starter acquisition method The sub folder has the format supplied compound name OptMS3 date time The data files have t
10. and get the overlaid XIC traces hold the Shift key down while clicking the script To update the processing preferences and get the overlaid XIC traces press the Ctrl key while clicking the script XIC From Base Peak Masses CIE XI Start Mass 100 amu Stop Mass 1000 amu Mass Tolerance 0 5 amu Threshold E 0 counts Overlay ADC Trace ADC Channel Number fi y E Cancel Figure 1 44 XIC From Base Peak Masses dialog When processing is started a progress bar indicates the current step When finished a new pane with overlaid XIC traces is the active pane in the Analyst software document If you want to use the Cycle Overlays feature and it is unavailable switch the active pane in the Analyst software to another one and then reselect the overlaid XIC pane Scripts User Guide Analyst 1 6 1 Software 85 of 88 Analyst Software Scripts XIC from Table Use this script to create and read start and stop masses from a file The file will have two columns separated by a tab The first column contains the start mass and second column contains the stop mass When working with full scan data the Analyst software can show an Extracted lon Chromatogram XIC for a subset of the scanned mass range If the subsets are repeatedly used on different data it is often convenient to store the subsets of the mass range consisting of a start and stop mass in an external file and have the Analyst software generate an XI
11. Acquisition method In the former case the script calculates one spectrum for each experiment by averaging all spectra acquired for the experiment In the latter case the script uses all dependent product spectra combining adjacent spectra with the same parent mass and charge state To use the script If you are planning to send just one spectrum to Mascot make sure that a centroided spectrum is active in the Analyst software before running the script If you are planning to send all spectra contained in a sample the TIC for the sample should be active 1 With a centroided spectrum in an active pane click Script gt Mascot The File Sample Periods and Scan type IDA data fields are read only and show information about the active sample Mascot Search version 1 6627 Search The current spectrum O AIMS MS spectra for current sample AI MS MS spectra from all samples for current file File C Analyst Data Projects E xampleND ata LIT Bromocriptine vall Sample Pos EPI 1000 amu s Q1 unt CE 35 Penods 1 Scan type Enhanced Product Figure 1 21 Mascot Search dialog 2 In the Search group you can do one of the following Ifthe active pane is a centroided spectrum either MS or MS MS click The current spectrum to search this spectrum on its own You can select this option only if the spectrum was centroided using the Centroid command on the Explore menu Ifthe active pane contains MS MS data c
12. After an intensity value falls below this threshold the sub sample is considered complete and it is sent to the file The Width field contains the width threshold value This value is used to prevent short periods of loud noise If noise exceeds the noise threshold value for a short period of time that is have a small width then without the width threshold this noise will be considered a new sample Only detected samples with a width that exceeds the width threshold will be exported to the file In the Decompose To field type the full path and the file name of the file in which the sample will be decomposed or navigate to a file If this file does not exist then the Make Subset File program will create it To start decomposing the sample click Extract Analyst 1 6 1 Software Scripts User Guide 44 of 88 Analyst Software Scripts Manually Integrate The Analyst software has various peak finding algorithms that determine and integrate peaks and then show the results in the peak list If required you can also use the Manually Integrate script to integrate a selected region of a graph because the software may not have detected the peak or perhaps because only a portion of the peak is of interest Use the script to draw a line on a chromatogram and have the area above the line integrated The integrated area is highlighted in the chromatogram and the calculated area of the region can be pasted on the graph The results are display
13. Chromatogram with You will be prompted for the name of the JCamp file and one or no selections every spectrum for the run exported to it Batch N A The name of the JCamp file is generated by appending the sample number to the name of the wiff file and changing the extension to jdx Every spectrum for the run is exported to the JCamp file For multiple period experiment data a separate file is exported for each experiment the period and experiment numbers are appended to the filename To use the Export to JCAMP do one of the following With either a chromatogram or a spectrum in an active pane click Script gt Export to JCAMP Inthe Batch Editor dialog type Analyst Data API Instrument Processing Scripts Export to Jcamp dll in the Batch Script field Scripts User Guide Analyst 1 6 1 Software 31 of 88 Analyst Software Scripts i JCamp Options x mr td ti rta Field Intensity Sum v v Deisotope Only show this dialog again if the control key is down DK Cancel Figure 1 11 JCAMP Options dialog 2 To select the centroiding options do one of the following e Check the Centroid Exported Spectra check box to centroid the spectra before exporting to the JCAMP format To have the JCAMP Options window appear only if the Ctrl key is pressed when clicking the script from the Script menu or when submitting the batch to the queue select the Only show
14. The half width of the Savitzky Golay smoothing filter from 0 to 20 when using the IntelliQuan algorithm MQ III Noise No The noise percentage when using the MQ III option of the Percent IntelliQuan algorithm This should be an integer from O to 100 MQ III Baseline No The baseline subtraction window from 0 to 10 minutes Sub Window when using the MQ III option of the IntelliQuan algorithm MQ III Peak No The peak splitting factor from O to 10 when using the MQ Splitting Factor Ill option of the IntelliQuan algorithm MQ III Use Largest No When using the MQ III option of the IntelliQuan algorithm specifies whether the largest peak within the retention time window or the peak whose retention time is closest to that expected is reported Select Yes to use the largest peak and No to use the closest Summation No When using the special window summation algorithm Baseline Sub specifies whether the area should be integrated to the intensity 0 line or to the intensity value of the least intense data point within the window The following table shows an example text file for full scan data Imagine that there are tabs between the columns and a hard return at the end of each line Table 1 2 Example Text File for Full Scan Data Peak Name First Mass Second Mass Bunching Factor Analyte Peak 1 500 10 500 70 Scripts User Guide Analyst 1 6 1 Software 19 of 88 Analyst Software Scripts Table 1 2 Example Text File
15. also provides an overview of the uses of each script and how to uninstall a script if required Related Documentation You can also create your own scripts using the Visual Basic Version 6 program Lor more information see the Analyst Automation Cookbook A guide to controlling Analyst 1 6 1 Software from Visual Basic NET Installing or Uninstalling Scripts Some scripts are automatically installed when the Analyst software is installed The remaining scripts are available in the Scripts folder When you have decided which scripts you want to use use the following procedure to install them versions To determine which scripts the software version installed on your instrument 7 Note This guide contains the scripts for all instruments and different software supports see the Analyst software release notes Installing a Script 1 Navigate to the following folder on your workstation lt drive gt Program Files Analyst Scripts 2 Open the required folder open the script folder and then double click ScriptRunner exe 3 Follow the on screen instructions to install the scripts The script is available on the Script menu e Note For some scripts if you hold down the Shift key while accessing a script on the Script menu a description of the script is displayed Uninstalling a Script e To uninstall a script do one of the following For processing scripts navigate to the lt drive gt Analyst Data Projec
16. and experiment number will be appended to the MatLab filename For example if a data file is called test wiff and contains two samples the MatLab files will be called test 1 mat test 2 mat If the acquisition method contained one period and two experiments four MatLab files would be generated two for each sample test 1 1 1 mat test 1 1 2 mat test 2 1 1 mat test 2 1 2 mat If the data file contains diode array data and the Save DAD data check box is selected then an additional file with the sample index and DAD is created For the previous example files called test 1 DAD and test 2 DAD mat are created Known Issues and Limitations When attaching this script to a batch make sure that the Acquisition Queue window is opened before submitting the batch This window must be open until the acquisition has completed to make sure that the script is working properly Related Scripts Export to JCamp Converts spectra from wiff format to JCamp format Table 1 8 Matrix Definitions Matrix Dimensions Type Descriptions Data Number of Float The raw intensities for all of the spectra masses or wavelengths x number of scans SparseData 2 x total number Double The raw masses first row and intensities of mass second row for all spectra This matrix is intensity pairs used only with the sparse format Masses Number of Float The actual m z values scanned by the masses x 1 instrument This m
17. fields type the sample number range to be processed Scripts User Guide Analyst 1 6 1 Software 11 of 88 Analyst Software Scripts 7 Torun the script on each data file in the list click Run 8 To stop the script click Close Analyst 1 6 1 Software Scripts User Guide 12 of 88 Analyst Software Scripts Change All Methods It is often necessary to change the ion source conditions of a method particularly with the NanoSpray ion source after changing the emitter tip This script modifies every method in a selected project with new values for lonSpray Voltage IS lon Source Gas 1 GS1 and Interface Heater Temperature IHT lt Note This script is used with any QTRAP system It is not used by QSTAR instruments P To use the script 1 Click Script change all methods ia Change All Methods xi Change all methods in the project Example r Source Parameters Iv lonSpray Voltage IS E 700 1 700 Vv lon Source Gas 1 651 5 5 E Interface Heater Temperature IHT E 00 E oo E update method with current instrument settings 4F3 EXB C2B Cancel Figure 1 2 Change All Methods dialog Select a project containing the methods that you want to modify Select the parameters that you want to change If the parameter is not in the method file it will be ignored Type a value for positive experiments Type a value for negative experiments Select the update me
18. file that will be used to create the quantitation method 6 To create multiple methods from multiple text files click Create Multiple Methods and then navigate to the folder A quantitation method is created for each text file in this folder To use the Create Text File from Quan Method script 1 Create and save a quantitation method in the Analyst software 2 Click Script gt Create Text File from Quan Method Analyst 1 6 1 Software Scripts User Guide 16 of 88 Analyst Software Scripts Options mfe x v Export all columns otherwise only if field value is not the same for all peaks Figure 1 5 Options dialog Select the Export all columns check box and then click OK Navigate to and select the quantitation method file qmf Navigate to and select the location of the text file The script will generate the text file Text File Format The text files used to create the quantitation methods Create Quan Methods from Text Files and generated from the methods Create Text File from Quan Method are in the following format Separate the various fields with tab characters and end each line with a carriage return line feed characters The very first row of the file should contain column headings All of the columns shown in the following table marked as Required must be present the remaining columns are optional The actual order of the columns is not important Each subsequent line should contain the information as s
19. of this field empty For internal standard peaks themselves the contents of this field are ignored Period No The period number for the peak from 1 to the number of periods in the data Experiment No The experiment number for the peak from 1 to the maximum number of experiments in the period Use Relative RT No For analyte peaks that are using an internal standard specifies whether or not the expected retention time is relative to that of the IS Select Yes if so otherwise select No The contents of this field are ignored for other peaks but must still contain either Yes or No Conc Units No The concentration units Calc Conc Units No The calculated concentration units Bkg Start No Start time in minutes for the peak background Note that this parameter does not affect the peak integration in any way however it does affect how the noise and hence S N is calculated Bkg End No End time in minutes for the peak background Expected RT No The expected retention time in minutes from O to 1666 RT Window No The retention time window in seconds from 1 to 1000 Algorithm No Specifies which peak finding and integration algorithm should be used If present this should be one of 0 Analyst Classic TurboChrom 1 IntelliQuan IQA II Automatic 2 IntelliQuan MQ III 3 Window Summation Note that the window summat
20. script generates the final MS methods If the Save Optimal Method Only option is clicked in the Advanced Settings dialog only an optimal MS3 method with 10 amu around the most intense second generation fragment ion is created If the Save All Final Methods option is clicked then the optimal method as well as an MS3 method for each of the top second precursors are created using a mass range from the user defined lower limit to an upper limit of second precursor 5 amu Analyst 1 6 1 Software Scripts User Guide 64 of 88 Analyst Software Scripts MS3 Quant Optimization Script The MS3 Quant Optimization script has been replaced by the MRM3 Optimization script Users who have upgraded from the Analyst 1 5 2 to 1 6 to 1 6 1 software versions may still have this script installed The MS3 Quant Optimization has not been tested with the Analyst 1 6 1 software therefore users should use the MRM3 Optimization script instead Scripts User Guide Analyst 1 6 1 Software 65 of 88 Analyst Software Scripts Multiple Batch Scripts Use this script to attach multiple acquisition scripts to a single batch that is submitted to the queue These acquisition scripts are used to immediately process the data either after a sample completes or after the batch finishes Using the Analyst software you can submit only one script with the batch but sometimes it is convenient to run two or more scripts to perform two or more different types of processing
21. text is blue and underlined Task completed no link text is blue Part of task completed hyperlink text is blue underlined and italic When the text is underlined you can click it like a web page hyperlink and the corresponding spectrum or chromatogram is displayed The text found under MS MS MS also displays the MS scan number that is being performed because it is possible to have between 1 and 10 scans The Overall Progress section also includes a Message area In this area a progress bar displays the current step progress Above the progress bar various messages are displayed such as the time and other statuses for the current optimization step MRM3 Optimization ep 059 19 CE 45 CES 20 0 053 to 0 16 Max 4 346 cps Enhanced Resolution Determine actual 1st precursor Q1 Multiple lon Optimize DP Optimize EP E Enhanced Product lon v Get 2nd precursors Multiple Reaction Monitoring O Anini LEs fer Sad precursor MS MS MS C Optimize AF2s for 2nd precursors 5 46 55 PM 2nd precursors and get 3id precursors 0 of 3 5 46 55 PM 301 001 amu 1 798 07 cps 5 46 55 PM 207 066 amu 9 98 06 cps Generate Final Methods 5 46 55 PM 346 021 amu 9 67E 06 cps JM 5 46 55 PM gt MRM scan of 2nd precursors e pin died rk es Intensity cps Creating method and subenting batch events seins EET D Figure 1 30 MRM3 Optimization window after EPI scan Item
22. window Fragments Formula Chemical formula of the fragment protonated deprotonated form Fragments Extract Start data processing according to the current settings Fragments Polarity Select the polarity of the fragment spectrum experiment where the specified fragment is expected to be found Fragments Clear Clear all fragments in the settings Options From Time min Start of time region to be processed Options To Time min End of time region to be processed Options Trace Width Da Width of XIC traces in resulting survey scan and dependent scan chromatograms and mass tolerance window for processing in case that the neutral losses or fragments are specified as chemical formulas Options Remove Unconfirmed Review all peaks in the survey XIC traces Peaks and retain only those that were validated based on the data in corresponding dependent scan Options Spectrum Peaks gt Minimum size of diagnostic peak in dependent scan in terms of signal to noise lowest measurable signal Options Label Peaks gt Peak label threshold applied to resulting survey and dependent scan chromatograms note that just the largest peak in each trace is labeled Scripts User Guide Analyst 1 6 1 Software 37 of 88 Analyst Software Scripts Table 1 6 Tab and Menu Parameters Continued Analyst 1 6 1 Software 38 of 88 Location Parameters Description Options Sho
23. Analyst Data Progects API Instrument Acquistion MethodsNLIT PosPPG dam Update MRM compound ID from file name Figure 1 25 Merging MRM methods dialog 2 To navigate to the original acquisition method file or template click the button to the right of Original method 3 To specify the name and location of the merged acquisition method file click the button to the right of New Method type the name of the method and then click Open 4 To add an acquisition method to the list of methods to be merged click Add to the right of Methods to be merged To remove a method from the list click Remove 5 Optional If both methods were created using Compound Optimization select the Update MRM compound ID from the file name check box to populate the compound ID column with the compound name in the merged method 6 To add all the mass ranges from the selected methods click Go Analyst 1 6 1 Software Scripts User Guide 54 of 88 Analyst Software Scripts MRM3 Optimization Script Use this script for quantitation on the QTRAP systems in order to provide increased specificity and therefore improved detection when quantifying analytes in complex matrices This script is designed to generate an optimal MS acquisition method on any QTRAP system with any source and at any flow using infusion The script performs the following optimization steps Confirm precursor mass Optimize transmission to collision cell e Determine the major
24. C based on this file The script generates the requested XICs either as one XIC per pane or all the XICs overlaid in one pane from the current Total lon Chromatogram TIC To use the script 1 With a TIC open in an active pane click Script gt XIC_from_table i XIC Preferences Ea Fie Table About H 7 2 3 4 5 6 iy 8 Jv Overlay Is Extract Cancel Figure 1 45 XIC Preferences dialog 2 Using the grid in the dialog type the mass ranges to be extracted into an XIC To populate the grid from a text file click File gt Open and then navigate to the text file 3 To save the current information in the grid to a file click File gt Save As Analyst 1 6 1 Software Scripts User Guide 86 of 88 Analyst Software Scripts To clear the grid of all entries click Table gt Clear To overlay all XICs into a single pane select Overlay XICs check box To create all the specified XICs click Extract NO de To close the dialog and close the script click Cancel Known Issues and Limitations When not loading XIC start stop masses from a file the maximum number of mass pairs you can type is 25 However when loading from a text file an unlimited number of start stop mass pairs can be specified Due to limited space on the screen you may have to select the Overlay XICs check box when creating more than six XICs Scripts User Guide Analyst 1 6 1 Software 87 of 88 Analyst Software Scripts AB SCIE
25. Driver With the Batch Acquisition Script Driver you can run an acquisition script on multiple data files wiff by attaching the acquisition script to a batch that is submitted to the queue These scripts are used to immediately process the data either after a sample completes or after the batch finishes The script will process the data files as if they were separate batches submitted to the queue Occasionally you might need to apply these acquisition scripts on previously acquired data because either the script did not exist at the time of acquisition or a rerun of the script is required To use the script 1 Navigate to C Program Files Analyst bin and then double click BatchScriptDriver exe i Batch Acquisition Script Driver xi Script Select Files to Process Add File Add Folder Remove Selected Remove All rm F from sample fi to fi Close Figure 1 1 Batch Acquisition Script Driver dialog To select an acquisition script click Select To add a data file click Add File Click Add Folder to add an entire folder of data files 4 Toremove a data file select it from the list and then click Remove Selected Click Remove All to remove all the data files 5 To process some of the samples contained in a wiff data file select the Process Limited Sample Number Range check box This field is available only if you have selected only one data file to process 6 In the from sample and to
26. File Name Ic Analyst Data Projects Example D ata TOF Neg MSMSTa Select Sample fi Time Offset Sample Control min fa v Overlay Sample Data v Overlay Control Data Cancel Figure 1 41 Subtract Control Data QS Preferences dialog Scripts User Guide Analyst 1 6 1 Software 79 of 88 Analyst Software Scripts Unit Conversion Use this processing script to convert from one set of concentration units to another To use the script 1 Click Script gt Unit Conversion Convert from g v E 11 0 y la 1 0 y Convert Figure 1 42 Unit Conversion dialog In the Convert from field type the concentration and units to be converted If the conversion is from a weight based concentration for example g L to a molar based concentration for example mol L then in the MW field type the molecular weight of the component In the Convert to field type the unit To perform the conversion click Convert The calculated values will be displayed in the Convert to field 6 To retrieve these values press Ctrl C to select and copy them to the clipboard These values can then be pasted into another application Analyst 1 6 1 Software Scripts User Guide 80 of 88 Analyst Software Scripts Wiff To MatLab Use this script to extract the data from a wiff file and creates the following matrices in a MatLab file Data Masses Wavelengths Scans and Filename For more information a
27. In the Algorithm drop down list select a peak finding algorithm The Window Summation algorithm sums all the intensities in the retention threshold and will not find any peaks Inthe Extraction Type drop down list select the type of data that will be integrated Inthe Period and Experiment drop down lists select the period number and experiment number The Default Analyst Classic Parameters Default General IntelliQuan Parameters Default IntelliQuan MQ III Parameters and the Default Window Summation Parameters groups contain the parameters that are used by the algorithm selected in the Algorithm field ss Note The Smoothing Width field in the Default General IntelliQuan Al Parameters group is half the smoothing width P 3 Select the Use Baseline Subtraction check box to have the Window Summation algorithm sum the intensities to the horizontal line at the minimum intensity of the data points within the summation window as opposed to summing down to the intensity zero 4 In the Regression Parameters group select the regression information The information specified here is applied to every analyte peak Unlike the previous parameters it is not possible to indicate this information in the text files therefore the same regression parameters are applied to all analytes For a full description of the parameters see the Help 5 To create one quantitation method click Create One Method and then navigate to the text
28. Loss or fragment spectrum experiment where the specified neutral loss or fragment is expected to be found im IDA Trace Extractor xi Fie Tools Help Clear Extract m F1 a Polarity TLL AUT LLL e Use Masses C Use Formulas Figure 1 15 IDA Trace Extractor dialog Neutral Losses tab Scripts User Guide Analyst 1 6 1 Software 35 of 88 Analyst Software Scripts im IDA Trace Extractor xi Fie Tools Help Neutral Losses f m z a n m m rn ishchehehehehshehehel Use Masses C Use Formulas Clear Extract Figure 1 16 IDA Trace Extractor dialog Fragments tab 5 Click Extract to find the survey XIC traces that give the selected neutral loss or fragment 6 If the precursor information was saved the found precursor mass time data can be converted to a compatible format using other scripts To convert the data click the Results tab and then select the Results File View and edit this file as required To make a format for the XICfromTable script click Make XIC Table The Results File will be converted into a file of the same name with the suffix XIC To make a format for the CreateQuantMethodFromText script on the Precursor XICs dialog click Make Quant Input The Results File will be converted into a file of the same name with the suffix Peaks 7 Click File gt Save Settings as Alternatively previously saved settings can be used Click Fi
29. Scan Neutral Loss MRM Q3 MS and Q3 MI DBS does not apply to the confirmation or dependant levels Known Limitations and Issues With the exception of DBS being on or off DBS parameters are not stored as part of the file information In order to recall which settings were used the user should document the parameters selected during the acquisition e DBS is meant for use during LC analysis therefore when performing IDA by infusion it is recommended to have DBS deactivated Analyst 1 6 1 Software Scripts User Guide 22 of 88 Analyst Software Scripts Define Custom Elements Use this script to select a custom isotope pattern when working with radio labeled compounds An experiment specific element pattern is used in the data interpretation in conjunction with the Analyst software calculators or the Metabolite ID application The custom isotope patterns are stored together with the information from the periodic table elements in the element definition file SAElements ini which is located in the Analystibin folder In the element definition file the custom elements must have a unique symbol and an atomic number of 104 or higher Prerequisites The following program is optional e Metabolite ID 1 3 for Analyst QS 2 0 software When the script is launched for the first time a backup copy of the SAElements ini file is saved in the API Instrument folder If needed the edited SAElements ini file in Analystibin folder can be repla
30. SplitGraph Note Make sure that the mass or time range for splitting is displayed If A necessary dock the graph to make sure that the whole graph can be seen 2 Doone ofthe following The current version of XICfromTable script can handle a maximum of 25 XIC mass ranges If you press the Ctrl key while clicking the script a dialog appears prompting you to select the number of panes to create The preset value is four If the Ctrl key is not pressed then the last number typed in the dialog is used Analyst 1 6 1 Software Scripts User Guide 78 of 88 Analyst Software Scripts Subtract Control Data from Sample Data Use this script when the sample data of interest are in the active graph The data may be any spectrum TIC or ADC trace The script determines the data type retrieves the corresponding data from the control file and then displays the subtracted data in a graph You may select to overlay the subtracted data with sample data or control data To use the script 1 To process the data with current preferences click Script gt SubtractControlData 2 Doone of the following To see the script description set the processing preferences and get the subtracted data hold down the Shift key when clicking the script To update the processing preferences and get the subtracted data press the Ctrl key when clicking the script iw Subtract Control Data QS Preferences CI xi Control Data
31. X Analyst 1 6 1 Software Scripts User Guide 88 of 88
32. a points between the specified background start and background end times Use this processing script to calculate the signal to noise ratio for the active chromatogram The script first subtracts the average background signal from the selected peak and then divides the subtracted signal by the peak to peak noise level It then differentiates the noise and peak regions based on the maximum intensities of each region Upon completion the active chromatogram is labeled with the signal to noise ratio To use the script With a noise region and the peak of interest selected in the active chromatogram click Script gt S to N The signal to noise ratio is calculated and the graph is labeled e Tip To remove the labels press the Ctrl key while clicking the script Related Scripts S NstdDevQS Calculates the signal to noise value with a method that uses the noise regions standard deviation Analyst 1 6 1 Software Scripts User Guide 76 of 88 Analyst Software Scripts Signal to Noise Using Standard Deviation Use this script to calculate the signal to noise ratio of chromatographic peaks and label them The script requires two regions to be selected on the chromatogram a selection region containing the noise region and a selection region containing the peak of interest The script will automatically determine which region contains the peak and the noise based on maximum intensities in each selection It subtracts the average backgr
33. a window where you can type compound information required for the optimization process Click Start to initiate the optimization process During optimization this button is renamed to Abort which you can click to stop the optimization process Scripts User Guide Analyst 1 6 1 Software 55 of 88 Analyst Software Scripts MRM3 Optimization Current Settings Enhanced Resolution Analyst Project ISB Kinase Determine actual Ist precursor Method Q1 dam Compound Name Peptide Q1 Multiple lon Q1 Resolution Unit Optimize DP Expected m z 581 0392 Da Optimize EP Polanty Posilive T Advanced Settings EJ ER Scan Rate 1000 Da s Q1MIDP Ramp 30V to 1504 with 5V step Enhanced Product lon EPI ScanRate 1000Da s C Get 2nd precursors gt MS3Scenfete S000 Da Multiple Reaction Monitoring Final Methods Save all final methods C Optimize CE s for 2nd precursors MS MS MS Optimize AF2s for 2nd precursors Select Settings button to modify the current settings and get 3id precursors 0 of 0 Select Advanced button in Settings to modily the advanced settings Generate Final Methods C Generate final MS MS MS Figure 1 26 MRM3 Optimization window Item Description 1 Status Window Log File 2 3 Main Controls 4 Overall Progress To set the preferences When the script is run for the first time the Settings dialog appears automatically Otherwise the MRM3 Optimizat
34. agments Clear Tools Menu Clear Neutral Losses See Neutral Losses Clear Tools Menu Make XIC Table See Results Make XIC Table Tools Menu Make Quant Input See Results Make Quant Input Table 1 7 Related Scripts Script name Description Export to JCamp Converts spectra from wiff format to JCamp format Scripts User Guide Analyst Software Scripts Table 1 7 Related Scripts Continued Script name MSMS Methods from MW Lists Description Allows lists of molecular weights obtained from text files to be used as the basis for creating a series of MS MS acquisition methods Multiple Batch Scripts Script Allows multiple batch acquisition scripts to be used at the same time the Batch Editor only allows a single batch script to be specified Unit Conversion Converts from one set of concentration units to another Wiff To MatLab Scripts User Guide Converts the data from a data file from the wiff format to MatLab mat format Analyst 1 6 1 Software 39 of 88 Analyst Software Scripts Label Selections Use this script to add missing labels to the selected peaks in the active graph or to remove them The script can be run when a pane containing a spectrum or a chromatogram is active in Explore mode and there are one or more selections in the active pane If neither peak mass spectrum nor peak retention time chromatogram is available the data will be marked with information for the selectio
35. alog Tip To display the Mascot search form defaults Web page click Set default parameters Ed 5 To set where the text file used as input to Mascot is located click Set search file location select one of the following options and then click OK Scripts User Guide Analyst 1 6 1 Software 49 of 88 Analyst Software Scripts Search File Location The script will create a fle containing the data for the Mascot search Specty the location for this fle In the Windows T emp folder with a random filename Always to the following fie O To the following deectory with a random flename Prompt me each time for the location Figure 1 23 Search File Location dialog To place the file in the Windows temporary folder with a random but unique file name click In the Windows Temp folder with a random filename To always write to a specific file overwriting the file for every search click Always to the following file To navigate to the folder click Set To create the file with a random but unique filename in the specified directory click To the following directory with a random filename To navigate to the folder click Set To be prompted to save every time a search is performed click Prompt me each time for the location 6 Inthe Default precursor charge states group when searching only the current spectrum values here are not available the precursor charge state should be set manually usin
36. alog DFT Tracker monitors the dynamic changes occurring during a real time run The system dynamically calculates the time required to fill the linear ion trap For abundant compounds a short fill time reduces the space charge effects by limiting the number of ions in the ion trap on the other hand the longer fill time increases weak signals by allowing the ions to accumulate Scripts User Guide Analyst 1 6 1 Software 27 of 88 Analyst Software Scripts Export IDA Spectra Use this script to export data in a format that can be searched using a third party application The Export IDA Spectra script exports every dependent product spectrum from an IDA Information Dependent Acquisition LC MS run to a series of text files These text files can then be submitted and searched using Sequest The export is optimized so that any spectra in adjacent cycles with the same precursor m z are combined into a single spectrum This optimization also applies to spectra with the same precursor but which reside in different experiments most likely using different values of the collision energy The charge state of the precursor ion is a required input Sequest tries to automatically determine this from the isotope spacing at the precursor m z in the IDA survey spectrum Note that while this determination is usually correct it is not always so To use the script It is assumed that the first experiment of the IDA method represents the survey spectrum and
37. ata folder loaded into the Data Files list ij Open in Workspace oix File Workspace Help r Workspace Data files Available samples Available workspaces 2 DataT utorials2 wiff Set selected workspace as default gt r Default Workspace Name of the Default Workspace Open file with default workspace Cancel Figure 1 35 Open in Workspace dialog To select a data file from another project click File gt Open Data File In the Data files list click a data file All the samples in the selected data file appear in the Available Samples list 4 After choosing the sample select a workspace in the Available workspaces list To select a different workspace click Workspace gt Open Workspace 5 To seta default workspace to be used each time this script is run select the Set Selected Workspace As Default check box 6 To open the sample click Open Sample in Workspace Known Issues and Limitations The Open in Workspace script is for the presentation of graphs only The script cannot handle data lists that are saved in the workspace and a Type Mismatch error will be displayed You must save your workspaces with the graphs only first and then after loading the workspace create the necessary data lists Scripts User Guide Analyst 1 6 1 Software 67 of 88 Analyst Software Scripts Peak List from Selection Use this script to determine the peak data for a selected region or region
38. atrix is only present when exporting MS data Scripts User Guide Analyst 1 6 1 Software 83 of 88 Analyst Software Scripts Table 1 8 Matrix Definitions Continued Analyst 1 6 1 Software 84 of 88 Matrix Dimensions Type Descriptions Wavelengths Number of Float The actual wavelengths acquired by the wavelengths x 1 diode array detector This matrix is present only when exporting DAD data Index 1 xnumber of Long scans Scans 1 x number of Float The retention times in seconds or the scan scans numbers for the spectra Filename 1 x length of Text This optional matrix specifies the filename filename of the original data file Scripts User Guide Analyst Software Scripts XIC from BPC Use this script to retrieve the list of base peak masses and overlay the corresponding extracted ion chromatograms in an Explore pane below the active pane Run the XIC From Base Peak Masses script after selecting a time region of interest in a chromatogram The largest peak in each XIC trace will be labeled with its mass if it is greater than 5 of the most intense XIC peak To use the script You may optionally select to overlay the XIC traces with an ADC trace The ADC trace will be normalized to the most intense XIC trace 1 To process the data with current preferences click Script gt XIC from BPC 2 Doone of the following To see the script description set the processing preferences
39. bout these matrices see Table 1 8 Matrix Definitions on page 83 This MatLab file can then be included in a MatLab script and used to compute and display the results Although you can use the Analyst software to perform various data manipulations you may use this specialized script Wiff to MatLab for data computations The Analyst software cannot extract data from a wiff file and then store it in a file that can be read and interpreted by MatLab In addition to exporting the mass spectral data this script can also export data from a diode array detector DAD The script will create a single MatLab mat file for each sample to be translated Depending on the type of mass spectra data and the user options the script can create data in two different formats The first format saves the intensities in a matrix of size number of masses x number of scans There is thus an entry in the matrix for every mass that was scanned and every spectrum This is the default format for quadrupole spectra and the format that is always used for diode array data The second format is sparse or compressed and allows data points that have an intensity of zero to be omitted depending on the number of such data points in the original wiff sample this can potentially greatly reduce the size of the MatLab file This format uses a matrix of size 2 x total number of mass intensity pairs The first row represents masses and the second row the intensities
40. can times not scan numbers field is cleared then the script will populate the Scans matrix with the time in seconds for each scan Otherwise the Scans matrix is populated with the scan numbers 8 Inthe Use fixed step size field type the step size of the data to extract If this field is cleared then the acquisition step size is used 9 To populate the wavelengths matrix with the DAD data when the script is attached to a batch select Save DAD data in batch mode if available 10 To save these settings and continue processing the data click Save 11 To discard any changes made to the settings click Don t Save Data processing will continue after you click this button Analyst 1 6 1 Software Scripts User Guide 82 of 88 Analyst Software Scripts 12 To automatically generate a MatLab file for every sample submitted as part of a batch run in the Batch Editor click Select Script to select the script before submitting the batch to the queue The script should be located in the Processing Scripts subproject of the API Instrument project A MatLab file is created for every different experiment for each sample in the batch The MatLab files are placed in the same location as the data files and will have the same names with the wiff extension replaced by mat however the index of the sample within the wiff file will be appended In addition for samples acquired using acquisition methods containing more than one experiment the period
41. ced with this file To update the element definition file successfully 1 Do not open the SAElements ini file in another text editor program while using the software 2 Make sure the file access must be set to read write To edit the Define Custom Elements table The custom element table cannot be edited in the dialog After the element definition file is updated the custom elements can be used with the Analyst software calculators 1 Click Script gt DefineCustomEl Scripts User Guide Analyst 1 6 1 Software 23 of 88 Analyst Software Scripts w Define Custom Elements cee yee pa oe ope is Jane Figure 1 6 Define Custom Elements dialog 2 Inthe table click the row containing the element Analyst 1 6 1 Software Scripts User Guide 24 of 88 Release Date March 2012 Analyst Software Scripts Symbol Abundance show OK Cancel Figure 1 7 Define Element dialog 3 Edit the fields and then click OK 4 To save the updated element definition file and exit the program click OK To view the custom element symbol custom element name and custom pattern To view the custom pattern in the mass relative intensity graph in the Define Custom Elements dialog click Show The Isotopic Distribution dialog appears The total of the individual isotope abundance for an element stored in the element definition file must be equal to one Therefore the abundances entered in the Define Element Window are rescal
42. d fragments for a compound class and load them to the settings at a later time Process just a selected time region in the chromatogram Display precursor survey scan or fragment dependent scan XIC trace that yield given fragment or neutral loss From the survey scan chromatograms the user can link to any survey scan spectra From the dependent scan traces the user can link to any dependent scan spectra Reduce the precursor XIC traces to show just peaks that have corresponding neutral losses or fragments e Save the precursor mass list in a text file for further processing build the list of precursors from a set of samples Save the list of masses in a format that can be loaded to the XICfromTable script Scripts User Guide Analyst 1 6 1 Software 33 of 88 Analyst Software Scripts Save the list of masses and peaks in a format that can be loaded to the CreateQuantMethodFromText script Note The script is compatible with MRM MIM IDA data and with the Analyst 1 6 1 software Note The script supports parallel data processing from positive and negative experiments Multiple survey and dependent experiments of any polarity can be used To use the script Note The Analyst software version for a specific file can be displayed in the file Al properties gt comments 1 With a chromatogram of IDA data open in an active pane click Script gt IDATraceExtractor You can process a
43. data file to their own data file and decomposing a single sample into many samples To use the script Section 1 General This section describes how to select a data file to work on 1 Click Script gt MakeSubsetFile Make Subset File LOL x File Decompose Transfer Samples to Transfer Samples to Exclude Extract To C Analyst Data Projects Example D ata E Add All Samples Remove All Samples Extract Figure 1 18 Make Subset File dialog 2 By default all the files in the current project data folder are loaded To add another file to this list click File gt Open and then navigate to the file 3 Click the file To exit the program click File gt Exit Section 2 Transfer This section describes how to transfer samples from one data file to another 1 Click the Transfer tab All the samples in the current working data file will automatically appear in the Samples to Transfer list 2 To exclude a sample from the transfer select it from the list and then click gt gt The unwanted sample appears in the Samples to Exclude list 3 To include an excluded sample in the transfer select it from the Samples to Exclude list and then click lt lt 4 To exclude all the samples click the Remove All type the full path and the file name of the file in which the sample will extract or navigate to a file If the file does not exist the Make Subset File script will create it 5 To
44. e check boxes that correspond to the information that you want to export and then click OK Analyst 1 6 1 Software Scripts User Guide 30 of 88 Analyst Software Scripts Export to JCamp You can use the Analyst software to export graph data to a tab delimited text file that can be read by most applications however some applications require a more specific format With the Export to JCAMP script you can export graph data in the JCAMP format The script works on both chromatograms and spectra For chromatograms depending on the number of selections made either all the spectral data of the chromatogram is exported or the averaged sum of the selected regions is exported If you are using this script on a single spectrum then only that data is exported This script can also be attached to a batch so that the export occurs automatically after the sample is acquired Table 1 5 shows an overview of the operation of the script When run interactively the exact behavior depends on the active Analyst software data Table 1 5 Script Operation To use the script 1 Modes Active data Operation Interactive Spectrum You will be prompted for the name of the JCamp file and the active spectrum exported to it Interactive Chromatogram with You will be prompted for the name of the JCamp file and an two or more averaged spectrum corresponding to each of the selections chromatogram s selections exported to it Interactive
45. ed before they are added to the Define Custom Elements dialog This Isotopic Distribution dialog cannot be edited You can zoom in the area of interest by dragging along the corresponding x or y axis region The application requires the gen01 wiff example file to display custom pattern If the gen01 wiff file is not in the Example folder in the Analyst Data Projects folder you will be prompted to find this file Scripts User Guide Analyst 1 6 1 Software 25 of 88 Analyst Software Scripts Delete Others Use this processing script to delete all panes except for the active one To use the script 1 With a sample file wiff with multiple panes open click a pane The pane becomes the active pane 2 Click Script gt DeleteOthers All the panes except for the active one are deleted Analyst 1 6 1 Software 26 of 88 Scripts User Guide Analyst Software Scripts DFT Tracker The Dynamic Fill Time DFT Tracker script tracks the DFT settings used during QTRAP instrument scans You can use the script to determine the optimal fill time for linear ion trap LIT mode to obtain high data quality over a wide dynamic range The DFT Tracker monitors the following LIT scan types Enhanced MS EMS Enhanced Resolution ER Enhanced Product lon EPI and MS MS MS MS3 To use the script Click Script gt DFTTracker B Dynamic Fill Time Tracker Fie EMS Time Intens DFT Time Figure 1 8 Dynamic Fill Time Tracker di
46. ed in the script window which can also be added to the graph or exported to a text file for storage To use the script 1 With a chromatogram opened click Script Manually Integrate The fields in the Result group are modifiable from the Options button Manual Integration 1 Text File Exporting Export fle test IC hat fv Automatically export after each selection Result if staying open Arex N A Height N ZA Retention Time N A Hall Width N A Figure 1 19 Manual Integration 1 dialog In the Text File Exporting group click Select to navigate to a text file Do one of the following Select Automatically export after each selection to automatically export the results to the specified text file Click Export Now to export the current results To remove the last exported results from the text file click Remove Last Entry To populate the fields in the Result group highlight a section of the graph The values are calculated using the selected area of the graph 6 To change the manual integration options click Options Scripts User Guide Analyst 1 6 1 Software 45 of 88 Analyst Software Scripts Manual Integration Options Lj Mode C Add sea as caption to graph then quit Add aea as caption to graph and stay open Don t add caption to graph and stay open Display Format Area Heaght Retention Time 10 00 Halt Width 10 0 Iv Show integrated area in graph fv Force zero intensity basel
47. eld Intensity Sum m Y Deisotope Only show this dialog again if the control key is down OK Cancel Figure 1 39 JCamp Options dialog 2 To centroid the exported spectra select the Centroid Exported Spectra check box This option reduces the size of the exported JCamp file 3 To select the threshold that will be applied to the exported spectra in the Threshold field type a value If you do not want to use a threshold type 0 in the Intensity Threshold field 4 Select the Only show this dialog again if the control key is down check box to have the JCAMP Options dialog appear if the Ctrl is pressed when selecting the script from the Script menu or when submitting the batch to the queue Analyst 1 6 1 Software Scripts User Guide 74 of 88 Analyst Software Scripts 5 Click OK to continue processing and to have the spectra exported These values are used as defaults until you change them again 6 To close the dialog without making any changes click Cancel In the case of interactive use canceling the dialog will also stop the export operation However in the case of batch operation the batch will still be acquired and JCamp files will be exported using the original parameters Scripts User Guide Analyst 1 6 1 Software 75 of 88 Analyst Software Scripts Signal to Noise Using Peak to Peak The Analyst software calculates the signal to noise ratio by taking the standard deviation of all the chromatographic dat
48. for Full Scan Data Continued Peak Name First Mass Second Mass Bunching Factor Analyte Peak 2 812 00 813 00 Analyte Peak 3 400 00 401 00 The following table shows one more example for MRM data The Analyte Peak 1 will be set up to use the specified internal standard and Analyte Peak 2 will not use an internal standard Table 1 3 Example Text File for MRM Data Peak Name Is IS IS Name First Mass Second Mass IS Peak 1 Yes 500 10 413 20 Analyte Peak 1 No IS Peak 1 600 20 382 10 Analyte Peak 2 No 400 00 312 1 The following table contains a mixture of full scan and MRM data in different experiments Table 1 4 Example Text File for MRM Data Peak Name Extraction Experiment First Mass Second Mass Type Analyte Peak 1 0 1 500 10 413 20 Analyte Peak 2 0 1 600 20 382 10 Analyte Peak 3 2 2 812 00 813 00 Analyte Peak 4 2 2 400 00 401 00 Analyst 1 6 1 Software Scripts User Guide 20 of 88 Analyst Software Scripts DBS Settings The DBS Dynamic Background Subtraction algorithm is a feature that ensures better selection of precursor ions in an IDA Information Dependant Acquisition experiment When DBS is activated IDA uses a spectrum that has been background subtracted to select the candidate ion of interest for MS MS analysis as opposed to selecting the precursor from the survey spectrum directly DBS enables detection of species as their signal increase
49. fragment ions e Optimize the Collision Energy CE for each fragment ions Perform MS scans on each fragment ion Optimize Excitation Energy AF2 for all MS scans Generate a report Save all data and acquisition methods The script can also be used in qualitative applications to generate collections of MS MS and MS3 spectra for compounds in a semi automated way that is one compound at a time MRM3 Optimization Window Overview You can use the controls in the MRM3 Optimization window to navigate through the script The window also displays the optimization results as they are generated The following is an overview of the various sections in this window Status Window When the script is first started this window displays the current optimization settings that will be used for optimization When the optimization is started spectral information is displayed in this window Log File Displays the results found during optimization in text format Each entry found in this section is also added to the generated Log txt file Overall Progress This is a visual display of the overall optimization progress e Main Controls Contains all of the main functions associated with the setting and execution of the optimization process After the optimization is completed a Results txt file is automatically generated and saved If you click View Results you can open and review it with Microsoft Notepad Click Settings to open
50. full chromatogram or just a selected region make a selection before running the script m IDA Trace Extractor x File Tools Help Neutral Losses Options From Time min 0 078 To Time min i201 Trace Width Da 0 5 Remove Unconfirmed Peaks Spectrum Peaks gt 3 S N Label Peaks gt s x Show Dependent Scan Chromatograms m Subtract Peaks Present in Control Sample No Control Sample Specified Get Sample Number NA To continue proceed to Neutral Losses or Fragments tab or use Tools menu Figure 1 13 IDA Trace Extractor dialog 2 Inthe Options tab of the script set the fields as required For more information see Table 1 6 Tab and Menu Parameters on page 36 3 To store the retention times and precursor masses found during processing click the Results tab type a filename and then select Save Precursor Information Analyst 1 6 1 Software Scripts User Guide 34 of 88 Analyst Software Scripts i IDA Trace Extractor xi File Tools Help Neutral Losses Results Result File 104 Mi Peaks tt XIC Peaks gt 5000 JV Append Results to an Existing File m Results Tools View Make XIE Table Make Quant Input Figure 1 14 IDA Trace Extractor dialog Results tab 4 To review or enter the mass information click the Neutral Losses or Fragments tab Type the neutral losses and fragments as masses or chemical formulas You can also specify the polarity of the Neutral
51. g it from 1 V to 12 V 12 V to 1 V for negative mode with 0 5 V step If the optimal EP is less than 10 V greater than 10 V for negative mode then DP is re optimized If the instrument is not a 4000 QTRAP instrument the CEP parameter is also optimized by ramping from 0 to 100 V 100 to O V for negative mode with 2 V step In determining the optimal voltage graphs are smoothed two times and the voltage yielding the greatest ion count is used Dwell Time for each scan is set to 100 ms Enhanced Product lon Scan This step selects the fragment ions that will be used for MS optimization This is performed using an EPI scan for three cycles at the selected scan rate You can specify an optimal CE for the compound to be analyzed If the optimal CE is unknown then you can specify a Collision Energy Spread CES value such that a range of CE settings are used The most intense second precursor peaks are then found excluding any peaks within 2 5 amu window of first precursor The number of second precursors to use is selected in the Advanced Settings The mass range from which the second precursors are selected is specified by the user Multiple Reaction Monitoring Scan This step optimizes the collision energy for each of the fragment ions selected from the EPI scan This is performed using MRM scans Use CE ramps of 5 to 130 V 130 to 5 V in negative mode Scripts User Guide Analyst 1 6 1 Software 63 of 88 Analyst Software Scripts w
52. g the resulting Mascot web form When searching product spectra for an IDA run the charge state is automatically determined by the script the values specified here are not used unless the charge state could not be automatically determined or the Try to determine charge state from survey scan check box is not selected When searching product spectra for a multiple period or multiple experiment LC MS run the specified charge states will always be considered for each product spectrum This option is only required for those using an older Mascot software version that does not accept charge states greater than 5 If you are using one of these older versions make sure that in the Default precursor charge states group the Discard ions with charge of 5 or higher check box is selected Some versions of the Mascot search engine cannot accept ions with a charge state of 5 or higher and show a warning for each ion exceeding this limit 7 To use the default precursor charge states as is clear the Try to determine charge state from survey scan check box Otherwise it will attempt to determine the charge state by examining the isotope spacing in the survey spectrum and for Analyst TF 1 5 software data it will use the charge state determined by the MS Acquisition Engine which is saved to the data file If this check box is selected but the charge state determination fails the default charge states are used 8 Inthe MS MS averaging of IDA dependents gro
53. he format supplied compound name scan type m z wiff Analyst 1 6 1 Software Scripts User Guide 62 of 88 Analyst Software Scripts Detailed Description of Script Logic This section describes each phase of the optimization process All scans are performed with the number of scans to sum set to 3 Initialization Before performing any optimization scans the MRM3 Optimization script first performs the following initialization steps If an error occurs during any of these steps the script will stop the optimization process 1 Ensure that the Analyst software is running 2 Load the starter acquisition method to see if it is valid and check the device type 3 Create a new Data sub folder to store the wiff files 4 Create the Log txt file Enhanced Resolution Scan This step confirms the mass of the ion used for optimization The ER scan is performed for 20 cycles at the specified scan rate The most intense peak within 1 amu of the expected first precursor m z is then selected Similar to the Analyst software this scan is performed with a 30 amu mass range around the specified m z For multiply charged species the C12 ion is determined in this step Q1 Multiple lon Scan This step optimizes transmission of the ion of interest up to the collision cell This is performed using a Q1MI scan The script first optimizes the DP parameter by performing the scan at the specified DP ramp Optimize the EP parameter by rampin
54. hich the purchaser may put the equipment described herein or for any adverse circumstances arising therefrom For research use only Not for use in diagnostic procedures The trademarks mentioned herein are the property of AB Sciex Pte Ltd or their respective owners AB SCIEX is being used under license AB SCIEX 71 Four Valley Dr Concord Ontario Canada L4K 4V8 AB Sciex LP is ISO 9001 registered 2012 AB SCIEX Printed in Canada REGISTERED COMPANY Contents Igor nn a a ee weed AAA 5 Related Documentation 000 cece eee ees 5 Technical SUpport uuu ise ncs de Seco a ea eta We bote Ue ER p Ee edo oi 5 Analyst Software ScriptS 0ooooooooooorrccana eee 7 Related Documentation 2 0 0 0 ccc eee 7 Installing or Uninstalling Scripts llli 7 Add Missing ZBI OS ccoo ci CER OR UR nb ac Roca e RU e oon E ee 8 Add Normalized ADC Traces a id xi ned tea Ree Rede Ne es 9 Analyst 1 2 Peak Finder Parameters 00 e eee eee eee 10 Balch Script DrlV8l scsi ear PE ah eRe ERS RAR RARA 11 Change All Methods o ooooccccccccc ee 13 Convert Meltliotdls 23 2 taco Raa a Rene REAR eee eee had 14 Creating Quantitation Methods and Text Files o ooooooooo 15 DBS Settings cdas ar PEA AS 21 Define Custom Elements 0 000 ccc cece ene ee eee ee 23 Delete Others 0 ccc tenet teens 26 DFI Tracker se ccm 27 Export IDA Spectra ia bei
55. hould be removed from the MS MS spectra before sending them to Mascot by selecting the De isotope MS MS data check box It is recommended that you enable this option Report peak area otherwise intensity lf selected the script uses the area of the peak Otherwise it uses the intensity at the apex Reject spectra if less than n peaks If a spectrum contains unreasonably few peaks after combining adjacent spectra if used and centroiding if used the spectrum can be eliminated Remove peaks within n Da of precursor m z Sets a window around the precursor ion m z and then removes any peaks within that window 10 In the Other group select Use original format for query titles if you are using a third party protein quantitation application and you would like to use the original title format 11 To display the Mascot search form defaults Web page click Set default parameters You can edit the various defaults so that you do not need to reset them manually every time before submitting a search After changing the parameters click Save defaults as cookie to close the Web page Scripts User Guide Analyst 1 6 1 Software 51 of 88 Analyst Software Scripts Mass Defect Filter The identification of drug metabolites in biological fluids with low concentrations from a total ion chromatogram TIC is challenging because of significant interferences from endogenous species A technique of filtering the data based on the mas
56. hown in the table for either one analyte or an internal standard peak Table 1 1 Text File Formats Column name Required Description Peak Name Yes The name of the analyte or internal standard peak First Mass Yes For MRM data the Q1 mass for the peak for full scan data the starting mass for the XIC to integrate for Q1 MI or Q3 MI data the mass Second Mass Maybe This field is required when integrating full scan or MRM data but not for Q1 MI or Q3 MI data For MRM data this is the Q3 mass for the peak for full scan data it is the ending mass for the XIC to integrate Extraction Type No The type of data to integrate If present this should be one of 0 MRM data 1 Q1 MI or Q3 MI data 2 full scan data Scripts User Guide Analyst 1 6 1 Software 17 of 88 Analyst Software Scripts Table 1 1 Text File Formats Continued Column name Is IS Required No Description Specifies whether the current peak is an internal standard or an analyte Select Yes if the peak is an internal standard otherwise select No If this column is not present then all peaks defined are assumed to be analytes Note Internal standard peaks should be defined first in the text file before any analyte peaks that use that IS IS Name No For analyte peaks specifies the name of the corresponding internal standard if any If a given analyte will not use an internal standard leave the contents
57. ine Y Report raw peak height otherwise interpolated fv Report centroid Retention Time V FmedRT widh 100 sec D ton Figure 1 20 Manual Integration Options 7 If required in the Mode group do one of the following Click Add area as caption to graph then quit to paste the area of a single selection on the graph and then exit the program Click Add area as caption to graph and stay open to display the area on the graph as well as in the program Click Don t add caption to graph and stay open to only display the results in the program and to leave the graph unaltered 8 Select the following as required In the Display Format group select how the results will be displayed in the Manual Integration 1 dialog Select the Show integrated area in graph check box to display the integrated area in the active chromatogram Select the Force zero intensity baseline check box to force the integrated area to start from the intensity 0 baseline In this case the starting and ending times from the manual selection are used but the y positions are ignored Select the Report raw peak height check box to report the peak height as the intensity of the largest point comprising the peak If the check box is cleared then the usual software algorithm is used to calculate the peak height a parabola is fitted to the three largest data points and the peak height is set to the y value of the parabola s apex Select the Report centr
58. ing the script Scripts User Guide Analyst 1 6 1 Software 41 of 88 Analyst Software Scripts Make Exclusion List from Spectrum Use this script to create a text file containing all of the peaks from the currently active spectrum The text file is in a format that can be directly imported into the Analyst software IDA Information Dependent Acquisition exclusion list To use the script If the spectrum has been previously manually centroided the resulting peak list is exported directly to the exclusion text file Otherwise the script will first centroid the spectrum 1 With a spectrum in an active pane click Script Make Exclusion List from Spectrum i Make Exclusion List from Spectrum x Exclusion List File N ame CAE xclusion List txt Options Threshold Figure 1 17 Make Exclusion List from Spectrum dialog In the Exclusion List File Name field type the name and path of the text file If required in the Threshold field type the threshold that will be applied to the centroided spectrum so Threshold field that small noise peaks are not included in the exclusion list Type 0 in the to not use a threshold 4 Click Export to export the exclusion list using the specified parameters Analyst 1 6 1 Software Scripts User Guide 42 of 88 Analyst Software Scripts Make Subset File Use this script to manipulate data files by transferring samples to another data file unpacking all samples in a
59. ion algorithm is not available from within the Analyst software user interface but can be enabled using the script Bunching Factor No The bunching factor for the peak from 1 to 100 when using the TurboChrom algorithm Num Smooths Analyst 1 6 1 Software 18 of 88 No The number of smooths from 0 to 10 when using the TurboChrom algorithm Scripts User Guide Analyst Software Scripts Table 1 1 Text File Formats Continued Column name Required Description Noise Threshold No The noise threshold from 1 to 1 when using the TurboChrom algorithm Area Threshold No The area threshold from 15 to 112 when using the TurboChrom algorithm Separation Width No The separation width from O to 5 when using the TurboChrom algorithm Separation Height No The separation height from O to 1 when using the TurboChrom algorithm Exp Peak Ratio No The exponential peak ratio from 1 to 18 when using the TurboChrom algorithm Exp Adjusted No The exponential adjusted ratio from 2 to 19 when using the Ratio TurboChrom algorithm Exp Valley Ratio No The exponential valley ratio from 1 to 16 when using the TurboChrom algorithm Min Height No The minimum allowed peak height from O to 116 when using the IntelliQuan algorithm Min Width No The minimum allowed peak width from O to 116 in seconds when using the IntelliQuan algorithm Smooth Width No
60. ion window appears displaying the last values and information used for optimization 1 Click Browse to navigate to the starter acquisition method This method predominantly contains the source conditions to be used for the optimization 2 Inthe Compound Name field type a descriptive compound name This name is used as a prefix to all of the acquisition methods and data files generated 3 In the Q1 Resolution field select a Q1 Resolution to be used for MS MS and MS3 In the Polarity group click a polarity which may differ from the starter method The Both Polarity option is currently not supported 5 Inthe Expected m z amu field type the expected mass to charge m z ratio for the compound If you do not know the m z of the compound then click Calculate from chemical formula to calculate it from the chemical formula of the compound See To calculate m z on page 57 6 To modify some of the settings used by the optimization process click Advanced See To use the Advanced Settings dialog on page 58 7 To verify and use the updated settings click OK Analyst 1 6 1 Software Scripts User Guide 56 of 88 Analyst Software Scripts To use the script 1 Build a starter acquisition method if one does not already exist The starter method should be a Q1 acquisition method created in Manual Tune and should contain the source conditions required for the tuning process because these are not optimized by the script 2 Save the me
61. ipt will work only once for each graph Use the following procedure if you want to run the script on the same graph more than once To calculate the average and standard deviation of a graph more than once 1 Copy the graph into a new pane Click Explore gt Duplicate Data gt Same Window 2 Make a selection in the new graph pane and run the Selection Average and StdDev script again Scripts User Guide Analyst 1 6 1 Software 73 of 88 Analyst Software Scripts Send to ACD SpecManager This script is similar to the Export to JCamp script However instead of prompting for the name of the JCamp file the spectra are exported to a temporary file and then sent directly to the ACD SpecManager application There is no need to open this temporary file directly Prerequisites You must have the SpecManager software installed To use the script The first time you run the script you are prompted to locate SpecManager You will not be prompted again unless you press the Ctrl key while clicking the script 3 Note This script cannot be used with batch acquisition 1 With either a chromatogram or a spectrum in an active pane click Script Send to ACD SpecManager The following figure shows the options dialog that appears when you run either of the scripts to process chromatographic data If you are interactively processing a single spectrum active in the Analyst software these options do not apply i JCamp Options x Fi
62. ith 2 V step and Dwell Time of 50 ms Each overlaid graph is then smoothed two times and the voltages yielding the greatest ion count are used as the optimal CE values MS MS MS Scan The script performs an MS scan for each chosen second precursor at the specified scan rate and with an AF2 ramp of 0 to 100 V with 2 mV step or 0 to 0 4 V with 0 01 V step on QTRAP 5500 system for both polarities The fill time of the scan is set and QOTrapping can be turned on for maximum sensitivity if required The lower limit of the mass range for the MS MS MS scan can be specified and the upper limit is second precursor 5 amu The generated graphs are smoothed twice and the optimal AF2 as shown in the Figure 1 33 How AF2 is determined is obtained when the residual intensity of the second precursor based on XIC is at 5 of its maximum intensity The spectrum at this AF2 value is then used to find the two most intense second generation fragment ions excluding peaks within 1 amu of the second precursor If the second precursor m z is greater than 10 of the total ion count no fragments from that spectrum will be used This condition exists because if the second precursor m z is greater than 10 there is insufficient fragmentation 100 Optimum AF2 XIC of 346 MS at Optimum AF2 P d 2 most intense ions 1335853553 Figure 1 33 How AF2 is determined Generate Final Methods After the optimization scans are performed the
63. le gt Load Settings to open previously saved settings Several functions are available in the Tools menu You can start processing without switching to a specific tab Table 1 6 Tab and Menu Parameters Location Neutral Losses Parameters Use Masses Description Select the required neutral loss es as mass Neutral Losses Use Formulas Select the required neutral loss es as formula Analyst 1 6 1 Software 36 of 88 Neutral Losses Start Low mass limit from mass for the neutral loss es Neutral Losses End High mass limit to mass for the neutral loss es Scripts User Guide Table 1 6 Tab and Menu Parameters Continued Analyst Software Scripts Location Parameters Description Neutral Losses Formula Chemical formula of the neutral loss Neutral Losses Extract Start data processing according to the current settings Neutral Losses Clear Clear all neutral losses in the settings Neutral Losses Polarity Select the polarity of the Neutral Loss experiment where the specified neutral loss is expected to be found Fragments Use Masses Describe the characteristic fragment s in terms of their m z Fragments Use Formulas Describe the characteristic fragment s in terms of their formulas Fragments Start Low mass limit from mass for the fragment m z window Fragments End High mass limit to mass for the fragment m z
64. le Data 0 0000 eee eee 79 Scripts User Guide Analyst 1 6 1 Software Release Date March 2012 3 of 88 Contents Unit COrHVverslOlis Last aan Se a esas e e e e o Bt 80 Witt To Matlab 21 ares ett eria a ia ds 81 XIG from BPO een id dire ae Sage IN sted ade cue Ra E Pie ERI eda 85 MIGHOMV TADS 2 5 een ei Rt Ri Mer de i 86 Analyst 1 6 1 Software Scripts User Guide 4 of 88 Foreword This guide provides information on how to use the Analyst software scripts and is intended for customers and FSEs Related Documentation The guides and tutorials for the instrument and the Analyst software are installed automatically with the software and are available from the Start menu All Programs gt AB SCIEX gt Analyst A complete list of the available documentation can be found in the online Help To view the Analyst software Help press F1 Technical Support AB SCIEX and its representatives maintain a staff of fully trained service and technical specialists located throughout the world They can answer questions about the instrument or any technical issues that may arise For more information visit the Web site at www absciex com Scripts User Guide Analyst 1 6 1 Software Release Date March 2012 5 of 88 Foreword AB SCIEX Analyst 1 6 1 Software Scripts User Guide 6 of 88 Analyst Software Scripts The purpose of this document is to explain how to install and use Analyst software scripts It
65. le clicking the script Otherwise the options previously specified are used Scripts User Guide Analyst 1 6 1 Software 81 of 88 Analyst Software Scripts PM Wiff to MatLab Conversion Options xi Matrix Names Data Data Save Sparse Data s parseData Scans Scans Masses Masses Index index Filename Filename Wavelengths wavelengths Threshold for sparse format o v Save original filename as matrix Use sparse format for quadrupole data v Save scan times not scan numbers Use fixed step size 0 5 amu Save DAD data in batch mode if available Figure 1 43 Wiff to MatLab Conversion Options dialog 3 In the Matrix Names fields type the names of the matrices that the script produces It is recommended that you keep these names as their default values 4 Inthe Threshold for sparse format field type a value that will be used to reduce the size of the output MatLab file For the sparse format only only mass intensity pairs with intensity larger than the specified value will be written 5 If the Save original filename as matrix field is selected then the script will create and populate the Filename matrix 6 To have the quadrupole spectra saved in the sparse format select the Use sparse format for quadrupole data check box Note that TOF spectra are always saved in the sparse format and that diode array data is always saved in the non sparse format 7 Ifthe Save s
66. lick All MS MS spectra for current sample to perform a single search using all spectra for the sample Ifthe active pane contains MS MS data and also contains a selected region click All MS MS spectra from selected region in TIC to perform a single search using only the spectra from this region This can be particularly useful to speed the generation of the search input if only a portion of the run is known to contain useful data Analyst 1 6 1 Software Scripts User Guide 48 of 88 Analyst Software Scripts If the active pane contains MS MS data and the associated data file contains more than one sample click All MS MS spectra from all samples for current file to perform a single search using all MS MS spectra from all samples in the data file 3 To open the Mascot search form and populate it with the appropriate information click Search If you are searching all product spectra contained in the sample this may take some time a progress bar will appear After the Web form appears click Start Search 4 To set the various search options click Options Mascot Search Options MS MS aw IDA us vene me S MS averaging ol IDA depender Set delauk parameters Set sesech file location O Local Mascot C Matrix Science public web site MS MS data processing O Protein Prospector IDA survey scan Default precursor charge states Other C Use original format for query titles Figure 1 22 Mascot Search Options di
67. m Text Files w Create Quantitation Methods from Text Files LX m Defaull Generic Parameters Algarthm vet Classic T uiboLhrom Extaction Type MAM Period h x Experiment h zl Expected RT it min RT window Do sec UseRelative RT Bk Steri min 0 BkgErd win O Conc Units Calc Conc Units r Default amp nalyst Classic TuiboChrom Parameters Bunching Factor h sz Noise Threshold foo Area Threshold ETS Num Smoothe 0 X Separation Width i2 Separation Heizht nn Esp Peak Ratio D Exp Adusled R atic ho Esp Valley A alio es m Default Genera nieliQuan Paraneiers_ Detauk Intel uan MU Ill Parameters Mn Peak Hegh jf ums Nase Peicent En x Mn Peak Width D sec Base Sub Window hh mn Smeotring wits o paint Poa Spltlina Factor 2 Report Largest Pesk Regression Parameters Delauk Window Summation Perameters Fit lins Use Bacelin Subtraction Weightng oe l Parameter lace El Create One Method Iterzta No canes Create Mutple Methods Figure 1 4 Create Quantitation Methods from Text Files dialog Scripts User Guide Analyst 1 6 1 Software 15 of 88 Analyst Software Scripts 2 Use the parameters in the Default Generic Parameters group to create a quantitation method The Algorithm Extraction Type Period and Experiment fields are not available in the Analyst software Set the following parameters as required
68. mooth is available The active graph in the Analyst software will be replaced by the smoothed graph The Savitzky Golay Smooth script smooths the current active graph using the Savitzky Golay smoothing algorithm The IntelliQuan algorithm in Quantitate mode performs the smoothing process To use the script 1 With the peaks of interest selected in the active spectrum click Script Savitzky Golay Smooth ij Savitzky Golay Smooth ICE XI Smoothing Half width 5 y points DK Figure 1 38 Savitzky Golay Smooth dialog 2 Use the Smoothing Half Width drop down list to set the half width for smoothing the data The total width will be twice this value plus one This parameter is the same as the smoothing parameter used with the IntelliQuan algorithm in the Analyst software 3 To perform the smoothing click OK Analyst 1 6 1 Software Scripts User Guide 72 of 88 Analyst Software Scripts Selection Average and Standard Deviation Use this script to calculate the average intensity and standard deviation of a selection in a graph for both spectral and chromatographic data The graph is labeled with both the average and standard deviation of the selection To use the script e Select either an active spectrum or active chromatogram and click Script gt Selection Average and StdDev script The graph will be labeled with the average and standard deviation of the selection Known Issues and Limitations This scr
69. n maximum the best performance synchronize the other font attributes manually in the Appearance Jl Note Only font type and label color are synchronized with the automatic labels For Options dialog a Note Labeling spectra with centroid mass charge state appends just the centroid Al mass Labeling chromatograms with base peak ion mass or base peak ion intensity is not supported To use the script Do one of the following To add labels to selections in active graph click Script gt LabelSelections Tosee the script description and add the labels to an active Explore pane hold the Shift key down while clicking the script Analyst 1 6 1 Software Scripts User Guide 40 of 88 Analyst Software Scripts Label XIC Traces Use this script when a pane containing one or more XIC traces is active in Explore mode There may be a time region selected in a trace If there is no selection a complete chromatogram will be considered for processing The script labels the largest peak in each XIC trace with mass XIC traces with a maximum point of less than 5 of the most intense trace will not be labeled Other types of traces TIC ADC in the overlay will be ignored Jl Note No user settings are required for this script To use the script Do one of the following Click Script LabelXICs To see the script description and add the labels to an active Explore pane hold the Shift key down while click
70. n sce aca ee eden das Qe Soda ec ea RR wd RR RR 28 Export Sample Information nnna anaana eae rs cc eee ee 30 Export to JCamp EU 31 IDA Trace Extractor sopua ina n a a eee 33 Label Selections sisisi at rn arar ee a a 40 Label AIC Traces 12 oen ER e RA EERE RARA AA ERS a 41 Make Exclusion List from Spectrum 0000 ce ee 42 Make Subset File ac ak ak s RR CC OCA ECT ot kG A ead ean be RE RES AA 43 Manually Integrate 22cccciacee sees edu rr aa Woe 2 45 Mascot pP 48 Mass Defect FING iu ues rdi ade c ee CIRC eR De RR c E A 52 Merge MRM usos i obice EROR RIA ee AOL RO ACE SIE NC R rac n 54 MRM3 Optimization Script au caca Vac XO e a CK od Qe s i kab eee 55 MS3 Quant Optimization Script 2 2 0 0 65 Multiple Batch Scripts us ie 1e dae e o e eile d e a I CR d 66 Open In Workspace 2 2 e200 0b bee ee ee he a eee XR RR TR RR ceed 67 Peak List from Selection 0 0 00 eee eens 68 Regression Calculator 0 00 eee 69 Remove Graph Selections 000 cece eee eens 70 Repeat IDA Method 22 2 0 cee 71 Savitzky Golay Smooth inicia ad a 72 Selection Average and Standard Deviation 0 0000 e eae 73 Send to ACD SpecManager 00 c eee eects 74 Signal to Noise Using Peak to Peak 0 0 0 0 000 cc nae 76 Signal to Noise Using Standard Deviation 0 00000 eee eee 77 Split Graph GMO oic adco 93 ade Ge Raw ae we AA 78 Subtract Control Data from Samp
71. number of charges To calculate the m z for the entered chemical formula and charge click Calculate To close the calculator and update the Expected m z amu field in the Settings dialog with the calculated m z click Use m z To use the Advanced Settings dialog In this dialog a description for each of the optimization steps is provided You can also modify some of the settings in order to customize the optimization 1 In the MRM3 Optimization window click Settings 2 In the Settings dialog click Advanced Analyst 1 6 1 Software Scripts User Guide 58 of 88 Advanced Settings Enhanced Resolution Finds the most intense peak wilhin a 2 Da window of expected 1st precursor molecular weight Mass range window defaulted to 30 Da around expected mass to charge ralio Scan Rate 1000 Da s Cycles Q1 Multiple lon Optimizes DP and EP DP re optimized if 10 lt EP lt 10 CEP is optimized only when applicable Smooths TIC 2 times and finds voltage yielding greatest ion count Stat Stop Step DP Ramp 30 150 5 0 200 Dwell Time ms Enhanced Product lon Finds the most intense 2nd precursor peaks excluding any peaks within a 5 Da window of 1st precursor Scan Rate 1000 v vas 2ndPrecusos E 110 Mass range 300 to i000 ce 30 ces fio Cycles Optimizes CE values for the most intense 2nd precursor peaks by cycling through each XIC overlay XIC graph smoothed 2 times and voltage
72. oid Retention Time check box to report the retention time using a centroid calculation If the check box is cleared then the usual software algorithm is used to calculate the retention time a parabola is fitted to the three largest data points and the retention time is set to the x value of the parabola s apex Analyst 1 6 1 Software Scripts User Guide 46 of 88 Analyst Software Scripts The Fixed RT width sec option is for MALDI workflows only If selected the total width of the resulting peak is fixed at the specified value and is centered at the apex retention time 9 To save changes and return to the Manual Integration 1 dialog click OK 10 On the Manual Integration 1 dialog click Close Scripts User Guide Analyst 1 6 1 Software 47 of 88 Analyst Software Scripts Mascot Use this script to send either the active spectrum or all product spectra contained in the active sample or all samples in the active data file to the Mascot protein search engine The script was co developed with Matrix Science Limited the creators of Mascot When sending only the active spectrum the script can work with either MS or MS MS data in the first case a peptide mass fingerprint search is conducted When sending all spectra the script works with data acquired using two distinct types of acquisition methods either a multiple period multiple experiment method containing any number of product experiments or an IDA Information Dependent
73. or Analyst 1 2 files check box 3 Dothe following Inthe Intensity Threshold field type the value as a percentage of the minimum intensity required to distinguish between noise and peak Inthe Centroid Height field type the value as a percentage to be used by the centroiding algorithm to find the peak and to determine the centroid m z value at this percentage height Inthe Centroid Peak Width min field type the minimum value in ppm to be used by the centroiding algorithm to find the peak width and to determine the centroid m z value at this width Inthe Centroid Peak Width max field type the maximum value in ppm to be used by the centroiding algorithm to find the peak width and to determine the centroid m z value at this width Inthe Centroid Merge Distance amu field type a value in amu to be used to determine whether two centroid peaks should be merged into one If two peaks are within this tolerance then they will be merged together Inthe Centroid Merge Distance ppm field type a value in ppm to be used to determine whether two centroid peaks should be merged into one If two peaks are within this tolerance then they will be merged together 4 Click OK 5 Toreturn to the Analyst 1 4 1 Peak Finder algorithm clear the Use Analyst 1 2 peak finder algorithm for Analyst 1 2 files check box Analyst 1 6 1 Software Scripts User Guide 10 of 88 Analyst Software Scripts Batch Script
74. ound signal intensity from the peak signal intensity and then divides the subtracted signal by a user specified factor times the standard deviation of the noise region To use the script 1 With a noise region and the peak of interest selected in the active chromatogram click Script gt S_NstdDevQS i S NxstdDev PHI E3 Help SINx 1 StdDev Erase Labels Figure 1 40 S NxstdDev dialog 2 To erase any labels current on the active chromatogram click Erase Labels 3 To calculate the signal to noise ratio and label the graph click Go Related Scripts S to N using Peak to PeakS Calculates the signal to noise for an active chromatogram The background subtracted signal is divided by the peak to peak noise level Scripts User Guide Analyst 1 6 1 Software 77 of 88 Analyst Software Scripts Split Graph Script Use this script to split a spectrum or chromatogram into a specified number of panes Each resulting pane displays a proportional fraction of the total mass or time range For example ifa spectrum displaying a mass range of 100 to 400 amu is split into three the original spectrum will display a range of 100 to 200 amu the second new spectrum will display 200 to 300 amu and the third new spectrum 300 to 400 amu The intention is to allow the maximum possible number of peaks to be labeled for subsequent printout To use the script 1 With either a chromatogram or a spectrum in an active pane click Script gt
75. s defect of the parent drug and a small tolerance value has been used to decrease the amount of interference Using this technique aids in the identification of phase and phase Il metabolites The Mass Defect Filter script filters either a TIC or a spectrum using this technique Only those data points in the spectrum whose centroid mass is within the tolerance range of the parent ion s mass defect applied at that nominal mass will be retained All other points are excluded Summing the intensity for each of the spectra generates the TIC A further filtering based on absolute mass can also be applied A new graph of the filtered TIC or spectrum will appear below the current graph To use the script 1 With either a spectrum or a TIC active click Script gt Mass Defect Filter m Mass Defect Parameters Parent Formula 0 Nominal Mass Da Mass Defect fp smal Mass Defect Tolerance D mDa Resolution Factor o v Use Dynamic Mass Defect Calculation Mass Range Parameters Start Mass m z Stop Mass m z Use Mass Range Filter Cancel Figure 1 24 Mass Defect Filter Settings dialog 2 Inthe Parent Formula field type the formula for the parent ion The Mass Defect and Nominal Mass fields are automatically updated 3 Ifthe parent formula is unknown then type values in the Nominal Mass and Mass Defect fields In the Mass Defect Tolerance field type the
76. s in intensity thus focusing on detection and analysis of the precursor ions on the rising portion of the LC peak up to the top of the LC peaks maximum intensity The DBS functionality is embedded in the Analyst 1 6 1 software for IDA experiments however the associated parameters are not accessible in the Analyst software The hidden parameters and their default values are as follows Average number of previous spectra 4 Smooth before subtract activated Smooth 5 data points Use this script to change the default parameters to ones that are more representative of the experimental conditions Depending on the cycle time and chromatography the default settings may result in an obvious candidate ion from being omitted for dependent MS MS analysis or the same candidate ion may be selected for MS MS analysis over the entire LC peak Therefore this script will be useful to customers who find that the embedded default values are not appropriate for their analysis After the script is installed the DBS feature uses the settings in the script The script will remember the last settings used Prerequisites e Analyst 1 6 1 Software installed Administrator rights on the computer To use the script 1 Activate the DBS feature by selecting the After Dynamic Background Subtraction of Survey scan check box on the IDA First Level Criteria tab Mire Edt View Acqure Tools Explore Window Script Help c E db A X Ba NSE acaire Mode
77. s in the chromatogram The area and height listed relate just to peaks in the selection The peaks are listed in a text pane below the active chromatogram The peak definition is shown in the chromatogram For more information see Figure 1 36 Data Processing with the PeakListFromSelection Script To use the script Do one of the following To run the script click Script gt PeakListFromSelection To see the script description and get the peak list hold the Shift key down while clicking the script To process the data make one or more selections in the chromatogram and then select the script from the drop down menu Wl XIC of MRM 2 pairs 400 0 200 0 Da from Sample 1 API2 012 of QuanData Wiff Unknown lon Source Max 64 0 Intensity cps 1 1 12 13 1 4 1 5 q 7 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 Time min Baseline Height Width min Height cps Time min Area counts Area Type 1 0 77 1 36E 01 100 00 3 00E 00 100 00 0 10 Base to Bas Figure 1 36 Data Processing with the PeakListFromSelection Script Analyst 1 6 1 Software Scripts User Guide 68 of 88 Analyst Software Scripts Regression Calculator Use this script to calculate the slope y intercept and r values for each mass speed dependent parameter To use the script 1 Click Script gt Regression Calculator Regression Calculator xi X Values Y Values Slope N A Intercept N A R N A o dem
78. s overlayed In case that several ADC traces are part of the wiff file all of them will be displayed Notes Ifthe data in the active pane came from several samples then the ADC data for the sample corresponding to the first data set not the active data set is shown To use the script Do one of the following e Click Script gt AddNormalizedADC To see the script description and add ADC data to an active Explore pane hold the Shift key down while clicking the script Scripts User Guide Analyst 1 6 1 Software 9 of 88 Analyst Software Scripts Analyst 1 2 Peak Finder Parameters The Analyst software uses an improved version of Peak Finder for better peak detection and ion abundance measurement If you are using the Peak Finder algorithm to analyze wiff files that were acquired using the Analyst software then you can use this script to set the peak finder algorithm parameters To install the script e Make sure that the Analyst Data Projects API Instrument Processing Scripts folder contains the Analyst12PeakFinderParams dll s Note This script is installed automatically when the Analyst 1 6 1 Al software is installed There is no separate installation program for it To use the script 1 Click Script gt Analyst12PeakFinderParams The Analyst 1 2 Peak Finder Algorithm Parameter dialog appears 2 To activate the Analyst 1 2 version of Peak Finder select the Use Analyst 1 2 peak finder algorithm f
79. ss range field type a mass range for the second precursors that will be selected for MS optimization Inthe CE field type a collision energy value and in the CES field type a collision energy spread CES value that will provide a good MS MS spectrum from which fragment ions can be selected To generate all of the final MS methods for each second precursor and the optimal MS3 method for quantitation in the Generate Final Methods group click Save All Final Methods Click Save Optimal Method Only to save only the optimal MS method most sensitive for quantitation Click OK to accept the updated Advanced Settings Optimization In Progress When the optimization is started Manual Tune in the Analyst software is automatically stopped While the script is running all of the functions in the software can still be used A Log txt file is also updated as each part of the optimization procedure is completed To stop the script at any time click the Abort button Examples of the script in progress are shown in the Figure 1 30 on Scripts User Guide Analyst 1 6 1 Software 59 of 88 Analyst Software Scripts page 60 and the Figure 1 31 on page 61 In the Overall Progress section the Checklist images and text fonts represent different statuses that are described in the following section L Task not performed yet text is black Task in a Text is blue and italic Task will not be performed text is grey MI eh MEL pedis
80. start the transfer click Extract Scripts User Guide Analyst 1 6 1 Software 43 of 88 Analyst Software Scripts Section 3 Unpack This section describes how to unpack every sample in one data file into its own data file 1 2 5 Click the Unpack tab In the Destination Directory group the location of the current working data file appears Use this tree to select the location for the unpacked data files To create a new directory right click the directory tree You will be prompted to type the new folder name and set the active folder In the Output File Name text field type the output file name Each sample unpacked from the working data file will begin with this name followed by the sample number in parentheses Do not give an extension to this file for example do not include wiff because the Make Subset File program will automatically append this To begin unpacking the samples click Extract Section 4 Decompose This section describes how to decompose a sample into different samples 1 2 Click the Decompose tab To use the Make Subset File script default values select the Use Defaults check box To provide threshold values deselect the Use Defaults check box and then type the values in the fields The Noise field contains the noise threshold value This value indicates when a sub sample begins and ends If the intensity value exceeds this threshold then it is considered a new sample
81. that all other experiments represent dependent product spectra Therefore this script cannot be used if there are multiple survey experiments 1 With an IDA chromatogram in an active pane click Script Export IDA Spectra iw Export IDA Spectra in Sequest Format xi Mass tolerance for combining MS MS spectra 0 1 amu MS MS intensity threshold 2 Minimum number of MS MS ions for export s Separate values in output with a space not a tab Use _ and not to delimit parent m z in dta filename Close Figure 1 9 Export IDA Spectra in Sequest Format dialog 2 Inthe Mass tolerance for combining MS MS spectra field type the tolerance to be used to determine if two precursor m z values should be considered identical If the precursors for two sequential product spectra differ by less than this value the spectra are added and a single text file is exported 3 In the MS MS intensity threshold field type the threshold that is applied to each product spectrum after it is centroided It is assumed that peaks below this threshold are most likely noise Type 0 in the field if you do not want to use a threshold 4 n the Minimum number of MS MS ions for export field type the minimum number of ions that must be present in a product spectrum after centroiding and thresholding in order for a text file to be exported If a spectrum does not contain the specified number of ions it is assumed that the quality of the spec
82. this dialog again if the control key is down check box 3 To continue processing and to have the spectrums exported click OK When prompted type the file name of the exported JCAMP file When a script is attached to the batch the file name is automatically generated using the following format WiffFileName Sample Period Experimentz jdx Analyst 1 6 1 Software Scripts User Guide 32 of 88 Analyst Software Scripts IDA Trace Extractor Use this script to review the survey data collected using IDA Information Dependent Acquisition based on the information in the corresponding dependent data The script searches the MS MS data for given neutral losses or fragments and then calculates the Extracted lon Chromatograms XICs for the precursor masses which give the specified losses or fragments The XICs are overlaid in Explore mode and their peaks are labeled with the precursor mass Figure 1 12 Time min oo ERA Fiano Pats EB ido Figure 1 12 Characteristic Traces in Dependent Experiment and XICs of the Survey Experiment The characteristic m z 387 1 detected in negative mode was converted to m z 389 1 in the positive survey scan You can use the script to do the following Specify a list of expected fragments or neutral losses in either the positive mode polarity the negative mode polarity or both in terms of fragment formula or mass e Save the list of masses an
83. thod in the Acquisition Methods folder of the required project where all generated files will be saved 3 Click Script gt MRM3 Optimization Settings Acquisition Method Browse Polarity Compound Name Compound Positive Expected m z Daj 1400 20 C Negative Calculate from ES chemical formula Q1 Resolution Unit y Cancel Advanced Figure 1 27 Settings dialog 4 Enter the compound information required for the optimization process and then click OK on the Settings dialog 5 To initiate the optimization process click Start in the MRM3 Optimization window To calculate m z The m z calculator is accessed through the Settings dialog 1 In the MRM3 Optimization window click Settings 2 Inthe Settings dialog click Calculate from chemical formula Scripts User Guide Analyst 1 6 1 Software 57 of 88 Analyst Software Scripts m Calculate m z Enter a formula i e C6H6 and charge Chemical Formula C32H40N505Br Num of charges 4 Calculated m z 654 229 amu Use m z Cancel Figure 1 28 Calculate m z dialog 3 Inthe Chemical Formula field type the chemical formula of the compound Use capital letters for elements The chemical formula for peptides is also entered into this dialog You can obtain the chemical formulas for peptides by typing the peptide sequence into the New Protein Sequence window in the BioAnalyst Software In the Num of charges field click the
84. thod with current instrument settings check box if you want to change the AF3 EXB and C2B parameters for all the methods in the selected project 7 Click Change All Scripts User Guide Analyst 1 6 1 Software 13 of 88 Analyst Software Scripts Convert Methods Use this script to convert methods from one type of instrument to another The script converts the method to the currently active hardware profile using appropriate values for each parameter Only the ion source and compound dependent parameters for mass ranges and experiments are shown The Convert Methods script automatically optimizes mass ranges and in addition to single period single experiment methods converts multiple periods multiple experiments and IDA criteria Prerequisites Net framework 3 5 SP2 will be automatically installed if required To install the script e To install the script navigate to the lt drive gt Program FilesVAnalystiScripts Convert Methods folder and then double click Convert Methods Setup exe To use the script Make sure a hardware profile is active 1 Click Script Convert Methods Convert Methods Jox Original Method Convert Converted Method EM Acquisition method Instrument QTRAP 5500 E yf ReserpineMSMS dam E lt Not Saved gt E Mass Spec E Mass Spec a 9 Period 1 a Period 1 a Si Product lon Bg Product lon Ej c3 Mass Range 192 00 19 E Mass Range 192 00 1 E DP EP CEP E CE CXP
85. tolerance value 5 If required type a value in the Resolution Factor field The Resolution Factor further filters the data by keeping only the centroid values whose resolution is greater than or equal to it Analyst 1 6 1 Software Scripts User Guide 52 of 88 Analyst Software Scripts 6 If required to allow the mass defect to be applied differently at each nominal mass in the spectrum click the Use Dynamic Mass Defect Calculation check box If you deselect the check box a constant value of the mass defect is added to each nominal mass 7 In the Mass Range Parameters group select the Use Mass Range Filter check box to set the mass range parameters Only masses in the spectrum between Start Mass and Stop Mass inclusively will be retained 8 Click OK to start processing Scripts User Guide Analyst 1 6 1 Software 53 of 88 Analyst Software Scripts Merge MRM Use this script to add all experiment data from the method to be merged to the base method Both methods should have only one period and one experiment The script does not limit the number of mass ranges in an experiment All mass ranges are saved to the base method This script can be used to merge multiple final methods created by Compound Optimization After you have merged the methods the LC information can be modified if necessary to reflect the analysis conditions To use the script 1 Click Script gt Merge MRM Merging MRM methods AE Help CA
86. trum is too low to merit exporting Analyst 1 6 1 Software Scripts User Guide 28 of 88 Analyst Software Scripts 5 To separate fields in the output files with a space character select the Separate values in output with a space not a tab check box otherwise a tab character is used Certain versions of Sequest require a space delimiter To export the text files click Go In the Save As dialog type a location and root file name for the exported text files Before being exported to a text file each of the product spectra is centroided The cycle number range and charge state is appended to this file for each exported spectrum Scripts User Guide Analyst 1 6 1 Software 29 of 88 Analyst Software Scripts Export Sample Information Use this script to extract sample information such as the name sample ID comment and acquisition method name for all samples in the wiff file You can define the information you would like exported and the script saves the information in an inf file located in the same folder as the Wiff file To use the script 1 With a chromatogram or spectrum in an active pane click Script gt ExportSamplelnformationFromMultipleSampleinOneWiff while pressing the Ctrl key im Export Sample Information x Export Full wiff path Short wiff name Sample Name Sample ID Comment Acquisition Method Name Figure 1 10 Export Sample Information dialog 2 Select th
87. ts API Instrument Processing Scripts folder and then delete the script dll or if applicable exe and bmp files manually For acquisition scripts navigate to the lt drive gt Analyst Data Projects API Instrument Acquisition Scripts folder and then delete the script dll or if applicable exe and bmp files manually Scripts User Guide Analyst 1 6 1 Software E se Date March 2012 7 of 88 Analyst Software Scripts Add Missing Zeros Use this script to add a value of zero intensity for the missing mass values in the spectrum To minimize storage requirements and to speed data display and processing the Analyst software does not store or display spectral points with an original intensity of zero If required for example when exporting a spectrum for subsequent processing by custom software you can add these data points back to the spectrum To use the script e Click the spectrum and then click Script gt Add Missing Zeros The script will add the zero values to all missing masses in the current spectrum Analyst 1 6 1 Software Scripts User Guide 8 of 88 Analyst Software Scripts Add Normalized ADC Traces Use this script to overlay the active chromatogram or chromatograms with normalized ADC data from a corresponding data file Run the script when a pane containing one or more chromatograms is active in Explore mode There may be a time region selected in a trace If no selection is made then the entire chromatogram i
88. up edit the parameters that pertain to the calculation of the product ion spectra for an IDA run Analyst 1 6 1 Software Scripts User Guide 50 of 88 Analyst Software Scripts The Precursor mass tolerance for grouping field is used to potentially combine adjacent product spectra into a single spectrum If two spectra have precursors with the same charge state and the same MW within this tolerance they will be combined In the Max number cycles between groups field spectra are not combined if the number of cycles between spectra with the same m z and charge state is greater than the specified value Use this option if you do not want to combine spectra with significantly different retention times In the Min num cycles per group field type the minimum number of spectra that need to be combined in order for the result to be kept Rm 1 Note If you have used the dynamic exclusion IDA option set this value to 9 In the MS MS data processing group select parameters that pertain to the filtering of product ion spectra Remove peaks if intensity lt _ Removes peaks that are either less than a specific count or a specific percent of the maximum peak intensity of the spectrum To centroid the MS MS spectra before sending them to Mascot for searching select the Centroid all MS MS data check box It is highly recommended that you enable the centroid option If the centroid option is used indicate whether isotope peaks s
89. w Survey Scan Display XICs original or filtered from the Chromatograms survey scan that correspond to parent masses yielding specified fragment or neutral loss Options Show Dependent Display neutral loss traces one for each Chromatograms neutral loss reconstructed from dependent scan data Options Subtract Peaks Present in Remove peaks that can be found in the Control Sample control sample XICs from the survey scan chromatograms Results Result File Select Results file containing identified peak times and masses will be saved Results Save Precursor Write processing results list of found Information peaks times and masses to selected results file Results XIC Peaks gt Minimum size of the peak in survey scan to be stored in the results file Results Append Results to an Do not overwrite existing results file Existing File Results View Open the selected results file Results Make XIC Table Use the selected results file to prepare settings file for XIC from Table script Results Make Quant Input Use the selected results file to prepare an input for CreateQuanMethodFromText script File Menu Load Settings Load previously saved script settings to the interface File Menu Save Settings As Save current script settings for future use File Menu Set Results File See Results Result File Tools Menu Extract Fragments See Fragments Extract Tools Menu Extract Neutral Losses See Neutral Losses Extract Tools Menu Clear Fragments See Fr
90. yielding greatest ion count is determined CE is ramped for its entre range with a 2V step size Dwell Time o ms Analyst Software Scripts MS MS MS XIC graph smoothed 2 times Finds 2 most intense 3rd precursors at 5 max intensity Exclude peaks within 2 Da window of 2nd precursor parent must be lt 10 total ion count AF2 is ramped for optimal sensitivity Scan Rate 11000 Da s v Use Q0 Trapping Fixed Fil Time 50 ms Mass range 100 ii Da Generate Final Methods Creates final MS MS MS methods with mass range of 50 Da to 2nd precursor 0 8 Da for each top 2nd precursor Creates optimal MS MS MS method with 20 Da mass range window around most intense 31d precursor Save All Final Methods C Save Optimal Method Only Figure 1 29 Advanced Settings dialog In the Scan Rate fields in the Enhanced Resolution Enhanced product lon and MS MS MS groups select a scan rate for ER EPI and MS In the Q1 Multiple lon group in the DP Ramp fields type the declustering potential DP range for optimization The range is expressed in absolute values and the appropriate polarity is automatically applied based on the selection made in the Settings dialog In the Enhanced Product lon group do the following Inthe 2nd Precursors field type the maximum number of second precursors fragment ions used for MS optimization Type a number between 1 and 10 Inthe Enhanced Product lon group in the Ma

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Analyst® 1.6.1 Software Scripts User Guide