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Protein Deconvolution 3.0 User Guide Version A

1. Thermo Scientific Protein Deconvolution User Guide 185 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples e The scan range that the reference spectrum was derived from e The retention time range that the reference spectrum was derived from The number of peaks in the reference spectrum The mass of the most abundant component in the reference spectrum You can view this information on the Sample Comparison page as shown in Figure 94 on page 195 To save a deconvolved spectrum as a reference spectrum 1 Be sure that the deconvolved spectrum that you want to save as a reference spectrum is currently visible in the Deconvolved Spectrum pane on the Process and Review page as shown in Figure 88 In this example the portion of the chromatogram in the IgG_source_cid raw file to be saved as a reference spectrum is between approximately 3 4 and 3 5 minutes 186 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 88 Deconvolved spectrum to be saved as a reference spectrum Protein Deconvolution DefauitMethodReSpect aa aa a ich iae SCIENTIFIC oG soune didra o Method Selection Run Queue o Parameters o Chromatogram Sample Comparison B Reporting i Deconvolution completed successfully Process Save Result As Reset Method Save as Reference Main Paramet
2. Isotopically Unresolved Load Results gt 5 In the Results pane select the results that you want and click Load Result The application transfers you to the Process and Review page which displays the requested saved results in its Source Spectrum Chromatogram and Deconvolved Spectrum panes and in its Results table The file name of the requested results appears in the Saved Xtract Results pane To load saved results from the Process and Review page 1 1 On the Method Selection page load a raw data file and select a method For instructions see Setting Up a Manual Xtract Protein Deconvolution on page 14 2 Click the Process and Review tab 3 In the Saved Xtract Results or the Saved ReSpect Results pane select the results that you want to load and click Load Result at the bottom of the page Thermo Scientific Protein Deconvolution User Guide 99 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Loading Saved Xtract Results The Protein Deconvolution application places the selected previous results in the Source Spectrum Deconvolved Spectrum and Results panes of the Process and Review page In addition it changes the report on the Reporting page 100 Protein Deconvolution User Guide Thermo Scientific Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm This chapter explains how to automat
3. Parameter Source Spectrum Description Determines whether to display the Source Spectrum section of the report For information on this section see Source Spectrum Section on page 92 e Default Selected Displays the Source Spectrum section of the report e Cleared Does not display the Source Spectrum section of the report Deconvolution Parameters Determines whether to display the Main Parameters Xtract and Advanced Parameters Xtract sections of the report For information on these sections see Main Parameters Xtract Section on page 93 and Advanced Parameters Xtract Section on page 93 e Default Selected Displays the Main Parameters Xtract and Advanced Parameters Xtract sections of the report e Cleared Does not display the Main Parameters Xtract and Advanced Parameters Xtract sections of the report Deconvolved Spectrum Components Table 34 Protein Deconvolution User Guide Determines whether to display the Deconvolved Spectrum section of the report For information on this section see Deconvolved Spectrum Section on page 94 e Default Selected Displays the Deconvolved Spectrum section of the report e Cleared Does not display the Deconvolved Spectrum section of the report Determines whether to display in the report the information that appears in the Results table on the Process and Review page For information on this section see Displaying th
4. Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Saving the Results of a Deconvolution Figure 33 SaveAs dialog box 1L Saves x Result Name test2 a Descriptions IgG intact Save Cancel c Click Save The name that you entered into the Result Name box appears in the Saved Xtract Results pane on the Process and Review page The Protein Deconvolution application saves the results of the deconvolution in a file with an sqlite suffix in the same directory where you stored the raw data files If you want to analyze another averaged spectrum from the same LC MS data file navigate back to the Chromatogram pane and follow the instructions in Selecting the Spectrum to Deconvolve on page 37 To display saved results Ifthe Saved Xtract Results pane and the Saved ReSpect Results pane are not visible on the Process and Review page click the double arrow s gt at the top of the Results pane If only the title bar of the Saved Xtract Results pane or the Saved ReSpect Results pane is visible click the side arrow g in the appropriate pane to expand the pane The Saved Xtract Results pane and the Saved ReSpect Results pane display any Xtract or ReSpect results respectively that you saved from a given raw data file If the raw data file includes both ReSpect and Xtract saved results
5. The default is 80 percent Remainder Threshold Specifies the height of the smaller overlapping isotopic cluster as a percentage with respect to the height of the most abundant isotopic cluster when the Xtract algorithm attempts to resolve overlapping isotopic clusters For example if one isotopic cluster in a spectrum has an abundance of 100 and you set the Remainder Threshold parameter to 30 the Xtract algorithm ignores any overlapping clusters with an abundance less than 30 The Remainder Threshold parameter is a percentage value that lies between 0 and 100 percent The default is 25 percent Consider Overlaps Determines whether the Xtract algorithm is more tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster Default Selected The Xtract algorithm is more tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster Because this option can lead to increased false positives select it only in cases where you expect overlapping isotopic clusters in a data set Cleared The Xtract algorithm is less tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster Charge Range Specifies the lowest and highest charge state to be deconvolved Low Specifies the lowest charge state High Specifies the highest charge state F
6. To assign a priority level to jobs in the queue on page 221 While the Protein Deconvolution application is analyzing data files in the run queue you can still use the manual workflow to create new methods If you start a job and then realize that you need to use another method change parameters or change the job s priority you can pause the run queue The Protein Deconvolution application completes the analysis of the current job and then pauses the run queue until you click Run again To pause a job see To temporarily stop the processing in the run queue on page 222 When you close the Protein Deconvolution application while the application is analyzing data files and restart the application later the application automatically opens the run queue and displays the same information that it did when you closed it even if analyses are still in the queue The samples remain queued but the application does not automatically start to process them Torun the jobs in the queue 1 Click the Run Queue tab to open the Run Queue page if it is not already opened 2 Click Run The Run button changes to a Pause button After a few moments the status of the job changes from Queued to Processing When the status in the Status column changes to Processing the Pause button becomes unavailable unless the queue contains other jobs with a Queued status During processing only the Method Selection and the Parameters tabs are availa
7. on page 196 Select the appropriate reference spectrum in the Reference Spectrum Library pane Click Add to Method Save the method as follows e To save the method under the existing name select the method from the library and click Save Method or 196 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples e Ifyou used the default method save the method under another name i Click Save Method As after you select a reference spectrum and add it to the reference library The Save dialog box appears ii Type the name of the method in the Method Name box iii Type a brief description of the method in the Descriptions box iv Click Save Comparing Samples in Saved ReSpect Results You can compare a source spectrum to the reference spectrum in previously saved ReSpect results When you load saved results the Sample Comparison page becomes available whether or not you used a reference spectrum If no reference spectrum appears on this page the method did not use a reference spectrum to generate the results When you load saved ReSpect results you can select only a reference spectrum generated by the ReSpect algorithm Similarly when you load saved Xtract results you can select only a reference spectrum that was generated by the Xtract algorithm You can select a new reference spectrum to show in th
8. Figure 61 DefaultMethodReSpect_2730_2752_3_9 2012_12_40_18_PM_RSP_AUTO ae a Method Start Date Time Experiment name scan type End scan All results generated from an analysis of a given raw data file are stored in the SQLite file associated with the raw data file The SQLite file and the raw data file have the same name but different suffixes For example if you analyze a file called IgG_source_cid raw the results are stored in the IgG_source_cid sqlite file All results for a given automated run on a given raw data file have the same date and time stamp For information on loading saved results see Loading Saved Xtract Results on page 115 Displaying an Xtract Deconvolution Report When you click Run on the Run Queue page the Protein Deconvolution application generates a report displaying several aspects of the deconvolution so that you can track the progression of the data For a description of this report see Displaying an Xtract Deconvolution Report on page 85 You can view this report on the Reporting page and save it as a PDF file If you selected the Concatenate All Reports parameter in the Automation Parameters section of the Parameters page the Protein Deconvolution application opens one report for all the chromatographic peaks If you did not select this parameter it opens a report for each peak To display the report e On the Run Queue page select the completed job with the report that you w
9. Index S Mirror Plot shortcut menu 85 200 parameters on 82 197 Reference Spectrum Library pane 78 80 83 193 195 198 Sample Information parameter 24 33 132 146 Sample Information report section description 89 203 setting parameter for displaying 24 33 132 146 Sample Volume uL column 90 203 Sample Weight column 90 203 Save a Copy dialog box 88 89 202 Save a Copy icon 89 Save As Reference Spectrum command 65 179 Save dialog box Parameters page 26 134 Sample Comparison page 75 81 82 190 196 197 Save File icon 88 202 Save Method As command Chromatogram page 47 112 161 227 Parameters page saving method changes in a file 16 121 saving parameter values to a new method 25 36 133 149 Process and Review page 112 227 Sample Comparison page changing reference spectrum in method 81 82 196 197 saving reference spectrum to new method 75 84 190 199 Save Method command Chromatogram page 47 112 161 227 Parameters page 16 25 36 121 133 149 Process and Review page 112 227 Sample Comparison page 75 84 190 199 Save Result As command Process and Review page 65 66 179 181 Sample Comparison page 85 200 Save to PDF parameter 35 104 148 218 SaveAs dialog box Parameters page 26 134 Process and Review page 66 72 181 187 197 Sample Comparison page 200 Saved ReSpect Results pane deleting results from 67 making visible 181 purpose 50 62 67 164 176 182 resizing 10 67 182 Saved Xtract Result
10. RT min x axis 46 Protein Deconvolution User Guide Displays the retention time of the spectrum which is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Table 6 Chromatogram page parameters for Xtract deconvolution Sheet 4 of 4 Parameter Source Spectrum pane Description Displays the spectrum that you selected Relative Intensity y axis m z x axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Displays the mass to charge ratio of ions formed from molecules This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion Chromatogram Page Menu Bar Commands Table 7 describes the commands on the menu bar of the Chromatogram page Table 7 Menu bar commands on the Chromatogram page for Xtract deconvolution Parameter Save Method Description Saves the current parameter values to the existing method overwriting any previous values Clicking Save Method automatically advances the application to the Process and Review page Save Method As Saves parameter values to a new method Clicking Save Method As automatically
11. Table 12 Sample Comparison page parameters for Xtract deconvolution Sheet 1 of 3 Parameter Description Method Reference Spectrum Displays information about the reference spectrum in the pane currently loaded method Reference Spectrum Displays the name of the reference spectrum in the currently loaded method as you specified it in the SaveAs dialog box Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Table 12 Sample Comparison page parameters for Xtract deconvolution Sheet 2 of 3 Parameter Descriptions Description Displays the description of the reference spectrum in the currently loaded method as you specified it in the SaveAs dialog box Reference Data File Displays the name of the raw data file that produced the reference spectrum in the currently loaded method Show Details Opens a Reference Spectrum Information box see Figure 41 on page 80 displaying the deconvolution parameters used to produce the reference spectrum that you selected in the currently loaded method Remove Removes a reference spectrum from the currently loaded method Reference Spectrum Library pane Displays commands that allow you to select reference spectra to show in the mirror plot display information about specific reference spectra add reference spectra to the method and delete reference spectra from t
12. User Guide Software Version 3 0 XCALI 97576 Revision A March 2014 DOCUMENTATION 2014 Thermo Fisher Scientific Inc All rights reserved ReSpect is a trademark of Positive Probability Ltd Exactive Plus is a trademark and Exactive Orbitrap Thermo Scientific and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc in the United States Microsoft Excel PowerPoint and Windows are registered trademarks of Microsoft Corporation in the United States and other countries Adobe Acrobat and Reader are registered trademarks of Adobe Systems Inc in the United States and other countries All other trademarks are the property of Thermo Fisher Scientific Inc and it subsidiaries Thermo Fisher Scientific Inc provides this document to its customers with a product purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser This document is not part of any sales contract between Thermo Fisher Scientific Inc and a purchaser This document shall in no way govern or modify any Terms and Condi
13. WO files ReSpect Q Exactive lgG_source_cid raw Queue will pause after the analysis is completed 2 Click OK 222 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue After the current analysis finishes the rest of the jobs remain in the run queue in the Queued state Click Run to have the Protein Deconvolution application process the remaining jobs To remove a selected job or jobs from the queue Select the job or jobs to remove in the queue Remove multiple jobs by doing the following e To select contiguous files names click the name of the first job hold down the SHIFT key and click the last job that you want to select e To select noncontiguous names click the name of the first job hold down the CTRL key and click each separate job On the Run Queue menu bar choose Queue Manipulation gt Remove Selected The confirmation box shown in Figure 112 appears Figure 112 Message box confirming the removal of completed jobs Protein Deconvolution X This action will remove the items from the queue permanently Do you want to continue Yes No Click Yes To remove all jobs from the queue In the Run Queue menu bar choose Queue Manipulation gt Remove All You can only remove all jobs when their status is Queued or Completed In the confi
14. 1 00727663 Specifies that the adduct was hydrogen K 38 9631585 Specifies that the adduct was potassium Na 22 9892213 Specifies that the adduct was sodium Protein Deconvolution User Guide 21 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Custom Specifies that the adduct was a charge carrier other than hydrogen potassium or sodium When you select this option a box opens so that you can type the mass of the custom charge carrier Note In negative mode these adduct ions correspond to deduct rather than adduct masses e Min Num Detected Charge Specifies the minimum number of charge states required to produce a component No components with less than this minimum number appear in the deconvolved spectrum This parameter must be an integer greater than or equal to 1 The default is 3 e Isotope Table Specifies the type of isotope table to use Isotope tables simulate the distribution of isotopic peaks in m z for different choices of the monoisotopic mass Xtract chooses the monoisotopic mass with the best fit between the theoretical and the observed isotope distribution To generate an isotope table the Protein Deconvolution application uses a chemical formula to describe the type of molecule You can choose one of the following formulas Default Protein Uses an averagine formula to generate the isotope table The default method
15. 132 147 spectral peak modeling 5 splitter bars 10 SQLite files contents of 8 deleting 69 184 loading previous results 98 212 location of saved methods 26 134 saving deconvolution results in 67 181 starting Protein Deconvolution application 8 Status parameter 106 111 221 225 238 Protein Deconvolution User Guide Submit Time parameter 110 224 Sum Intensity column 63 66 68 system requirements viii T Target Mass parameter 127 138 TIC chromatogram See total ion current chromatogram Time Limits parameter 38 44 151 158 top down proteomics 5 70 185 total ion current chromatogram 38 39 45 150 151 159 Types parameter 39 45 151 159 U Use Auto Spectral Averaging parameter 39 46 152 160 Use Relative Intensities parameter 129 141 Use Restricted Time parameter 38 44 151 158 V Vial column 90 203 X Xcalibur data system 6 41 154 Xtract algorithm adjusting deconvolution results 61 automatic deconvolution 101 centroid data 43 65 69 centroid spectra 42 comparing samples 4 changing a reference spectrum in a method 81 comparing a source spectrum to a reference spectrum 75 deleting a reference spectrum from a method 81 displaying parameter settings of reference spectrum 79 in saved Xtract results 82 mirror plot 70 purpose 70 saving deconvolved spectrum as a reference spectrum 71 saving reference spectrum 70 spectra compared 70 185 creating a method 18 deconvolving a spectrum 49 deleting
16. 151814 578 354372024 36 B 11 19 100 0000 151976 766 338471159 45 48 2 41 15 87 162 19 95 5129 152138 906 239189716 31 42 3 05 20 06 324 33 67 4968 151653 891 228202029 69 41 3 01 19 86 160 69 64 3962 75907 445 180495932 79 33 0 87 11 43 75907 13 50 9340 75988 602 170404048 70 24 1 15 15 15 75825 98 48 0862 76067 8567 123293404 71 31 1 52 20 02 75746 71 34 7921 152300 750 120421083 16 34 9 22 60 52 486 17 33 9815 50604 863 117219475 80 17 0 67 13 32 101209 71 33 0781 50658 328 114688695 77 16 0 87 17 19 101156 25 32 3639 152430 281 93105208 16 27 3 73 24 44 615 70 26 2733 37953 980 89820328 56 16 0 60 15 93 113860 60 25 3463 37994 043 86495221 14 14 0 71 18 62 113820 54 24 4080 152267 953 84902414 50 21 4 15 27 24 453 38 23 9586 50713 191 80914625 09 19 1 45 28 51 101101 39 22 8332 151496 734 79224722 66 34 4 52 29 81 317 84 22 3564 50550 672 77486182 63 18 0 97 19 28 101263 91 21 8658 152588 625 69964645 00 26 7 05 46 21 774 05 19 7433 38034 102 62737189 86 15 0 86 22 60 113780 48 17 7038 152471 328 54662426 88 18 6 92 45 39 656 75 15 4252 157045 484 49126154 38 10 6 66 42 41 5230 91 13 8629 149480 688 46514292 28 20 8 51 56 95 2333 89 13 1258 Source Spectrum Evidence Section The Source Spectrum Evidence section shown in Figure 105 displays a table and an accompanying graph for every component in the sample This section appears only if you select the Component Detail Tables parameter and the Component Source of Evidence Plots pa
17. 59 Calculated Monoisotopic m z column 54 57 64 CDL peak detection algorithm 5 centroid data 43 65 69 155 180 183 Charge Carrier parameter 21 30 125 135 charge deconvolution 3 Charge Normalized Intensity column 54 64 Charge Range parameter 23 32 Charge State column 54 55 64 168 169 178 Charge State Range parameter 121 127 139 218 charge state peaks 168 Choice of Peak Model parameter 128 139 Chromatogram m z Range parameter 39 46 152 160 Chromatogram page Chromatogram pane 38 150 Chromatogram Parameters pane 38 150 exporting chromatograms 68 183 Protein Deconvolution User Guide 231 Index D menu bar commands 47 161 parameters on 44 158 selecting a spectrum to deconvolve 37 149 shortcut menus 48 162 Source Spectrum pane 38 151 Chromatogram pane adjusting the view 40 152 creating spectrum in Source Spectrum pane 41 154 displaying extracted ion chromatogram in 171 displaying results in 58 59 170 header 48 162 parameters on 46 62 160 176 purpose 38 150 zooming 40 152 Chromatogram Parameters pane parameters on 38 44 151 158 purpose 38 150 Chromatogram Parameters report section displaying 24 33 132 146 204 information in 90 Chromatogram With Parameters parameter 24 33 132 146 chromatographic peak detection 4 5 Column Chooser dialog box 53 167 Column Chooser icon 53 167 Completion Time parameter 106 111 221 225 Component Detail Tables parameter 25 35 133 148 Component Detecti
18. Isotopically Unresolved Several more tabs appear along the top of the Protein Deconvolution window 216 Protein Deconvolution User Guide Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up an Automatic ReSpect Protein Deconvolution Figure 107 Initial Method Selection page Auto ReSpect Isotopicall Protein Deconvolution a aati SCIENTIFIC v Run Queue Parameters i Chromatogram a Process and Review im Sample Comparison Reporting gt Select an experiment type a data file and a method i Experiment Types Methods Load Raw Data File Description Raw Data Directory C Program Files Protein Deconvolutior a Manual Xtract Isotopically Resolved DefaultMethodReSpect Default method for ReSpect Read Only Method for Native MS Select Raw Data Files ExampleMethodNativeMS Manual ReSpect Isotopically Unresolved Auto Xtract Isotopically Resolved gt Auto ReSpect Isotopically Unresolved gt Load Results 4 In the Methods pane specify the extraction method to use by doing one of the following e If one of the existing methods contains the appropriate parameters and you do not want to make any changes to it select the name of the method of interest go to the Load Raw Data File pane and follow the instructions from step 8 on page 219 e Ifyou want to create a new m
19. Negative Charge False Minimum Peak Significance 1 Charge Carrier H Noise Rejection 95 n Rel Abundance Threshold 0 Use Relative Intensities True Quality Score Threshold 0 Peak Width 0 151813 623 m z Range 1000 4000 Feature Width 0 151976 123 Output Mass Range 10000 160000 Degree of Fit 0 Y Mirror Plot Source Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid ra 100 151655 323 Mass Tolerance 30 Ppm Number of Iterations 3 Si Target Mass 150000 Da Noise Compensation True A 151494 623 151896 223 152062 Charge State Range 10 100 4 Minimum Adjacent Charges 6 10 g 151735 823 5 Choice of Peak Model Intact Protein Number of Peak Models 1 23 452728 123 151571 423 d 8 2 0 nm Resolution at 400 m z 12374 151615 453 151753 953 151932 653 157 andia Peak Shape 22 jog 152636 153 152755853 g sog 151495353 152274 753 152428 353 151654 253 151975 653 152138 353 4 853 100 Mass Reference Data File C Program Files Protein Deconvolution source files ReSpect O Exactive lgG_source_cid raw From Library NL 6 13E 008 7 B To delete a reference spectrum from the reference spectrum library 1 Click the Sample Comparison tab if you are not already on the Sample Comparison page 2 Select the appropriate reference spectrum in the Reference Spectrum Library pane and click Delete 3 In the confirmation box that appears clic
20. Open Result Transfers you to the Process and Review page after the Protein Deconvolution application finishes processing a job Open Report Transfers you to the Reporting page after the Protein Deconvolution application finishes processing a job Queue Record Number Displays a sequential number that identifies each job Priority Submit Time 224 Protein Deconvolution User Guide Displays the priority level that each job in the queue has e Low Processes the job after jobs that are assigned a Normal or High priority level e Normal Processes the job before jobs that are assigned a Low priority level but after jobs that are assigned a High priority level e High Processes the job before jobs that are assigned a Low or Normal priority level Displays the time that you placed the job into the run queue Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue Table 45 Run Queue page parameters for automatic ReSpect deconvolution Sheet 2 of 2 Parameter Description Method Name Displays the method used to deconvolve the averaged spectrum Raw Data File Displays the path and name of the raw data file used to deconvolve the averaged spectrum Experiment Type Displays the type of algorithm used to deconvolve the averaged spectrum e XT_AUTO Deconvolves the spectrum with the automatic Xtract algorithm e RSP_A
21. Parameter Resolution at 400 m z continued Description The range of values for the Resolution at 400 m z parameter is 6000 to 240 000 The default is not a fixed number but varies from raw data file to raw data file In cases where the mass spectrometer measured the resolution in the raw data file at an m z value other than 400 the Xtract algorithm scales it as follows to account for the variation in instrument resolution versus m z R R x M measured converted measured 400 where Roonverted iS the resolution to be converted e Ro measured iS the resolution at the measured mass to charge ratio e M measured is the measured mass to charge ratio other than 400 Left Right Peak Shape Defines the sharpness of a peak Default for both the left and the right peak shape 2 Reporting Parameters pane Determines which parts of the generated report that the Protein Deconvolution application displays Sample Information Determines whether to display the Sample Information section of the report For information on this section see Sample Information Section on page 203 e Default Selected Displays the Sample Information section of the report e Cleared Does not display the Sample Information section of the report Chromatogram with Parameters 146 Protein Deconvolution User Guide Determines whether to display the Chromatogram Parameters section of the report For information on t
22. Parameter Set As Reference Component Results table only Description Resets the value in the Delta Mass Column of the Results table to 0 for the chosen component and recalculates the delta mass value for all other components in the Results table Export Top Level Results table only Exports just the top level results in the Results table to a file The top level results are the data in these columns Monoisotopic Mass Sum Intensity Number of Charge States Average Charge Delta Mass Relative Abundance Fractional Abundance RT Range and Apex RT They do not include the data in the columns that appear when you click the symbol to the left of the No column Export All Results table only Exports all results in the Results table to a file Saving the Results of a Deconvolution 66 You can save the results of the deconvolution in a file in the same directory where you stored the raw data files You can also copy and paste any one of the views in this window to a Microsoft PowerPoint presentation file To save the results of the deconvolution 1 Click Save Result As on the menu bar 2 In the SaveAs dialog box do the following a In the Result Name box type the name of the results file or select an existing file to overwrite from the dropdown menu b Optional In the Descriptions box type a brief description of the results The dialog box should resemble that shown in Figure 33
23. The default values for both limits depend on the data in the raw data file The default is the entire chromatogram for the given spectrum e Use Auto Spectral Averaging Determines whether the Protein Deconvolution application ignores all chromatographic peak picking when averaging spectra Default for the ExampleMethodNativeMS method Selected Creates the averaged spectrum from all spectra in the specified retention time range so it creates only one deconvolution result from an automated run To support Native MS data resulting from direct infusion into the mass spectrometer in automated workflows use this option to automatically average scans from either the entire run or within a specified retention time range By default the Protein Deconvolution application selects the full retention time range of a given data set so it produces the averaged spectrum for the full run Default for the default method Cleared Averages and deconvolves all spectra in the run This parameter is only available in the Auto ReSpect experiment type If you want to return the parameters in the Chromatogram Parameters pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any parameters in this pane but do not click Apply and then click another tab the message box shown in Figure 66 on page 128 appears Click Yes to apply the parameter changes or No to restore the par
24. in Saved ReSpect Results on page 197 You can use one of two ways to load the results of previous deconvolutions e Choose the Load Results command on the Method Selection page e Click the applicable results in the Saved ReSpect Results pane on the Process and Review page Note When you load results generated in version 1 0 any parameters added since version 1 0 are set to 0 To load saved results from the Method Selection page 1 Click the Method Selection tab 2 In the Experiment Types pane click Load Results 3 In the Raw Data Directory box of the Load Result File pane type the path and name of the SQLite file containing the saved results or click the Browse button to browse to the location of the file 4 In the Select Result Files area of the Load Result File pane select the name of the SQLite file containing the results and click Load The Protein Deconvolution application displays the results found in the Results pane as shown in Figure 106 212 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Loading Saved ReSpect Results Figure 106 Saved results in the Results pane Protein Deconvolution oad Results Help SCIENTIFIC IgG_source_cid SQUTE Method Selection 4 Run Queue Parameters Chromatogram Process and Review Sample Comparison Reporti a iP td gt Select an experiment type a data file
25. on page 37 To use the Process and Review page follow the instructions in Deconvolving the Spectrum on page 49 e Ifyou want to make changes to an existing method do the following i Select the name of the method of interest and click Load Method ii From the Chromatogram or the Process and Review page click the Parameters tab iii Change the appropriate parameters on the Parameters page click Apply in the appropriate pane or panes and click Save Method to save the changes to the file or Save Method As to save the changes as a different method Clicking Save Method or Save Method As automatically advances the application to the Chromatogram or Process and Review page Note You cannot overwrite a default method If you change the parameters in the default method use the Save Method As command on the Parameters page to save the changed method to a new name e Ifthe existing methods do not contain the appropriate extraction parameters or if there are no existing methods click Create Method to create a new method The Protein Deconvolution application automatically transfers you to the Parameters Pp y y page Follow the instructions in Creating an Xtract Method on page 18 to set the parameters on the Parameters page To delete a method from the Methods pane 1 In the Methods pane of the Method Selection page select the method that you want to delete 2 Press the DELETE key 3 Click Yes in the co
26. page reduce the value of the Mass Tolerance parameter to make the results cleaner As noted in the description of the Mass Tolerance parameter in Table 27 on page page 135 specifying this value using the PPM option might also improve results When you decrease this value the delta mass value for each charge state also drops e In the Advanced Parameters ReSpect pane of the Parameters page raise the values of the Minimum Adjacent Charges parameter Thermo Scientific Protein Deconvolution User Guide 157 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Chromatogram Page Parameters for the ReSpect Algorithm Table 29 lists the parameters that are available on the Chromatogram page for ReSpect deconvolutions Table 29 Chromatogram page parameters for ReSpect deconvolution Sheet 1 of 4 Parameter Chromatogram Parameters pane Description Displays the parameters that govern the appearance of the chromatogram in the Chromatogram pane Use Restricted Time Time Limits Determines whether the Protein Deconvolution application zooms the part of the chromatogram that you define with the Time Limits parameters e Selected Zooms the specified part of the chromatogram e Default Cleared Displays the entire chromatogram Specifies the beginning and the end of the chromatogram that you want to zoom The default values for both limits dep
27. rom_protein_micr F FTMS p ESI Full ms 300 00 2000 00 100 848 5562 otrap_11min_OT_60K_1 148 178 RT 3 27 3 88 AV 30 893 1164 942 7287 80 a o 8 Relative Intensity 8 829 8488 873 5784 921 9993 842 5079 852 7046 863 3128 a i 885 2114 897 3220 910 4350 Hy 927 5053 938 4501 951 2798 959 2963 230 840 850 860 870 880 890 900 910 920 930 940 950 960 mz To shrink the view of the entire spectrum right click and choose Zoom Out e To reset the view to the original spectrum right click and choose Reset Scale Unlike adjustments in the Chromatogram pane which you use to select a spectrum for processing adjustments in the Source Spectrum pane do not affect the spectrum that the Protein Deconvolution application deconvolves In particular they do not change the m z range that the deconvolution algorithm uses 6 When the spectrum is suitable for Xtract processing click the Process and Review tab and follow the instructions in Deconvolving the Spectrum on page 49 Chromatogram Page Parameters for the Xtract Algorithm Table 6 lists the parameters that are available on the Chromatogram page for Xtract deconvolution Table 6 Chromatogram page parameters for Xtract deconvolution Sheet 1 of 4 Parameter Description Chromatogram Parameters Displays the parameters that govern the appearance of the pane chromatogram in the Chromatogram pane Use Restricted Time Determines whether the Protein Deconvolu
28. 0 67 0 83 1 11 1 22 1 37 1 46 163 1 80 205 229 243 S 851 S 916 r TT al Aah i 05 10 15 20 25 40 45 5 0 55 60 30 RT min Draw a line under the baseline of the peak of interest When you zoom a region of the chromatogram or the spectrum the Protein Deconvolution application recalculates the values on the y axis so that 100 percent represents the highest abundance in the displayed region as shown in Figure 71 The abundances shown on the y axis remain at values relative to the most abundant component in the plot Drawing a box rescales the chromatogram or spectrum to the relative height of the box Figure 71 Chromatogram automatically scaled to the maximum height of the peak in the Chromatogram pane Chromatogram gG_source_cid NL 3 10E9 RT 3 41 S G S 1171 F 342 s 8 2 G RT 3 31 S 1093 3 32 Relative intensity a Y 8 3 24 325 3 27 3 42 4 34 RT min Thermo Scientific Protein Deconvolution User Guide 153 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve 154 If there is no obvious chromatographic peak change the limits of the m z Range parameter on the Parameters page to find it To shrink the view of the entire spectrum right click and choose Zoom Out To reset the view to the original spectrum right click and choose Reset Scale 4 Create a spectrum in th
29. 004 Reference Data File C Program Files Protein Deconvolution source files Xtract WPSA_240 raw From Library Table 20 lists the parameters in the Mirror Plot section Table 20 Mirror Plot section parameters for Xtract deconvolution Parameter Relative Intensity y axis Description Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis Displays the mass of the ions formed from molecules Thermo Scientific Protein Deconvolution User Guide 95 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Xtract Masses Table Section The Xtract Masses Table section shown in Figure 52 displays the results of the deconvolution It contains the same columns as those in the Results table on the Process and Review page For information on the columns in this table see able 9 on page 62 Figure 52 Xtract Masses Table section for Xtract deconvolution Xtract Masses Table 21 19 83 0 0000 100 0000 16941 0121 325937 34 16923 0450 223517 12 16 20 60 17 9671 68 5767 16922 8886 33607 63 4 14 97 18 1235 10 3111 16905 0232 30910 95 13 21 11 35 9889 9 4837 16568 8504 26801 33 14 20 35 372 1617 8 2228 16955 9318 13608 61 10 20 10 14 9197 4 1752 16963 9448 9684 77 10 18 15 22 9327 2 9714 16981 9743 9225 90 10 19 17 40 9622 2 8306 16971 9609 8844 52 8 20 26 30 9488 2 7136 168
30. 1060 4414 5 1131 0669 20 1304 9241 300 400 500 600 14136704 1542 1908 700 800 900 1000 1696 0152 48187929 1918 3286 T poy iaa p4 T 1500 1600 1700 1800 1900 2000 T f Thr 1100 1200 1300 1400 mz Draw a box around the peaks of interest Keeping the left mouse button pressed draw a line beneath the baseline of the peaks of interest as shown in Figure 20 Figure 20 Enlarging an area by drawing a line beneath the baseline of the peaks of interest Source Spectrum Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 148 178 RT 3 27 3 88 AV 30 F FTMS p ESI Full ms 30000 2000 00 100 808 1993 e 391 2844 mai ge 20 70 998 1281 60 50 4313 40 30 20 10 893 1164 Relative Intensity 1060 4414 1131 0669 13049241 4413 6704 15421908 1696 0152 4818 7929 1918 3286 300 400 500 600 700 T sages T T piere 200 300 1000 1100 1200 1700 1800 1900 2000 miz T eae ago 1300 1400 1500 1600 Draw a line under the baseline of the peaks of interest Right click and choose Zoom In to enlarge the view of the entire spectrum Figure 21 gives an example of the enlarged isotopic clusters Thermo Scientific Protein Deconvolution User Guide 43 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Figure 21 Enlarged peaks in the Source Spectrum pane for Xtract deconvolution Source Spectrum Myoglobin_30pmol_michi
31. 9 06 9 33 10 00 10 27 10 58 0 5 1 0 15 20 25 3 0 3 5 40 45 5 0 5 5 6 0 65 7 0 7 5 8 0 85 9 0 9 5 10 0 10 5 RT min Source Spectrum Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 161 180 RT 3 56 3 91 AV 19 F FTMS p ESI Full ms 300 00 2000 00 893 1132 1007 848 5578 90 4 80 737 9652_771 5054 704 607 507 391 2846 679 0464 40 30 204 942 7307 707 2971 998 1244 1060 3788 Relative Intensity 1131 0698 628 7847 1211 7144 la 536 1646 k 1304 9192 10 427 3785 1304 1413 6649 1542 1348 1696 0084 1855 0795 1918 3208 0 merpet a mr 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 miz The Source Spectrum pane shows the spectrum either single scan or averaged to be deconvolved It displays apex information for major peaks and m z information for deconvolved components It also shows peak apex information as a marker along with an accompanying label that describes the m z value for the most abundant peak from the spectrum at that retention time In single scan processing mode the most abundant m z value for a component should agree with the m z value shown for the corresponding peak in the source spectrum In averaged scan processing mode the two values might be different because of the way the application displays averaged spectra However this difference should be small on the order of 0 001 The Xtract algorithm can dec
32. Determines whether to display the spectrum of each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 210 e Selected Displays the spectrum of each component in the Source Spectrum Evidence section of the report e Default Cleared Does not display the spectrum of each component in the Source Spectrum Evidence section of the report Apply Implements the parameter settings that you selected in the Reporting Parameters pane This button is only available if you changed any parameter settings in the Reporting Parameters pane Automation Parameters pane Displays parameters that control automatic deconvolution You cannot set the parameters in this pane unless you select Auto ReSpect Isotopically Unresolved in the Experiment Types pane Save to PDF Determines whether to save the automatically generated deconvolution report to a PDF file e Default Selected Saves the automatically generated deconvolution report to a PDF file e Cleared Does not save the automatically generated deconvolution report to a PDF file Auto Print 148 Protein Deconvolution User Guide Determines whether to automatically print the generated deconvolution report When you select this option the Select a Printer box becomes available so that you can select the printer e Selected Automatically prints the generated deconvolution re
33. Does not display the Source Spectrum section of the report Deconvolution Parameters Determines whether to display the Main Parameters ReSpect and Advanced Parameters ReSpect section of the report For information on this section see Main Parameters ReSpect Section on page 207 and Advanced Parameters ReSpect Section on page 207 Default Selected Displays the Main Parameters ReSpect and Advanced Parameters ReSpect sections of the report Cleared Does not display the Main Parameters ReSpect and Advanced Parameters ReSpect sections of the report Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method e Deconvolved Spectrum Determines whether to display the Deconvolved Spectrum section of the report For information on this section see Deconvolved Spectrum Section on page 208 Default Selected Displays the Deconvolved Spectrum section of the report Cleared Does not display the Deconvolved Spectrum section of the report Components Table Determines whether to display in the report the information that appears in the Results table on the Process and Review page For information on this section see Displaying the Results on page 165 Default Selected Displays the information that appears in the Results table on the Process and Review page Cleared Does not di
34. Fit values scores display confidence values for the listed isotopic clusters e The Calculated Monoisotopic m z column displays the mass to charge ratio of the calculated monoisotopic mass for a specific charge state When you click on a value in this column the application highlights the value in the Source Spectrum pane as shown in Figure 28 This blue bar is not labeled Do not confuse it with nearby peak labels Thermo Scientific Protein Deconvolution User Guide 57 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 28 Calculated Monoisotopic m z value highlighted in the Source Spectrum pane Thermo Protein Deconvolution DefauitMethodXtract SEE Pore hye lana Help X SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_LRAW Method Selection Q RunQueue Parameters Chromatogram f DESSAS sample Comparison Reporting gt Deconvolve the spectrum Process Save ResultAs Reset Method Save as Reference gt Main Parameters Xtract Source Spectrum Chromatogram Saved Xtract Results 301 2844 893 1164 arabe 1003 7 ii S 166 S Ili 1060 4415 ee SS ma auaa u uIuauauaummmmmmmmmmmuMi a 536 1664 60 coupe 1606 2001 1918 3288 7 7 7 368 O75 7 ET pectral line 500 1000 1500 2000 corresponding to mz RT min a selected value Deconvolved Spectrum sealants in the Calculated 100 J P gt 00 Mono
35. Header on page 163 for the source and reference spectrum If the reference spectrum and the source spectrum do not cover the same mass range the Protein Deconvolution application automatically expands the range in the mirror plot to include the lowest limit and the highest limit of both spectra For example if the reference spectrum has a range of 25 000 to 160 000 m g and the source spectrum has a range of 15 000 to 100000 m z the application expands the range to 15 000 to 160 000 m z in the mirror plot 4 Optional Right click in the Mirror Plot pane and choose Zoom In to enlarge the plot as shown in Figure 93 or hold down the left mouse button and drag the cursor beneath the part of the spectrum that you are interested in 192 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples The zoom setting in the mirror plot remains the same until you change the deconvolution mass range or load a previous result This behavior is the same as that of the deconvolved spectrum in the Deconvolved Spectrum of the Process and Review page Figure 93 Enlarged mirror plot H H Manual ReSpect sotopically Unresolved Protein Deconvolution defauitmethoarespect 5 sou Help Xt SCIENTIFIC 19G_source cid raw o Method Selection Run Queue z Parameters B Chromatogram o Process and Review Sample Comparison m Reporting o Co
36. In addition the Process and Review page features an RT Range column in the Results table which displays the retention time range of the averaged source spectrum for the given component This retention range is also shown on the chromatogram as a red box for the given component Figure 3 on page 7 shows the workflow to follow to manually deconvolve mass spectra with the Protein Deconvolution application The workflow is nearly the same for the Xtract algorithm as it is for the ReSpect algorithm Protein Deconvolution User Guide 5 1 Introduction Workflow You can select the chromatogram to deconvolve either in the Protein Deconvolution application or in the Qual Browser utility which is part of the Xcalibur data system When transferring data for example in an e mail to submit to the Protein Deconvolution application you might want to reduce the amount of data to transfer by using Qual Browser to select either a single spectrum or an averaged spectrum For instructions on using Qual Browser to select a chromatogram refer to the Thermo Xcalibur Qual Browser User Guide 6 Protein Deconvolution User Guide Thermo Scientific Figure 3 Protein Deconvolution manual workflow Generate chromatogram TIC base peak of the compound of interest Zoom chromatogram peak of interest Extract an average spectrum across the peak or choose a single scan from peak Is spectrum suitable for deconvolution Apply the Xtract o
37. ReSpect The scan range that the reference spectrum was derived from The retention time range that the reference spectrum was derived from The number of peaks in the reference spectrum The mass of the most abundant component in the reference spectrum You can view this information on the Sample Comparison page as shown in Figure 41 on page 80 1 1 To save a deconvolved spectrum as a reference spectrum Be sure that the deconvolved spectrum that you want to save as a reference spectrum is currently visible in the Deconvolved Spectrum pane on the Process and Review page as shown in Figure 35 In this example the portion of the chromatogram in the PSA_240 raw file to be saved as a reference spectrum is between approximately 7 3 and 8 3 minutes Protein Deconvolution User Guide 71 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 35 Deconvolved spectrum to be saved as reference spectrum Protein Deconvolution DefauitMethodXtract Monal Nirac Dsptopically Recods aie SCIENTIFIC PSA_240 raw o Method Selection Run Queue K Parameters o Chromatogram Sample Comparison Reporting i Deconvolution completed successfully Process Save ResultAs Reset Method Save as Reference Y Main Parameters Xtract Output Mass M OMH m z Range Min 600 Max 2000 Apply Resolution at400 m z 240000 Charge Carrier H 100727663 S N Threshold 3 K 38 963158
38. Review page Reset Method Thermo Scientific Returns the parameters on the Chromatogram Parameters pane to the settings that you first chose when you loaded the raw data file Protein Deconvolution User Guide 161 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Chromatogram Pane Shortcut Menus When you right click in the Chromatogram or Source Spectrum pane on the Chromatogram page a shortcut menu appears that contains the commands listed in able 31 Table 31 Chromatogram page shortcut menu Parameter Description Mode Chromatogram pane Determines whether dragging the cursor zooms or selects a only range of scans to average e Default Averaging Averages the spectra for all the scans in the region that you drag the cursor over in the Chromatogram pane and displays them in the Source Spectrum pane e Auto Zooming Enlarges the area that you drag the cursor over in the Chromatogram pane without changing the view displayed in the Source Spectrum pane Reset Scale Restores the original chromatogram that first appeared in the Chromatogram pane Copy Bitmap Copies the view in the Chromatogram pane to the Clipboard Zoom Out Shrinks the view in the Chromatogram pane by a factor of two Zoom In Enlarges the view in the Chromatogram pane two times Chromatogram Pane Header The header in the Chromatogram pane displays the follo
39. Selected Opens one report for a single raw data file e Cleared Opens a report for each chromatographic peak in the raw data file Select a Printer Displays a list of available printers that can print the deconvolution report PDF file This option becomes available when you select the Auto Print option Senko M W Beu S C McLafferty FW Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions J Am Soc Mass Spectrometry 1995 Vol 6 226 233 Menu Bar Commands on the Parameters Page Table 5 describes the commands on the menu bar of the Parameters page Table 5 Menu bar commands on the Parameters page for Xtract deconvolution Parameter Description Save Method Saves the current parameter values to the existing method overwriting any previous values Clicking Save Method automatically advances the application to the Chromatogram or Process and Review page Save Method As Saves the parameter values to a new method Clicking Save Method As automatically advances the application to the Chromatogram or Process and Review page Reset Method Returns the parameters on the Parameters page to the settings that you first chose when you loaded the raw data file 36 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Selecting the
40. Spectrum to Deconvolve When you click Save Method or Save Method As after you have set the parameters on the Parameters page or when you load an existing method and click Load Method on the Method Selection page the Protein Deconvolution application automatically transfers you to the Chromatogram page if the raw data file contains a chromatogram Use the Chromatogram page to select the best possible spectrum for the target protein for deconvolution To select the spectrum to deconvolve 1 Click the Chromatogram tab if it is not already selected Figure 14 shows the Chromatogram page Figure 14 Chromatogram page for Xtract deconvolution Thermo Protein Deconvolution nefauitmethodxtract oaaae a Hep X SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_1imin_OT_60K_LRAW z Method Selection Qu o Parameters g Chromatogram j Process and Review Sample Comparison Reporting gt Select the chromatogram to deconvolve Save Method Save Method As Reset Method Chromatogram Parameters Use Restricted Time E Types TIC Z Time Limits 0 01 to 10 98 Sensitivity High xj z Rel Intensity Threshold i Chromatogram m z Range 600 0000 to 2000 0000 Use Auto Spectral Averaging Chromatogram Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 NL 8 43E7 F 3 65 RT 3 66 100 S 166 Relative Intensity D i RT 5 90 3 97 4 19 __462 491 534 S 268 603 6 59 7 06 7 2
41. The data was acquired in positive charge mode IMPORTANT Do not select the Negative Charge check box if your data was acquired in positive mode Results will not be usable Calculate XIC Determines whether the Protein Deconvolution application calculates an extracted ion chromatogram for each detected component Default Selected Calculates an extracted ion chromatogram for each detected component Cleared Does not calculate an extracted ion chromatogram for each detected component The application saves this setting to the method Using the Calculate XIC parameter can result in a much longer analysis time so you might avoid using it with complex data or with data where the displayed extracted ion chromatograms are unnecessary m z Range Specifies the portion of the input spectrum that the Xtract algorithm processes Min Specifies the lowest end of the input spectrum Max Specifies the highest end of the input spectrum For example if the total mass range of the spectrum is mass 100 to 2000 a setting of 300 to 500 for the m z Range parameter means that the Xtract algorithm processes only peaks with masses between 300 and 500 m z The valid range is 300 through 25 000 The default minimum is 600 The default maximum is 2000 Charge Carrier Specifies the adduct ions used during ESI processing Adduct ions bring the charge to the molecule and this charge converts it to an ion Default H
42. This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion Chromatogram pane Relative Intensity y axis Protein Deconvolution User Guide Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Table 32 Process and Review page information for ReSpect deconvolution Sheet 2 of 4 Parameter RT min x axis Description Displays the retention time of the spectrum which is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Deconvolved Spectrum pane Displays the deconvolved spectrum Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis Results table Displays the mass of the ions formed from molecules Displays the masses and intensities of the peaks that the ReSpect algorithm detected during the deconvolution along with their quality scores Average Mass Displays the calculated mass of a molecule based on the average atomic weight of each element Intensity Displays the sum of the intensities of the peaks for a
43. User Guide Indicates whether the data was acquired in positive charge mode or negative charge mode during the ESI process You might want to use this option when processing compounds that contain nucleotides like those found in DNA and RNA When these compounds are acquired in negative mode the resulting mass spectra are often clearer Deprotonation of nucleotides which are acidic occurs when the compound is dissolved in a basic solution and negative voltage is applied to produce negatively charged ions e Selected The data was acquired in negative charge mode e Default Cleared The data was acquired in positive charge mode IMPORTANT Do not select the Negative Charge check box if your data was acquired in positive mode Results will not be usable Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 3 of 10 Parameter Calculate XIC Description Optional Determines whether the Protein Deconvolution application calculates the extracted ion chromatogram from a range of deconvolved spectra This check box is not available for single spectra because there is no chromatogram e Default Selected Calculates the extracted ion chromatogram e Cleared Does not calculate the extracted ion chromatogram The application saves this setting to the m
44. a brief description of the method in the Descriptions box iv Click Save Comparing Samples in Saved Xtract Results You can compare a source spectrum to the reference spectrum in previously saved Xtract results When you load saved results the Sample Comparison page becomes available whether or not you used a reference spectrum If no reference spectrum appears on this page the method did not use a reference spectrum to generate the results When you load saved Xtract results you can select only a reference spectrum generated by the Xtract algorithm Similarly when you load saved ReSpect results you can select only a reference spectrum that was generated by the ReSpect algorithm You can select a new reference spectrum to show in the results but you cannot save it to the method You cannot delete the original reference spectrum that was saved in the results You cannot save the mirror plot that was generated with the new reference spectrum but you can copy its contents to the Clipboard to use in third party applications such as PowerPoint To view the original mirror plot that was saved with the results you must reload the results The Protein Deconvolution application does not update the report to be consistent with the contents of the modified mirror plot Sample Comparison Page Parameters for the Xtract Algorithm 82 Table 12 lists the parameters that are available on the Sample Comparison page for Xtract deconvolution
45. as input to the chromatogram The ReSpect algorithm ignores the portions of the spectrum outside this range The default values for both limits depend on the data in the raw data file The default is the entire chromatogram for the given spectrum Use Auto Spectral Averaging Determines whether the Protein Deconvolution application ignores all chromatographic peak picking when averaging spectra e Default for the ExampleMethodNativeMS method Selected Creates the averaged spectrum from all spectra in the specified retention time range so it creates only one deconvolution result from an automated run To support Native MS data resulting from direct infusion into the mass spectrometer in automated workflows use this option to automatically average scans from either the entire run or within a specified retention time range e Default for the default method Cleared Averages and deconvolves all spectra in the run This parameter is only available in the Auto ReSpect experiment type Apply Chromatogram pane Implements the parameter settings that you selected This button is only available if you changed any parameter settings in the Chromatogram Parameters pane Displays the chromatogram contained in the raw data file Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity RT min x axis Displays the retention time of
46. as shown in Figure 31 If you did not select the Calculate XIC check box on the Parameters page the application displays a chromatogram in the Chromatogram pane of the Process and Review page but does not display the extracted ion chromatogram for a given component The extracted ion chromatogram which is the shaded peak in Figure 31 indicates where the compound eluted It is unique to the Chromatogram pane on the Process and Review page Figure 31 Chromatogram pane showing an extracted ion chromatogram Chromatogram 100 NL 8 43E7 80 60 NL 1 847 40 20 Relative Intensity 2 4 6 8 10 RT min The Chromatogram pane also highlights in a red box the range of spectra used to produce the source spectrum for the deconvolved result Thermo Scientific Protein Deconvolution User Guide 59 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results When you enlarge the chromatogram on the Chromatogram page to include a specific retention time range the application likewise enlarges the extracted ion chromatogram to include the same retention time range on the Process and Review page However changes that you make in the Chromatogram pane are not reflected in the chromatogram on the Chromatogram page When you click an individual charge state of a selected isotopic cluster the chromatogram in the Chromatogram pane does not change The Chromatogram pane on the Process and Review pag
47. charge state Number of Charge States Displays the number of components in the list Mass Std Dev Displays the standard deviation in daltons of the delta masses for all the charge states of a component for example the standard deviation of Delta Mass Da PPM Std Dev Delta Mass Displays the standard deviation in parts per million of the delta masses for all the charge states of a component for example the standard deviation of Delta Mass PPM Displays the difference between the mass of a specific compound and the mass of the highest intensity compound Protein Deconvolution User Guide 171 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Table 32 Process and Review page information for ReSpect deconvolution Sheet 3 of 4 Parameter Relative Abundance Description Displays the peaks in the sample that are above the relative abundance threshold set by the Relative Abundance Threshold parameter on the Parameters page for information on this parameter see Table 27 on page 135 The Protein Deconvolution application assigns the largest peak in a deconvolved spectrum a relative abundance of 100 percent An abundance number in the Relative Abundance column represents the intensity in the same row of the Intensity column divided by the greatest intensity in the Intensity column multiplied by 100 For example if the largest peak in a deconvolved
48. deleting reference spectrum from 81 196 displaying 102 216 ExampleMethodNativeMS 121 218 existing 15 103 120 217 loading 15 103 120 217 parameters on Advanced Parameters ReSpect pane 128 139 parameters on Advanced Parameters Xtract pane 23 31 parameters on Automation Parameters pane 35 104 148 218 parameters on Main Parameters ReSpect pane 125 135 parameters on Main Parameters Xtract pane 19 27 parameters on Reporting Parameters pane 24 33 132 146 saving 25 26 133 134 saving isotope tables in 22 31 234 Protein Deconvolution User Guide Methods pane parameters on 17 123 purpose 14 102 119 216 Min Num Detected Charge parameter 22 30 Minimum Adjacent Charges parameter 121 130 144 157 218 Minimum Intensity parameter 23 33 Minimum Peak Significance parameter 128 139 mirror plot 76 77 191 192 Mirror Plot pane 76 84 85 191 199 200 Mirror Plot report section 95 209 Mode command 48 162 monoisotopic mass 2 19 64 Monoisotopic Mass column 66 68 Monoisotopic Mass for This Charge column 54 56 64 Monoisotopic Mass parameter 63 monoisotopic MH mass 19 20 Most Abundant Mass column 83 198 Mostabund m z column 54 55 64 multiple scans 41 154 Name column 17 123 Native MS data 121 218 Native MS method 4 Negative Charge parameter 21 28 125 135 Noise Compensation parameter 130 141 143 Noise Rejection parameter 128 140 Normal command 107 110 221 224 nucleotides deprotonation of
49. generates a report displaying several aspects of the deconvolution so that you can track the progression of the data For a description of this report see Displaying a ReSpect Deconvolution Report on page 200 You can view this report on the Reporting page and save it as a PDF file If you selected the Concatenate All Reports parameter in the Automation Parameters section of the Parameters page the Protein Deconvolution application opens one report for all the chromatographic peaks If you did not select this parameter it opens a report for each peak 228 Protein Deconvolution User Guide Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Loading Saved Results To display the report e On the Run Queue page select the completed job with the report that you want displayed and click Open Report From any other page click the Reporting tab The Protein Deconvolution application transfers you to the Reporting page which displays the report or reports Loading Saved Results You can reload the results of a previous deconvolution For instructions see Loading Saved ReSpect Results on page 212 Thermo Scientific Protein Deconvolution User Guide 229 Index A Abort All command 110 Abundance y axis label 94 208 Add to Method command 74 84 189 199 adduct ions 19 21 27 30 125 135 adjusting pane size 10 Adobe Acrobat toolbar 88 89 202 Advanc
50. highlighted in orange If you assign the same priority level to multiple jobs the Protein Deconvolution application prioritizes the jobs by the date and time that you submitted them to the queue To temporarily stop the processing in the run queue 1 Click Pause The Pause button is only available when the queue contains two or more jobs and the Status column shows Processing as shown in Figure 110 You cannot pause only a single job running in the queue The pause affects the next sample to be processed not the sample currently being processed Figure 110 Pausing a ReSpect job Protein Deconvolution ab reat rvinicim balance a i SCIENTIFIC C Method Selection f ERED C Parameters J chromatogram J Process and Review Sample Comparison J Reporting gt Process methods defined in the work queue Queue Manipulation Set Priority Open Result Open Report ERORE Number of Number Of Submit Time Method Name Raw Data File Chromatographic Components Completion Time me Peaks Detected 1 Normal 10 14 2013 4 24 08 PM i DefaultMethodReSpect C Program Files Protein Deconvolution source files RSP_AUTO g i _ E Processing el 2 Normal 10 14 2013 4 24 12 PM _DefaultMethodReSpect C Program Files Protein Deconvolution source files RSP_AUTO Queued Pause A confirmation box similar to that shown in Figure 111 appears Figure 111 Confirmation box i Analyzing C Program Files Protein Deconvolution source
51. in a charge state peak Source Spectrum 848 5576 100 942 7305 998 1242 737 9650 50 653 0065 1060 3785 1304 9187 1696 2056 1918 3199 1 E otal EAEE A or S 500 1000 1500 2000 miz Chromatogram pane When you click a charge state peak in the Results table and you selected the Calculate XIC check box on the Parameters page the Chromatogram pane displays the chromatogram overlaid onto the charge state peak s extracted ion chromatogram as shown in Figure 84 If you did not select the Calculate XIC check box on the Parameters page the application displays a chromatogram in the Chromatogram pane of the Process and Review page but does not display the extracted ion chromatogram for a given component The extracted ion chromatogram which is the shaded peak in Figure 84 indicates where the compound eluted It is unique to the Chromatogram pane on the Process and Review page Figure 84 Chromatogram pane showing an extracted ion chromatogram Chromatogram 100 NL 3 10E9 2 50 NL 1 089 w amp w 0 ie 1 2 3 4 5 6 RT min Protein Deconvolution User Guide 171 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results The Chromatogram pane also highlights in a red box the range of spectra used to produce the source spectrum for the deconvolved result When you enlarge the chromatogram on the Chromatogram page to include a specific
52. list by confidence level and m z range It deconvolves charge states in the list of filtered peaks to identify patterns of peaks that could represent individual components with series of associated charge states The ReSpect charge state deconvolution is somewhat liberal on purpose In particular it accepts a significant false positive rate to be sure that there are no false negatives For this reason the ReSpect algorithm leaves the scoring to a separate scoring algorithm that can distinguish which components are most reliable For more information on this capability see Calculating a Protein Quality Score on page 173 ReSpect accepts several parameters that control the peak detection process and the charge state deconvolution but the following are the most important M z Range Output Mass Range Target Mass Number of Peak Models Mass Tolerance Noise Rejection Resolution at 400 m z For information on these parameters see Creating a ReSpect Method on page 123 or Parameters Page Parameters for the ReSpect Algorithm on page 135 In addition for more information on the Output Mass Range parameter see Specifying the Output Mass Range on page 175 Setting Up a Manual ReSpect Protein Deconvolution First select the ReSpect deconvolution algorithm a raw data file and a method 1 bd 6 To set up a manual protein deconvolution with the ReSpect algorithm Start the Protein Deconvolution applicati
53. mass range these appearing and disappearing components can make the results unreliable For the initial processing select an output mass range that includes all of the expected components and their immediate neighbors You can narrow the mass range in subsequent runs For more information on the Output Mass Range parameter see Creating a ReSpect Method on page 123 or Parameters Page Parameters for the ReSpect Algorithm on page 135 Adjusting the Deconvolution Results Thermo Scientific If you are not satisfied with the deconvolution results you can adjust them To adjust the ReSpect deconvolution results 1 Do one of the following e Adjust the parameters in the Main Parameters ReSpect pane on either the Process and Review page or the Parameters page and click Apply or Protein Deconvolution User Guide 175 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results e Return to the Parameters page adjust the parameters in the Advanced Parameters ReSpect pane and click Apply 2 When you finish adjusting the parameters click Process on the Process and Review page again Obtaining the Best Results with the ReSpect Algorithm on page 157 offers some suggestions for obtaining better results when you use the ReSpect algorithm If you are satisfied with the results you can save them by using the procedure in Saving the Results of a Decon
54. negatives For this reason the Protein Deconvolution application applies a scoring algorithm to the ReSpect results to determine which components are the most reliable This scoring algorithm reduces the false positive rate by calculating a Bayesian fitness measure that distinguishes reliable components from components that could represent harmonics fortuitous fits to noise or both The algorithm compares the probability that a predicted component might produce peaks with an observed set of m z positions and an intensity profile to the probability that this particular pattern could occur by chance It then applies an additional set of penalties to exclude low intensity components harmonics and superfluous fits to successive charge states of successive valid components Thermo Scientific Protein Deconvolution User Guide 173 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results The score calculation algorithm does not require you to set any control parameters It copies any parameters that it might need from the corresponding control parameters used by the ReSpect algorithm or derives them from the ReSpect results themselves However you can use the Quality Score Threshold parameter to set a threshold value and exclude components with scores below the threshold value Protein scores are absolute rather than relative so that you can compare scores between spectra as well as compare scores
55. no obvious chromatographic peak change the limits of the m z Range parameter on the Parameters page to find it To shrink the view of the entire spectrum right click and choose Zoom Out e To reset the view to the original spectrum right click and choose Reset Scale 4 Create a spectrum in the Source Spectrum pane by doing one of the following e Fora single scan In the Chromatogram pane place the red cross shaped cursor on the chromatogram to select a single scan and to display the associated mass spectrum at that time point as shown in Figure 18 on page 42 You can use the left and right arrow keys to move to the previous or next time point in the chromatogram The spectrum window automatically updates or e For multiple scans Select a region of the chromatogram to display an averaged spectrum for all the scans in the selected region in the Source Spectrum pane i Right click and choose Mode gt Averaging if it is not already selected ii Drag the red cross shaped cursor across the area of interest The horizontal line of this cursor aids in assessing peak height The Protein Deconvolution application calculates an average spectrum for this interval The averaging method is better suited to complex data than the single scan method Averaging spectra produces higher signal to noise ratios so higher quality spectra are highly recommended for optimal deconvolution results Tip Optional You can perform step on
56. of mismatches between the model and the actual peak profiles Mass Tolerance is an important parameter in the ReSpect algorithm because it controls the tolerance for peaks in m z when the algorithm uses these peaks to fit to a particular component As you increase the value of this parameter the ReSpect algorithm expands the plausible set of charge state peaks but at the same time it correspondingly increases the false positive rate As described in Calculating a Protein Quality Score on page 173 the algorithm can exclude these false positives to some degree For most purposes the Ppm setting provides better results than the Da setting A Ppm setting between 20 and 50 is a good starting point e Ppm Specifies the mass tolerance in parts per million e Da Specifies the mass tolerance in daltons Default for the default method 20 ppm Default for the ExampleMethodNativeMS method 30 ppm Default for the default method 0 05 Da Default for the ExampleMethodNativeMS method 0 05 Da Target Mass 138 Protein Deconvolution User Guide Specifies an expected target mass in daltons to use in calculating the peak model This parameter is critical but does not have to be exact a value within 5 and 10 percent of the actual target is sufficient for best performance For samples where the range of masses is broad choose a mass somewhere in the middle of the range For example if the IgG light 20 kDa heavy chains 50 kDa an
57. on the Sample Comparison page You can collapse each of these panes by clicking the down arrow To expand a pane click the side arrow i You can also vertically resize many of the panes on the pages of the application except for those just listed To adjust the pane size vertically Drag the white splitter bar above each pane up or down Figure 6 shows the location of the splitter bars 10 Protein Deconvolution User Guide Thermo Scientific 1 Introduction Exiting the Protein Deconvolution Application Figure 6 Splitter bars used to size panes vertically Splitter bar Source Spectrum Chromatogram 2761 2698 100 100 NL 3 10E9 50 3535 4383 Relative Intensity o Oo 1000 1500 2000 2500 3000 3500 4000 1 2 3 4 a 6 miz RT min Deconvolved Spectrum 151814 375 Nu 47564 80 NL 4 7584 60 40 51560 414 149480 875 72950 086 707 695 100996 875 147350 594 Relative Intensity 20 16583 094 33177 289 20000 40000 60000 80000 100000 120000 140000 160000 Mass Results Splitter bar Exiting the Protein Deconvolution Application To exit the Protein Deconvolution application 1 Click the Close button amp in the upper right corner of the Protein Deconvolution window 2 If you clicked Process to produce deconvolution results and have not saved them the following prompt box appears Figure 7 Prompt to save results before exiting Save R
58. on these four panes see able 27 on page 135 To create a ReSpect method 1 Click the Parameters tab if it is not already selected The default settings for the ReSpect algorithm automatically populate the parameter boxes on the Parameters page as shown in Figure 65 Figure 65 Parameters page for ReSpect deconvolution e pa jicall FMO Protein Deconvolution DefaultMethodReSpect PE aa E E SCIENTIFIC IgG_source_cid raw Method Selection YJ Run Queue Chromatogram Process and Review Sample Comparison Reporting gt Set the parameters for the deconvolution Save Method Save Method As Reset Method Main Parameters ReSpect Negative Charge m z Range Min 1000 Max 4000 Apply a Charge Carrier H 1 00727663 Output Mes Pane Min Nex 2H 2 013553 5 z 10000 160000 Na 22 9892213 Mass Tolerance 30 D Custom Target Mass 150000 Da Rel Abundance Threshold 0 nn Charge State Range 10 S to 10 Calculate XIC V E Choice of Peak Model intact Protein Quality Score Threshold 0 X Advanced Parameters ReSpect Peak Filter Parameters Deconvolution Parameters Apply A Minimum Peak Significance 1 Standard Deviations Number of Iterations 3 Noise Rejection No Noise Rejection Noise Compensation _ 50 Confidence 68 Confidence Minimum Adjacent Charges 6 a to 10 5 95 Confidence 5 99 Confidence Peak Mo
59. or when you load an existing method and click Load Method on the Method Selection page the Protein Deconvolution application automatically transfers you to the Chromatogram page if the raw data file contains a chromatogram Use the Chromatogram page to select the best possible spectrum for the target protein for deconvolution To select the spectrum to deconvolve 1 Click the Chromatogram tab if it is not already selected Figure 68 shows the Chromatogram page Thermo Scientific Protein Deconvolution User Guide 149 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Figure 68 Chromatogram page for ReSpect deconvolution Thermo Protein Deconvolution pdefauimethoarespect Soiree atta iaie SCLENTLELG IgG_source_cid raw v Method Selection Run Queu o Parameters KA Chromatogram Process and Review 5S Sample Comparison Reporting gt Select the chromatogram to deconvolve Save Method Save Method As Reset Method Chromatogram Parameters Use Restricted Time Types TIC x Api Time Limits 0 00 to 6 23 Sensitivity High x Rel Intensity Threshold 1 Chromatogram m z Range 1000 0000 to 4000 0000 Use Auto Spectral Averaging F Chromatogram IgG_source_cid NL 3 10E9 F Relative Intensity 0 67 0 83 1 11 1 22 1 37 1 46 1 63 1 80 2 05 2 29 2 43 RT min Source Spectrum The Chromatogram page displays
60. permissible percentage of error allowed in calculating the ratio of the most abundant isotope to the next isotope higher in mass in the isotope series Default 3 Apply Implements the parameter settings that you selected in the Advanced Parameters Xtract pane This button is only available if you changed any parameter settings in the Advanced Parameters Xtract pane Reporting Parameters pane Determines which parts of the generated report the Protein Deconvolution application displays Sample Information Chromatogram With Parameters Thermo Scientific Determines whether to display the Sample Information section of the report For information on this section see Sample Information Section on page 89 e Default Selected Displays the Sample Information section of the report e Cleared Does not display the Sample Information section of the report Determines whether to display the Chromatogram Parameters section of the report For information on this section see Chromatogram Parameters Section on page 90 e Default Selected Displays the Chromatogram Parameters section of the report e Cleared Does not display the Chromatogram Parameters section of the report Protein Deconvolution User Guide 33 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 8 of 10
61. retention time range the application likewise enlarges the extracted ion chromatogram to include the same retention time range on the Process and Review page However changes that you make in the Chromatogram pane are not reflected in the chromatogram on the Chromatogram page When you click an individual charge state of a selected charge state peak the chromatogram in the Chromatogram pane does not change The Chromatogram pane on the Process and Review page does not display peak labels such as the retention time and scan number nor the header information for the source spectrum and chromatogram However it still displays the intensity of the most abundant peak in the entire LC MS run for the chromatogram e Deconvolved Spectrum pane When you click a charge state peak the Deconvolved Spectrum pane displays the deconvolved spectrum of the selected charge state peak as shown in Figure 85 Figure 85 Deconvolved Spectrum pane showing the deconvolved spectrum of a charge state peak Deconvolved Spectrum a 151814 500 NL 4 1261 NL 4 126 x aanas 50631 078 76266 430 6266 16583 es i _ z r 20000 40000 60000 80000 100000 120000 140000 160000 Mass 100999 445 Relative Intensity a When you click an individual charge state of a selected isotopic cluster the deconvolved mass of the specific component in the Deconvolved Spectrum pane changes It shows the mass for that single charge instead of the mass of th
62. saved from a given raw data file If the raw data file includes both ReSpect and Xtract saved results the Saved Results panes display both types of saved results The result file names appear in alphabetical order To load these results see Loading Saved ReSpect Results on page 212 To delete the results that appear in these panes see Deleting the Results of a Deconvolution on page 184 You can collapse each pane by clicking its down arrow When the list of files becomes longer than the visible pane can accommodate a vertical scroll bar appears so that you can scroll down to find a results file Exporting the Results of a Deconvolution You can export the results in the Results table to an Excel file a chromatogram source spectrum or deconvolved spectrum to a third party software file or the deconvolved spectrum mass and intensity data to an Excel or a CSV file To export the results in the Results table to an Excel file 1 Select a result then right click anywhere in the Results table 2 Choose one of the following Export All to store all the results in the Results table in a file or Export Top Level to store just the top level results in the Results table in a file The top level results are the data in these columns Average Mass Intensity Number of Charge States Mass Std Dev PPM Std Dev Delta Mass Relative Abundance Fractional Abundance RT Range Apex RT and Score They do not include the da
63. spectrometry The Use Relative Intensities parameter mainly acts as a confidence filter to distinguish the peaks in a peak table that are likely to be true signals from those likely to be noise artifacts For this purpose relative intensities perform at least as well as intensities and sometimes better Relative intensities are not suitable for quantitative comparisons of signal strengths so you should use intensities instead The Use Relative Intensities parameter is therefore not the default option for deisotoping This parameter is particularly useful with spectra where the noise level varies significantly across the spectrum especially for highly complex spectra for 150 kDa antibodies This noise is not a product of the instrument but is instead due to the high complexity of the sample e Default Selected Calculates the intensity of each peak relative to the noise level of the spectrum in the vicinity of the peak e Cleared Calculates global noise that is the noise across the entire spectrum Protein Deconvolution User Guide 141 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 8 of 15 Parameter Description Baseline Correction Displays parameters that identify and remove a baseline from the spectrum They perform three functions on a raw data file e Remove unwanted positiv
64. spectrum has an intensity of 1000 the Protein Deconvolution application assigns it a relative abundance of 100 percent If the next most abundant peak has an intensity of 500 the application assigns it an abundance of 50 percent 500 1000 x 100 50 Fractional Abundance RT Range Displays the fractional abundance of a component which is the abundance for that peak relative to the total abundance of all peaks in the spectrum expressed as a percentage The sum of all fractional abundances of all peaks in a deconvolved spectrum is 100 percent Displays the retention time range of the averaged source spectrum for the given component Apex RT Score Charge State Displays the retention time for the chromatographic peak when a component has a calculated extracted ion chromatogram XIC Displays the quality score of the deconvolved component For more information on how the Protein Deconvolution application calculates the quality score see Calculating a Protein Quality Score on page 173 Displays the imbalance between the number of protons in the nuclei of the atoms and the number of electrons that a molecular species or adduct ion possesses If the species possesses more protons than electrons its charge state is positive If it possesses more electrons than protons its charge state is negative Measured Average m z 178 Protein Deconvolution User Guide Displays the mass to charge ratio of
65. spectrum that you want to compare to the reference spectrum For best results with the automatic ReSpect workflow use the method to which you saved the reference spectrum to generate the source spectrum Figure 91 shows aa different portion of the spectrum that was deconvolved from the chromatogram in the IgG_source_cid raw file This portion is between approximately 3 5 and 3 6 minutes It is the source spectrum 190 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 91 Portion of the chromatogram used as a source spectrum Protein Deconvolution defauitmethodRespect thie eae otopini Aca eR SCLENTLELS IgG_source_cid raw _ Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting i Deconvolution completed successfully Process Save ResultAs Reset Method Save as Reference Y Main Parameters ReSpect Negative Charge m z Range Min 1000 Max 4000 rare a Charge Carrier H 1 00727663 R hin ics 2H 2 013553 REE PASS en ge aa 160000 Na 22 9892213 Mass Tolerance 30 Custom C Target Mass 150000 Da Rel Abundance Threshold 0 Charge State Range 10 E to 100 lal Calculate XIC v Quality Score Threshold 0 Choice of Peak Model Intact Protein v Results v Saved Xtract Results 2761 2705 2618 5390 323
66. the Run Queue page select the completed job with the results that you want displayed 2 Click Open Result The Protein Deconvolution application transfers you to the Process and Review page which displays the output spectrum in the Deconvolved Spectrum pane and the component list in the Results pane as shown in Figure 77 on page 166 It also displays the source spectrum in the Source Spectrum pane and the chromatogram in the Chromatogram pane In the Saved ReSpect Results pane it displays a result for each 226 Protein Deconvolution User Guide Thermo Scientific Result Names Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results peak shown in the Number of Chromatographic Peaks column on the Run Queue page For example if the Number of Chromatographic Peaks column displays 4 the Results pane on the Process and Review page displays four results unless one or more of the peaks did not deconvolve For more information on this page see Displaying the Results on page 165 To adjust the size of the panes on this page see Adjusting the Pane Size on page 10 The Save Method Save Method As and Result Method commands are not available when you access the Chromatogram page and the Process Save Result As and Reset Method commands are not available when you access the Process and Review page from the Open Result command on the Run Queue page
67. the peak in the source spectrum Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Table 32 Process and Review page information for ReSpect deconvolution Sheet 4 of 4 Parameter Measured Average Mass Description Displays the mass calculated from the measured mass to charge ratio and the charge state It represents the deconvolved mass for a particular charge state Delta Mass Da Displays the difference between the average mass for a component and the calculated mass for that charge state in daltons Delta Mass PPM Displays the difference between the average mass for a component and the calculated mass for that charge state in parts per million Process and Review Page Menu Bar Commands Table 33 describes the commands on the menu bar of the Process and Review page Table 33 Menu bar commands on the Process and Review page for ReSpect deconvolution Parameter Process Description Deconvolves the spectrum and displays the results in the Results table Save Result As Saves the current deconvolution results in an SQLite file Reset Method Returns the parameters on the Process and Review page to the settings that you first chose when you loaded the raw data file Save As Reference Thermo Scientific Saves the selected deconvolved spectrum to the reference spectrum library along with the associ
68. the spectrum which is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Source Spectrum pane 160 Protein Deconvolution User Guide Displays the spectrum that you selected Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Table 29 Chromatogram page parameters for ReSpect deconvolution Sheet 4 of 4 Parameter Relative Intensity y axis Description Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion Chromatogram Page Menu Bar Commands Table 30 describes the commands on the menu bar of the Chromatogram page Table 30 Menu bar commands on the Chromatogram page for ReSpect deconvolution Parameter Save Method Description Saves the current parameter values to the existing method overwriting any previous values Clicking Save Method automatically advances the application to the Process and Review page Save Method As Saves parameter values to a new method Clicking Save Method As automatically advances the application to the Process and
69. to 0 for the chosen component and recalculates the delta mass value for all other components in the Results table for a given deconvolved spectrum This parameter does not affect other results that might be concurrently loaded Export Top Level Results table only Exports the top level results in the Results table to a file The top level results are the data in these columns Average Mass Intensity Number of Charge States Mass Std Dev PPM Std Dev Delta Mass Relative Abundance Fractional Abundance RT Range Apex RT and Score They do not include the data in the columns that appear when you click the symbol to the left of the No column Export All Results table only Protein Deconvolution User Guide Exports all results in the Results table to a file Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Saving the Results of a Deconvolution Saving the Results of a Deconvolution Thermo Scientific You can save the results of the deconvolution in a file in the same directory where you stored the raw data files You can also copy and paste any one of the views in this window to a PowerPoint presentation file To save the results of the deconvolution 1 Click Save Result As 2 In the SaveAs dialog box do the following a Inthe Result Name box type the name of the results file or select an existing file to overwrite from the dropdown menu b
70. was acquired in positive charge mode IMPORTANT Do not select the Negative Charge check box if your data was acquired in positive mode Results will not be usable Charge Carrier Specifies the adduct ions used during ESI processing Adduct ions bring the charge to the molecule that converts it to an ion Default H 1 00727663 Specifies that the adduct was hydrogen 2H 2 013553 Specifies that the adduct was deuterium Na 22 9892213 Specifies that the adduct was sodium Custom Specifies that the adduct was a charge carrier other than hydrogen deuterium or sodium When you select this option a box opens so that you can type the mass of the custom charge carrier Note In negative mode these adduct ions correspond to deduct rather than adduct masses Rel Abundance Threshold Specifies a threshold below which the Protein Deconvolution application filters out data for data reporting This option sets a relative threshold as a percentage of the most abundant component in the spectrum The most abundant peak in the deconvolved spectrum has a relative abundance of 100 percent and all other peaks are calculated relative to that one In the Results pane on the Process and Review page the application shows only those components that are greater than or equal to this relative abundance threshold in the deconvolved spectrum For example if the highest peak has an absolute abundance of 1000 the relati
71. 0 resizing 10 Main Parameters Xtract report section 24 34 93 manual protein deconvolution methodology 4 ReSpect algorithm 17 117 122 setting up with ReSpect 118 setting up with Xtract 14 Xtract algorithm 13 17 122 manual workflow for Protein Deconvolution application 5 Mass Std Dev column 177 180 182 Mass Tolerance parameter 121 127 138 157 218 Mass x axis label Deconvolved Spectrum pane of the Process and Review page 62 177 Deconvolved Spectrum report section 94 208 Protein Deconvolution User Guide 233 Index N Mirror Plot section of report 95 209 Sample Comparison page 84 199 Measured Average m z column 168 178 Measured Average Mass column 168 179 Method Name parameter 110 225 Method Reference Spectrum pane deleting reference spectrum from method 81 196 parameters on 82 197 populating fields of 75 190 Sample Comparison page 75 190 Method Selection page Experiment Types pane parameters on 17 122 ReSpect 119 216 Xtract 14 102 Load Raw Data File pane parameters on 17 123 216 ReSpect 119 Xtract 14 102 Methods pane 102 119 parameters on 17 123 ReSpect 216 Xtract 14 parameters on 16 122 selecting algorithm raw data file and method 14 102 119 216 methods adding reference spectrum to 74 189 changing reference spectra in 196 creating in ReSpect algorithm 121 123 in Xtract algorithm 16 18 103 217 creating while running automatic deconvolution 105 220 default 15 16 19 85 119 121
72. 0727663 Min Num Detected Charge 1 Advanced Parameters Xtract Section The Advanced Parameters Xtract section shown in Figure 49 displays the parameter settings that you selected in the Advanced Parameters Xtract pane of the Parameters page for the deconvolution For information on these parameters see Table 4 on page 27 Figure 49 Advanced Parameters Xtract section for Xtract deconvolution Advanced Parameters Xtract Fit Factor 44 Remainder Threshold 25 Consider Overlaps True Charge Range 1 50 Minimum Intensity 1 Expected Intensity Error 3 Thermo Scientific Protein Deconvolution User Guide 93 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Deconvolved Spectrum Section The Deconvolved Spectrum section shown in Figure 50 displays the same information that appears in the Deconvolved Spectrum pane of the Process and Review page Figure 50 Deconvolved Spectrum section for Xtract deconvolution Deconvolved Spectrum 16940 9862 100 abundance 0 5000 10000 15000 20000 25000 30000 35000 40000 mass Table 19 lists the parameters in the Deconvolved Spectrum pane Table 19 Deconvolved Spectrum section parameters for Xtract deconvolution Parameter Description Abundance y axis Displays the relative peak abundance Mass x axis Displays the actual mass of an ion in atomic mass units 94 Protein Deconvolutio
73. 1 S 5 16568 8381 26967 16 3 6055 3 303 3 969 3619 Output values of Calculated Mana ic Mass ee Normalized a the deconvolution 1184 4963 16568 7910 1185 2076 45195 83 10 0 00 0 00 15 1105 5970 16568 8037 1106 2642 935 84 91 20 0 00 0 00 Values for individual 16 1036 5602 16568 7782 1037 1814 1 659 96 93 20 0 00 0 00 r isotopic clusters e The Charge State column lists the charge states of the individual isotopic clusters that constitute the total number shown in the Number of Charge States column The Mostabund m z column displays the mass to charge ratio of the most abundant isotope or the height of the tallest peak in the isotopic distribution When you click a value in this column the application automatically highlights the corresponding spectral line in the Source Spectrum pane as shown in Figure 26 You might have to right click and choose Reset to Scale to see the spectral line In Figure 26 a blue spectral line marks the selected value of 1129 8672 in the Mostabund m z column Protein Deconvolution User Guide 55 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 26 Highlighted spectral line in the Source Spectrum pane corresponding to a selected value in the Mostabund m z column SCIENTIFIC Protein Deconvolution DefaultMethodxtract isla o Help 3 Myoglobin_30pmol_michrom_protein_microtrap_limin_OT_60K_LRAW Deconvolve the spec
74. 1 534 6 00 4 an T 7 or oT 1000 1500 2000 2500 3000 3500 4000 7 2 3 4 5 6 miz RT min Deconvolved Spectrum NL is 151814 672 sped ML 80 2 09E8 5 Y Saved ReSpect Results g 40 DefaultMethodReSpect_1089_1 amp 3 n 51560 344 149505 563 DefaultMethodReSpect_1129_1 20 en maw wazia le Soe7y 424 60244 000 79440 102 149505 DefaultMethodReSpect_36_57_5 od 18827 Aes al l ote 3 f E 1 DefauttMethodReSpe TE 20000 40000 60000 ra 100000 20000 140000 160000 DefaultMethodReSpect_844_87 DefaultMethodReSpect_893_90 umber o z F D jelative ractional eT Average Mass Intensity Chavet Mass Std Dev PPM Std Dev Delta Mass prerane rrr RT Range S Eh 151814672 368 946 048 00 46 175152 3 304 3 630 0 tout v al eso fth e 151977 781 311 738 33600 44 14 7993 3 304 3 630 utpu u 152138 ARR PRARATAARND AA 113769 3304 3 63N deconvolution he 166 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results The range shown in the Deconvolved Spectrum pane is determined by the setting of the Output Mass Range parameter on the Parameters or Process and Review page Using the same settings of this parameter you can deconvolve several spectra with the exact same range and compare them For information about this parameter see Specifying the Output Mass Range on page 175 and Creating a ReSpect Me
75. 151814 672 Soas w 80 3 69E8 2 2 S 60 E 40 3 oe 51560344 149595 563 Y Saved ReSpect Results 30895424 37290301 maia 100997 500 ese 16527 43 S 51560843 60244 000 2902 140505 4 DefaultMethodReSpect_1089_109 i kga 7 fi la 7 r r i 20000 40000 60000 80000 100000 120000 140000 160000 DefaultMethodReSpect_1129_1314 Mass DefaultMethodReSpect_36_57_10 Results DefaultMethodReSpect_68_356_10 Number of Relative Fractional Charge States benii Wie Abundance Abundance 368 946 048 00 46 1240 0 00 100 0000 17 5152 311 738 336 00 44 1481 163 11 84 4943 14 7993 239 647 968 00 46 21 78 324 02 64 9547 11 3769 238 401 136 00 47 19 58 159 77 64 6168 113178 39 3889 498 151651 140 3 766 3792 848 151673 615 3611 796 151653 143 3527 813 151652 635 lt Average Mass Intensity DefaultMethodReSpect_844_874_1 G 3 304 3 630 3 304 3 630 3 304 3 630 3 304 3 630 151814 672 151977 781 152138 688 151654 906 Charge State DefaultMethodReSpect_893_900_1 DefaultMethodReSpect_900_942_1 amp DefaultMethodReSpect_942_1020 ReSpect3537 Line highlighting the measured average m z corresponding to an individual charge state Line highlighting the calculated mass corresponding to an individual charge state Selected charge state in the Charge State column For more information on the columns in the Results table see Table 32 on page 176 To display the results in the Source Spectrum Chromato
76. 151977 781 311 738 336 00 44 225 1481 163 11 844943 14 7993 3 304 3 630 340 0 utp ut va ues of 3 152138 688 239 647 968 00 46 331 2178 324 02 64 9547 11 3769 3304 3630 340 i a 4 151654 906 238 401 136 00 47 297 19 58 159 77 64 6168 113178 3 304 3 630 340 the deconvol ution a 5 152300 922 105 077 056 00 46 24 486 25 28 4803 4 9884 3 304 3 630 339 m 3542 872 152300 197 0 725 4 762 44 3462 689 152313 984 13 062 85 762 45 3385 528 152303414 2492 16363 Charge states of the 45 3311 708 152292 245 8677 56 973 7 output values 168 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results e The Charge State column lists the individual charge states e The Average Mass column displays the calculated mass of a molecule based on the average atomic weight of each element When you click on a value in this column the application automatically highlights the corresponding mass in the Deconvolved Spectrum pane with a blue line as shown in Figure 80 You might have to zoom in to see the highlighted line for instructions see Selecting the Spectrum to Deconvolve on page 37 In the Source Spectrum pane the application highlights all the charge states corresponding to the selected average mass as shown in Figure 80 Figure 80 Highlighted average mass and corresponding charge states rmo Protein Deconvolution sa Cacia Data e
77. 1min_OT_60K_1 166 RT 3 65 NL 4 19E5 F FTMS p ES Full ms 300 00 2000 00 ae 892 2206 90 2 707 3400 998 0697 70 A so 1060 3777 S 40 628 8972 0 1211 8618 at i 1413 6667 1696 0089 4995 3561 0 400 600 300 1000 1200 1400 1600 1800 2000 m z Table 18 lists the parameters in the Source Spectrum section Table 18 Source Spectrum section parameters for Xtract deconvolution Parameter Description Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion 92 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Main Parameters Xtract Section The Main Parameters Xtract section shown in Figure 48 displays the parameter settings that you selected on the Parameters page for the deconvolution For information on these parameters see Table 4 on page 27 Figure 48 Main Parameters Xtract section for Xtract deconvolution Main Parameters Xtract Output Mass M Resolution at 400 m z 60000 S N Threshold 3 Rel Abundance Threshold 0 Negative Charge False m z Range 300 2000 Charge Carrier H 1 0
78. 20 1 448 60 2531 273 151815 940 1 393 9 179 61 2489 798 151816 255 1 708 11 250 62 2449 636 151814 980 0 433 2 853 63 2410 739 151813 115 1 432 9 433 64 2373 096 151813 706 0 841 5 538 65 2336 606 151813 946 0 601 3 958 Source Spectrum Evidence lgG_source_cid 1059 1340 RT 3 26 3 62 AV 281 A 2761 2685 100 90 20 70 2 2 60 S E 50 B 40 4 U 20 10 1659 484 Vy 3535 4365 1383 1405 i ANAI AWWA a bn 1000 1500 2000 2500 3000 3500 miz Thermo Scientific Protein Deconvolution User Guide 211 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Loading Saved ReSpect Results Table 43 lists the parameters in the Source Spectrum Evidence section Table 43 Source Spectrum Evidence section parameters for ReSpect deconvolution Parameter Description Relative intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules Loading Saved ReSpect Results If you saved the results of a ReSpect deconvolution you can reload them at a later time You can load multiple results as long as they are from a single SQLite file You can load multiple Xtract results or multiple ReSpect results but not both types of results at the same time For information on comparing samples using saved ReSpect results see Comparing Samples
79. 21 125 135 generating isotope tables 22 31 specifying peak models for use with 128 139 Number of Charge States column 63 66 68 177 180 182 Number of Chromatographic Peaks parameter 106 111 220 225 Number of Components parameter 106 111 221 225 Number of Iterations parameter 129 143 Number of Peak Models parameter 130 144 Number of Peaks column 83 198 0 Open Report command 110 114 224 225 Open Result command 110 112 224 226 Orbitrap mass spectrometers defining resolution of source spectrum 20 27 131 145 specifying peak model for data produced by 128 139 using the Protein Deconvolution application with 8 Original File Name column 90 203 Thermo Scientific Output Mass parameter 19 27 Output Mass Range parameter default and ExampleMethodNativeMS settings in method 121 218 obtaining best results in ReSpect 157 specifying the output mass range 126 137 167 175 outputs of Protein Deconvolution application 8 P pane size 10 Parameters page Advanced Parameters ReSpect pane 123 128 139 Advanced Parameters Xtract pane 18 23 31 Automation Parameters pane ReSpect 124 148 218 Xtract 18 35 104 Main Parameters ReSpect pane 123 125 135 Main Parameters Xtract pane 18 19 27 menu bar commands on 36 149 Reporting Parameters pane ReSpect 123 132 146 Xtract 18 24 33 setting parameters for ReSpect deconvolution 123 135 setting parameters for Xtract deconvolution 18 27 Pause butt
80. 216 752 20 16 20 60 17 97 68 6566 28 9794 3 303 3 969 3 653 33 454 02 3 303 3 969 3 658 14 20 34 372 17 8 5419 Calculated Monoisotopic Mass Charge Normalized Stat si z Charge State joncisotopic m z for This Charge Mostabund m Intensity Fit Fit Left Fit Right 1184 4963 16568 7910 1185 2076 451 95 83 10 0 00 0 00 1105 5970 16568 8037 1106 2642 935 84 91 20 0 00 0 00 1036 5602 16568 7782 1037 1814 1 659 96 93 20 0 00 0 00 z 56 Protein Deconvolution User Guide e The Monoisotopic Mass for This Charge column displays the calculated monoisotopic mass for each charge state When you click a value in this column the application automatically zooms the mass to charge ratio of the selected charge state in the Deconvolved Spectrum pane as shown in Figure 27 highlighting it with a blue line You might have to zoom in to see the highlighted line for instructions see Selecting the Spectrum to Deconvolve on page 37 Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 27 Highlighted mass to charge ratio of the selected charge state Protein Deconvolution detauitmethoaxtract Hairaani Hep SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_LRAW Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting Deconvoive the spectrum Process Save Res
81. 3 Custom Min Num Detected Charge 3 Isotope Table Protein Chromatogram 893 1164 RT 5 90 S 268 6 75 7 40 8 47 8 94 9 64 5 6 7 RT min 1304 9242 1696 2001 1918 3288 1500 2000 1 Saved ReSpect Results Y S 16941 0113 16568 8381 15635 2961 T T 16000 T T T T T T T T 7000 9000 10000 12000 13000 14000 15000 17000 po 11000 Mass Load Result Thermo Scientific Relative Abundance Fractional Abundance lonoisotopi Monoisotopic Delta Mass Number of Charge Average Charge Sum Intensity mene RT Range Output values of the deconvolution 16941 0113 16923 0410 16922 8878 315 704 75 216 752 20 33 454 02 3 303 3 969 3 303 3 969 3 303 3 969 The Source Spectrum pane on the Process and Review page shows the same spectrum as the Source Spectrum pane on the Chromatogram page The Chromatogram pane on the Process and Review page shows the same chromatogram as the Chromatogram pane on the Chromatogram page R Kod To display the results in the Results table 1 Optional If necessary expand the Results table by following the instructions in Adjusting the Pane Size on page 10 2 Optional Select the columns that you want to display as follows a Click the Column Chooser icon Eg b In the Column Chooser dialog box shown in Figure 24 select the check boxes corresponding to
82. 304 3 630 120026405 elected average mass in 152477 828 19 926 696 00 663 16 z 3 304 3 630 149635 219 446 253 97 t 2 17945 3 304 3 630 f the Average Mass col umn 154314 063 1 384 044 25 2 499 39 3 304 3 630 RT Range DefaultMethodReSpect_893_900_1 ReSpect3537 When you click one of the charge states in the Charge State column the application highlights the corresponding calculated mass in the Deconvolved Spectrum pane as shown in Figure 81 In the Source Spectrum pane it highlights the corresponding value of the Measured Average m z column Thermo Scientific Protein Deconvolution User Guide 169 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Figure 81 Highlighted measured average m z corresponding to an individual charge state LL The Protein Deconvolutio Manual t sotopically Unresolved ual ReSpect Isotopically Ten IgG source cid raw Thermo Protein Deconvolution SCIENTIFIC E Process and Review C Method Selection Run Queue Parameters _ Chromatogram Sample Comparison _ Reporting Deconvolve the spectrum Process Save Result As Reset Method Save as Reference Main Parameters ReSpect Results Source Spectrum Chromatogram NL 3 1060 Saved Xtract Results i 2761 2696 109 NL 8 0868 io AN 4 35 i IN 0 4 2 3 4 5 6 RT min Deconvolved Spectrum ML aii
83. 3630 2574 1153 3102 5458 3231 1631 2339 1700 3374 6319 2169 7441 3535 4380 1199 9394 1383 1409 1508 7423 1659 4854 1947 3279 3707 8040 3952 6229 T T T T T T T T T T T 4 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 miz The Source Spectrum pane shows the spectrum either single scan or averaged to be deconvolved It displays apex information for major peaks and m z information for deconvolved components It also shows peak apex information as a marker along with an accompanying label that describes the m z value for the most abundant peak from the spectrum at that retention time In single scan processing mode the most abundant m z value for a component should agree with the m z value shown for the corresponding peak in the source spectrum In averaged scan processing mode the two values might be different because of the way the application displays averaged spectra However this difference should be small approximately 0 001 The Xtract algorithm can deconvolve centroid spectra and profile spectra The ReSpect algorithm can deconvolve only profile spectra e Centroid data represents mass spectral peaks in terms of two parameters the centroid the weighted center of mass and the intensity the normalized area of the peak The data is displayed as a bar graph of relative intensity versus m z Thermo Scientific Protein Deconvolution Use
84. 4 4772 1659 4858 2141 4542 RT min 151813 578 Saved ReSpect Results Relative Intensity 149771 016 74697 758 30956 70 39559 047 100992 430 25176 420 85311 563 Number of T a Relative Fractional Average Mass Intensity Chaos SiMe Mass Std Dev PPM Std Dev Delta Mass Aes res RT Range 151813 578 196 401 808 00 46 100 0000 13 9820 3 497 3 604 0 151976 141 167 505 696 00 41 85 2872 11 9248 3 497 3 604 340 7 2 Click the Sample Comparison tab 3 Select the appropriate reference spectrum from the Reference Spectrum Library by double clicking the spectrum or selecting the spectrum and then clicking Select The Mirror Plot pane now displays a mirror plot with the reference spectrum in the negative direction and the source spectrum in the positive direction as shown in Figure 92 Text at the top of the pane identifies the name of the source data file and text at the bottom of the pane identifies the name of the reference data file Thermo Scientific Protein Deconvolution User Guide 191 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 92 Mirror plot showing the source spectrum and the reference spectrum M ReSpect topically Us solved MO Protein Deconvolution cefauitmethoarespect os peonia reste ai SCIENTIFIC IgG_source_cid raw Method Selection Run Queue _ Parameters Chromatogram Proces
85. 4 6136 spectrum 25715 8758 n displayed as a source spectrum Relative Intensity The Sample Comparison page contains three panes e Method Reference Spectrum Displays information about the reference spectrum in the currently loaded method e Reference Spectrum Library Displays commands that allow you to select reference spectra to show in the mirror plot display information about specific reference spectra add reference spectra to the method and delete reference spectra from the library e Mirror Plot Displays the mirror plot of the selected reference spectrum and the source spectrum For detailed information on the commands and parameters on the Sample Comparison page see Table 12 on page 82 7 If you want to make this reference spectrum available in the method that you used to deconvolve the reference spectrum select the spectrum in the Reference Spectrum Library and click Add to Method in the Reference Spectrum Library pane 74 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples You cannot add more than one reference spectrum to a method To delete the reference spectrum in a method see To delete a saved reference spectrum from a method on page 81 To change the reference spectrum in a method see To change a reference spectrum in a method on page 81 The reference spect
86. 5 Na 22 9892213 Rel Abundance Threshold 0 Custom Negative Charge F Min Num Detected Charge 3 Calculate XIC F Isotope Table Protein x Y Saved Xtract Results 1543 9414 1634 8177 RT 9 38 RT 15 15 RT 28 72 S 101 S 154 17 85 22 71 25 91 S 281 31 77 15 20 RT min 27756 7570 Y Saved ReSpect Results 26266 2459 Relative Intensity Relative Fractional Abundance Abundance 27756 7570 7 268 8 403 27934 8388 108 i 7 268 8 403 2 Click Save As Reference in the upper right corner of the Protein Deconvolution window Note The Save As Reference command is unavailable if a deconvolved spectrum is not visible in the Deconvolved Spectrum pane or if you have not yet deconvolved the spectrum The SaveAs dialog box appears as shown in Figure 36 72 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 36 SaveAs dialog box 4 Saves x Name Descriptions Cancel 3 In the Name box of the dialog box type the name of the reference spectrum 4 In the Descriptions box type a brief description of the reference spectrum 5 Click Save 6 Optional Click the Sample Comparison tab Figure 37 shows the Sample Comparison page The reference spectrum that you just saved appears in the Referen
87. 5 90 7 27 840 27756 757 01 02 2014 15 53 47 Select Reference Spectra Raw Data File Scan Range RT Range Creation Time Descriptions Delete Add to Method Show Details Mirror Plot Source Data File C Program Files Protein Deconvolution source files Xtract PSA_240_highpl raw NL 7 85E 005 400 28412 9569 80 28121 8589 60 vi Source 2 a i be at 25714 8120 spectrum 2 J 2 20 25715 8758 EE a Reference g ag 26266 2459 c 60 80 spectrum 100 27756 7570 Mass Reference Data File C Program Files Protein Deconvolution source files Xtract PSA_240 raw From Library NL 6 71E 004 Note If you try to compare a spectrum generated by an Xtract algorithm to a spectrum generated by a ReSpect algorithm an error message appears The mirror plot is scaled to 100 percent in both directions but the actual intensities of the spectra can be completely different You can tell the different intensities of the peaks in each spectrum by looking at the NL values see Source Spectrum Pane Header on page 49 for the source and reference spectrum If the reference spectrum and the source spectrum do not cover the same mass range the Protein Deconvolution application automatically expands the range in the mirror plot to include the lowest limit and the highest limit of both spectra For example if the reference spectrum has a range of 25 000 to 160000 m g and the source spectrum has a range of 15 000 to 100000 m z the appl
88. 6 7 55 8 14 8 47 8 81 9 06 9 33 10 00 10 27 10 58 85 90 95 10 0 10 5 Bd BB T 05 1 0 15 2 0 25 3 0 35 40 45 5 0 55 6 0 65 7 0 75 8 0 RT min Source Spectrum Thermo Scientific Protein Deconvolution User Guide 37 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve 38 The Chromatogram page displays three panes Note To size these panes vertically see Figure 6 on page 11 e Chromatogram Parameters Contains parameters that you can use to adjust the view in the Chromatogram pane Chromatogram Displays a chromatogram of the data in the raw data file A chromatogram view shows the intensities of one or more masses as a function of time By default the Chromatogram pane displays a total ion current TIC chromatogram as shown in Figure 14 The application s internal peak selection mechanism selects the chromatographic peaks as described in Chromatographic Peak Detection and Spectral Peak Modeling on page 5 The chromatogram is fully magnified You can use the zooming and averaging functions in this pane to generate a spectrum for instructions see step 3 on page 40 Source Spectrum Displays the spectrum to deconvolve either single scan or averaged that you selected in the Chromatogram pane The mass spectrum in this pane is empty until you select a region in the chromatogram 2 Optional Use the parameters in the Chromatogra
89. 65 9744 7016 24 6 19 98 75 0377 2 1526 16941 2754 6766 36 2 14 56 0 2633 2 0760 16849 9721 5527 58 8 22 05 91 0400 1 6959 16879 9855 5474 79 6 19 19 61 0266 1 6797 16904 9683 4928 46 3 15 24 36 0438 1 5121 5626 6690 4747 77 4 7 14 11314 3431 1 4567 16882 9969 4633 39 4 21 83 58 0152 1 4216 5622 3267 4239 29 3 7 67 11318 6854 1 3006 16884 0017 3892 93 4 21 34 57 0104 1 1944 16907 9693 3881 22 3 17 11 33 0428 1 1908 17020 9404 3732 06 7 20 61 79 9283 1 1450 5616 6621 3705 58 4 7 17 11324 3500 1 1369 16998 9656 3559 40 5 19 81 57 9535 1 0921 16895 0100 3480 40 4 23 44 46 0021 1 0678 Source Spectrum Evidence Section The Source Spectrum Evidence section shown in Figure 53 displays a table and an accompanying graph for every component in the sample This section appears only if you select the Component Detail Tables parameter and the Component Source of Evidence Plots parameter on the Parameters page The table shows all the charge states that the Protein Deconvolution application detected for that component It displays the same parameters as those displayed in the Results pane on the Process and Review page For information on these parameters see Table 9 on page 62 The graph shows the isotopic clusters that are associated with a particular component The table shown in Figure 53 shows only a partial list of values 96 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra wit
90. DefaultMethodXtract Default method for Xtract 7 Select Raw Data Files Manual ReSpect Isotopically Unresolved Auto Xtract Isotopically Resolved gt Auto ReSpect Isotopically Unresolved Load Results 4 In the Methods pane specify the extraction method to use by doing one of the following e Ifthe default method or one of the existing methods contains the appropriate parameters and you do not want to make any changes to it select the name of the method of interest go to the Load Raw Data File pane and follow the instructions from step 8 e Ifyou want to create a new method select the Manual Xtract Isotopically Resolved experiment type and follow the instructions in Creating an Xtract Method on page 18 to set the parameters on the Parameters page Before you leave the Parameters page follow the instructions from step 5 5 On the Parameters page set the parameters that control the presentation of the output a Click the Parameters tab Figure 11 on page 19 shows the Parameters page for Xtract deconvolution Thermo Scientific Protein Deconvolution User Guide 103 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up an Automatic Xtract Protein Deconvolution 104 b C In the Automation Parameters pane of the Parameters page set the following output e Save to PDF Saves the automatically generated deconvolution r
91. Figure 1 Isotopically resolved mass spectrum Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 161 180 RT 3 57 3 93 AV 19 F FTMS p ESI Full ms 300 00 2000 00 808 1964 J 204 808 1053 808 2442 J 807 3462 a 60 807 4373 808 0575 2 807 4849 808 3397 407 808 3831 a J 807 2032 807 5326 808 0098 TE 2 w 807 1599 807 9621 808 4352 807 6280 7 805 7276 805 9135 806 2508 OTe 806 7701 asn we A ree 808 8132 809 1958 809 5265 EE EN OO A LID DDD Aone poe AAA F oe to DDO ALARA A Pr pens AA 8058 806 0 8062 8064 806 6 8068 807 0 807 2 807 4 807 6 807 8 808 0 808 2 8084 8086 808 8 8090 8092 8094 miz ReSpect Algorithm The ReSpect algorithm from Positive Probability Ltd PPL is a robust and efficient data fitting method that deconvolves isotopically unresolved complex mass spectra from biomolecules such as small and large proteins to the neutral average mass of each molecule It determines the m z ratio of every peak in an ESI mass spectrum and evaluates all possible charge states for any particular peak as determined by the mass ranges For analyzing spectra the ReSpect algorithm includes an optional spectrum preconditioning method including automated baseline subtraction and a number of automated and semiautomated peak modeling facilities The ReSpect algorithm first performs a baseline subtraction Next it performs a peak deconvolution to produce a list of peaks and then it filters these peaks Lastly it performs a c
92. For information on this section see Sample Information Section on page 89 Default Selected Displays the Sample Information section of the report Cleared Does not display the Sample Information section of the report e Chromatogram with Parameters Determines whether to display the Chromatogram Parameters section of the report For information on this section see Chromatogram Parameters Section on page 90 Default Selected Displays the Chromatogram Parameters section of the report Cleared Does not display the Chromatogram Parameters section of the report e Source Spectrum Determines whether to display the Source Spectrum section of the report For information on this section see Source Spectrum Section on page 92 Default Selected Displays the Source Spectrum section of the report Cleared Does not display the Source Spectrum section of the report e Deconvolution Parameters Determines whether to display the Main Parameters Xtract and Advanced Parameters Xtract sections of the report For information on these sections see Main Parameters Xtract Section on page 93 and Advanced Parameters Xtract Section on page 93 Default Selected Displays the Main Parameters Xtract and Advanced Parameters Xtract sections of the report Cleared Does not display the Main Parameters Xtract and Advanced Parameters Xtract sections of the report 24 Pro
93. Isotopically Automatically detects chromatographic peaks creates an Resolved averaged spectrum from all the mass spectra within the chromatographic peak and deconvolves an isotopically resolved mass spectrum with the Xtract algorithm Auto ReSpect Isotopically Automatically detects chromatographic peaks creates an Unresolved averaged spectrum from all the mass spectra within the chromatographic peak and deconvolves an isotopically unresolved mass spectrum with the ReSpect algorithm Load Results Loads the saved results of a previous deconvolution 122 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Load Raw Data File Pane Parameters Table 25 lists the parameters in the Load Raw Data File pane of the Method Selection page Table 25 Load Raw Data File pane parameters Parameter Description Raw Data Directory Specifies the directory where the raw data file containing the spectrum to deconvolve is located Select Raw Data Files Specifies the name of the raw data file containing the spectrum to deconvolve Load Loads the specified raw data file Methods Pane Parameters Table 26 lists the parameters in the Methods pane of the Method Selection page Table 26 Methods pane parameters Parameter Description Name Specifies the name of the method to use in the deconvolution Descripti
94. MS Select Raw Data Files ExampleMethodNativeMS IgG_5ug_SIM qb raw IgG_source_cid qb raw IgG_source_cid raw Create Method 5 In the Methods pane specify the extraction method to use by doing one of the following 120 Protein Deconvolution User Guide If one of the existing default methods contains the appropriate parameters select the name of the method of interest and click Load Method The Protein Deconvolution application automatically transfers you to the Chromatogram page if the raw data file contains a chromatogram or to the Process and Review page if it contains a spectrum only To use the Chromatogram page follow the instructions in Selecting the Spectrum to Deconvolve on page 149 To use the Process and Review page follow the instructions in Deconvolving the Spectrum on page 163 If you want to make changes to an existing method do the following i Select the name of the method of interest and click Load Method ii From the Chromatogram or the Process and Review page click the Parameters tab Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up a Manual ReSpect Protein Deconvolution iii Change the appropriate parameters on the Parameters page click Apply in the appropriate pane or panes and click Save Method to save the changes to the file or Save Method As to save the ch
95. Optional In the Descriptions box type a brief description of the results The dialog box should resemble that shown in Figure 86 Figure 86 SaveAs dialog box 41L SaveAs x Result Name test2 Descriptions IgG intact Save Cancel c Click Save The name that you entered into the Result Name box appears in the Saved ReSpect Results pane on the Process and Review page The Protein Deconvolution application saves the results of the deconvolution in a file with an sqlite suffix in the same directory where you stored the raw data files 3 If you want to analyze another averaged spectrum from the same LC MS data file navigate back to the Chromatogram pane and follow the instructions in Selecting the Spectrum to Deconvolve on page 149 To display saved results 1 If the Saved Xtract Results pane and the Saved ReSpect Results pane are not visible on the Process and Review page click the double arrow s gt at the top of the Results pane Protein Deconvolution User Guide 181 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Exporting the Results of a Deconvolution 2 If only the title bar of the Saved Xtract Results pane or the Saved ReSpect Results pane is visible click the side arrow g in the appropriate pane to expand the pane The Saved Xtract Results pane and the Saved ReSpect Results pane display any Xtract or ReSpect results respectively that you
96. Resolved Mass Spectra with the Xtract Algorithm Deleting the Results of a Deconvolution The Protein Deconvolution application exports the mass x axis and intensity y axis data from the Deconvolved Spectrum pane to the Excel or the CSV file For Xtract deconvolutions the saved data consists of a centroid spectrum Figure 34 gives an example of the mass and intensity data in an Excel file Figure 34 Mass and intensity data from the deconvolved spectrum in an Excel file fia a Os ca m Home Insert Page Layout Formulas B amp Calibri j ia 5a Paste p B E aA Clipboard Font IFJ tl Ml iil Mass Intensity 3373 199 5517 949 5612 34 5612 627 5612 986 5616 34 5616 669 5622 005 5622 322 5622 644 5626 5626 329 5626 664 5627 673 5630 658 5631 989 5632 318 5635 34 5635 657 5652 329 5660 986 Deleting the Results of a Deconvolution You can manually delete the results of a deconvolution outside of the Protein Deconvolution application To delete the results 1 Navigate to the directory containing the SQLite files 2 Select the SQLite file or files containing the results that you want to delete The SQLite file has the same name as the original raw data file Thermo Scientific Protein Deconvolution User Guide 69 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples 3 Right click and choose Delete The Protein Deconvolu
97. Restricted Time Determines whether the Protein Deconvolution application zooms the part of the chromatogram that you designate with the Time Limits parameters Selected Enlarges the designated part of the chromatogram Default Cleared Displays the entire chromatogram e Time Limits Specifies the beginning and the end of the chromatogram that you want to zoom The default values for both limits depend on the data in the raw data file This parameter is only available when you select the Use Restricted Time check box e Rel Intensity Threshold Sets a lower intensity threshold for peaks in the chromatogram as a percentage The Protein Deconvolution application ignores peaks with relative intensities below this threshold The values are 0 to 100 inclusive The default is 1 This parameter is different from the Relative Abundance parameter on the Parameters page which sets a lower intensity for signals in the spectrum not in the chromatogram e Types Specifies the type of chromatogram to display in the Chromatogram pane Default TIC Displays a total ion current TIC chromatogram BPC Displays a base peak chromatogram The base peak is the largest peak in a spectrum For information on these types of chromatograms see able 29 on page 158 e Sensitivity Specifies the sensitivity with which the chromatographic peak detector identifies peaks Low Directs the chromatographic peak detector t
98. Results Displaying the Results on the Process and Review Page The Protein Deconvolution application displays the results of the deconvolution in the Deconvolved Spectrum pane and in the Results table of the Process and Review page To display the results on the Process and Review page Click the Process and Review tab if the Process and Review page is not already open The Process and Review page displays the deconvolved spectrum in the Deconvolved Spectrum pane as shown in Figure 23 52 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 23 Deconvolved spectrum on the Process and Review page for Xtract deconvolution SCIENTIFIC Thermo Protein Deconvolution vetauitmethoaxtract Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting Manual Xtract isotopically Resolved Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_ RAW Help 2 Rel Abundance Threshold Negative Charge Calculate XIC Y Saved Xtract Results i Deconvolution completed successfully Process SaveResultAs Reset Method Save as Reference Output Mass M MH Resolution at 400 m z 60000 S N Threshold 3 Source Spectrum 1004391 2844 m z Range Min 600 Max 2000 Apply Charge Carrier H 100727663 K 38 9631585 Na 22 989221
99. Review page information for Xtract deconvolution Sheet 3 of 3 Parameter Description RT Range Displays the retention time range of the averaged source spectrum for the given component Apex RT Displays the retention time for the chromatographic peak when a component has a calculated extracted ion chromatogram XIC Charge State Displays the imbalance between the number of protons in the nuclei of the atoms and the number of electrons that a molecular species or adduct ion possesses If the species possesses more protons than electrons its charge state is positive If it possesses more electrons than protons its charge state is negative Calculated Monoisotopic m z Displays the mass to charge ratio of the calculated monoisotopic mass for a specific charge state Monoisotopic Mass for This Charge Displays the detected monoisotopic mass for a specific charge state Mostabund m z Displays the mass to charge ratio of the most abundant isotope or the height of the tallest peak in the isotopic distribution Charge Normalized Intensity Displays the quotient of the intensity divided for this charge by the relevant charge Fit Displays the quality of the match between a measured isotope pattern and an averagine distribution of the same mass This column displays a value between 0 and 100 percent e 0 requires only a poor fit between the measured pattern and the averagine pattern e 100
100. Saved Xtract Results SS Saved ReSpect Results Thermo Scientific Source Spectrum Chromatogram 893 1164 iT 3 1004291 2844 RT 3 66 100 gt RT 0 58 50 1060 4415 i sie A gt 13049242 1696 2001 1918 3288 is 0 1000 1500 2000 6 miz RT min Process Save Result As Reset Method Save as Reference Deconvolved Spectrum 16941 0113 100 80 60 40 3 20 16568 8381 15635 2961 si 0 sia aa T T T T r aeaa a T set T T peere pereerT T T T T 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 Mass Results Monoisotopic Number of Charge Relative Fractional 2 No hee Sum Intensity sabes Average Charge Delta Mass trai poeme RT Range Apex RT 1 16941 0113 315 704 75 20 19 82 0 00 100 0000 42 2091 3 303 3 969 3 671 2 16923 0410 216 752 20 16 20 60 17 97 68 6566 28 9794 3 303 3 969 3 653 d Output values of 3 16922 8878 33 454 02 14 98 10 5966 44727 3 303 3 969 3 658 Calculated Monoisotopic Mass a Normalized a S Fi Charge State peeraa for This Charge Mostabund m z Intensity Fit Fit Left it Right the deconvolution 1302 7694 16922 9025 1303 5380 3 762 86 96 00 0 00 ips 14 1209 7864 16922 8779 1210 4987 647442 9730 0 00 0 00 Values for individual 15 1129 2011 16922 8745 1129 8672 9 814 38 96 90 0 00 0 00 A 16 1058 6890 16922 8981 1059 3154 1340235 96 30 0 00 710 isotopic clusters a 4 16905 0254 31 121 70 2116 35 99 9 8578 41609 3303 3969 364
101. UTO Deconvolves the spectrum with the automatic ReSpect algorithm Number of Displays the number of chromatographic peaks detected in Chromatographic Peaks the spectrum If the Protein Deconvolution application finds Number of Components Detected Completion Time no chromatographic peaks or proteins the value is 0 Displays the number of components detected from the deconvolution of each averaged spectrum from all chromatographic peaks If the Protein Deconvolution application finds no chromatographic peaks or proteins the value is 0 Displays the time that the processing finished for a given raw data file Status Thermo Scientific Displays the real time status of the deconvolution e Queued The data file is awaiting analysis e Processing The Protein Deconvolution application is in the process of analyzing the data file e Completed The Protein Deconvolution application has finished analyzing the data file and has generated the report e Completed Report Not Concatenated The Protein Deconvolution application has finished analyzing the data file but has not generated the report because of size constraints Protein Deconvolution User Guide 225 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Generated with the Automatic ReSpect Algorithm Comparing Samples Generated with the Automatic ReSpect Algorithm To compare a reference spectrum generated b
102. UTO Indicates the automatic Xtract algorithm e XT_RSP Indicates the automatic ReSpect algorithm Number of Peaks Creation Time Protein Deconvolution User Guide Displays the number of peaks in the selected reference spectrum Displays the date and time that the selected reference spectrum was generated Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Table 35 Sample Comparison page parameters for ReSpect deconvolution Sheet 3 of 3 Parameter Description Descriptions Displays the description of the selected reference spectrum as you specified it in the SaveAs dialog box Select Selects the highlighted reference spectrum to use to compare to the source spectrum Delete Deletes the selected reference spectrum Add to Method Adds the selected reference spectrum to the method that you used to generate the reference spectrum Show Details Opens a Reference Spectrum Information box see Figure 94 on page 195 displaying the deconvolution parameters used to produce the reference spectrum that you selected in the reference spectrum library Mirror Plot Pane Source Data File Displays the mirror plot of the selected reference spectrum and the source spectrum Displays the path and name of the raw data file used to produce the source spectrum Reference Data File From Displays the path and name of the raw data fil
103. Xtract pane of the Parameters page For information on the parameters on this page see Table 4 on page 27 Apply Implements the parameter settings that you selected Saved Xtract Results pane Displays each set of Xtract results that you saved from a given raw data file Saved ReSpect Results pane Displays each set of ReSpect results that you saved from a given raw data file Load Result Source Spectrum pane Loads the selected previous results into the Source Spectrum Deconvolved Spectrum and Results panes Displays the selected spectrum before deconvolution This spectrum is the same as that shown on the Chromatogram page Relative Intensity y axis m z x axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Displays the mass to charge ratio of ions formed from molecules This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion Chromatogram pane Relative Intensity y axis Displays the same chromatogram as that shown on the Chromatogram page before deconvolution After deconvolution this pane displays the chromatogram of the component that you select in the Results table Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity RT min x axis Displays the retention time of the spectrum w
104. You cannot open the results if the Protein Deconvolution application has not yet analyzed the raw data file or is in the process of analyzing it Opening the results does not stop the application from analyzing subsequent data sets To adjust the chromatogram Click the Method Selection tab Click Manual ReSpect Isotopically Unresolved and reload the raw data file by following the instructions in Creating a ReSpect Method on page 123 Adjust the chromatogram by following the instructions in Selecting the Spectrum to Deconvolve on page 149 Resubmit the job to the job queue for automatic processing To copy a chromatogram After your job has finished processing click Open Result on the Run Queue page Click the Chromatogram tab From the Windows Start menu choose All Programs gt Accessories gt Snipping Tool Drag the cursor that appears around the area of the chromatogram that you want to capture Right click in Snipping Tool and choose Copy Open a third party software file and paste the copied picture into it The Protein Deconvolution application creates averaged spectra from the start scan to the end scan for each chromatographic peak It saves each deconvolution result from a given chromatographic peak The name of each deconvolution result is as follows method_name_start_scan_end_scan_date_time_experiment_type Protein Deconvolution User Guide 227 5 Automatically Deconvo
105. a Mass Displays the difference between the mass of a specific compound and the mass of the highest intensity component Relative Abundance Displays the peaks in the sample that are above the relative abundance threshold set by the Relative Abundance Threshold parameter on the Parameters page for information on this parameter see Table 4 on page 27 The Protein Deconvolution application assigns the largest peak in a deconvolved spectrum a relative abundance of 100 percent An abundance number in the Relative Abundance column represents the intensity in the same row of the Sum Intensity column divided by the greatest intensity in the Sum Intensity column multiplied by 100 For example if the largest peak in a deconvolved spectrum has an intensity of 1000 the Protein Deconvolution application assigns it a relative abundance of 100 percent If the next most abundant peak has an intensity of 500 the application assigns it an abundance of 50 percent 500 509 1000 100 50 Fractional Abundance Displays the fractional abundance of a component which is the abundance for that peak relative to the total abundance of all peaks in the spectrum expressed as a percentage The sum of all fractional abundances of all peaks in a deconvolved spectrum is 100 percent Protein Deconvolution User Guide 63 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Table 9 Process and
106. a Thermo Xcalibur raw data file When you export a mass spectrum to a new raw data file the data system appends the qb suffix to the original file name The new raw data file contains only a single mass spectrum The status log view in the Qual Browser window states that the data file was created by using the Qual Browser application and provides more information about the mass spectrum such as the source raw data file the scan number or averaged scan numbers and the subtraction settings if applicable In deriving the resolution the behavior of the manual and the automatic Xtract algorithms follows the behavior of the manual and the automatic ReSpect algorithms described for the Resolution at 400 m z parameter in Table 27 on page 135 although the resolution does not affect the results S N Threshold Specifies a signal to noise S N threshold x above which the Xtract algorithm considers a measured peak to be a real accepted peak The Xtract algorithm ignores peaks below this threshold Any spectral peak must be x times the intensity of the calculated noise for that spectrum before the Xtract algorithm considers it The minimum value is 0 and there is no maximum The recommended value is 2 to n The default is 3 Rel Abundance Threshold Specifies a threshold below which the Protein Deconvolution application filters out data for data reporting This option sets a relative threshold as a percentage of the most abundant co
107. acaay A 27498 5165 27620 5748 27716 5895 27790 6337 27862 6864 27976 7711 28105 8257 AMO DOr e ara 28370 9318 l 28513 0207 5 3 0 E j g i i H 8307 28414 9952 28472 0486 OHIEN 277147390 37796 7844 27853 8096 L 28058 8692 28266 9389 28307 9693 S 50 27976 8125 28162 9090 iio 27934 8388 28121 8884 Mass Reference Data File C Program Files Protein Deconvolution source files Xtract PSA_240 raw From Library NL 6 71E 004 lt _ To delete a reference spectrum from the reference spectrum library 1 Click the Sample Comparison tab if you are not already on the Sample Comparison page 2 Select the appropriate reference spectrum in the Reference Spectrum Library pane 3 Click Delete 4 In the confirmation box that appears click Yes 80 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples To delete a saved reference spectrum from a method Click the Sample Comparison tab if you are not already on the Sample Comparison page The Method Reference Spectrum pane displays the reference spectrum name and the name of the reference data file It also displays a description of this reference spectrum if there is one In the Method Reference Spectrum pane click Remove The deleted reference spectrum is removed from the Method Reference Spectrum pane and the mirror plo
108. aces or non alphanumeric characters It can include underscores In the Description box briefly describe the method For example you might want to describe the sample and the proteins analyzed Figure 13 shows a completed SaveAs dialog box Figure 13 Completed SaveAs dialog box 1 Saves Method Name test2 hd Descriptions IgG intact Save Cancel Click Save Note The Protein Deconvolution application saves all methods that you create in the database in C ProgramData ThermoScientific ProteinDeconvolution methods sqlite You cannot save individual methods to a directory that you choose The next time that you access the Method Selection page and click Manual Xtract Isotopically Resolved you will see the name of the method that you saved in the Methods pane The application transfers you to the Chromatogram page so that you can select the spectrum to deconvolve For information on this process see Selecting the Spectrum to Deconvolve on page 37 Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Parameters Page Parameters for the Xtract Algorithm Table 4 describes the parameters on the Parameters page for an Xtract deconvolution Table 4 Parameters page parameters for Xtract deconvolution Sheet 1 of 10 Parameter Main Parameters Xtract pane Description Displays basic parameters that migh
109. ad Result at the bottom of the page Thermo Scientific Protein Deconvolution User Guide 213 The Protein Deconvolution application places the selected previous results in the Source Spectrum Deconvolved Spectrum and Results panes of the Process and Review page In addition it updates the report on the Reporting page Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm This chapter explains how to automatically deconvolve isotopically unresolved mass spectra with the ReSpect algorithm For information about manually deconvolving these spectra with the ReSpect algorithm see Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm on page 117 Contents e Setting Up an Automatic ReSpect Protein Deconvolution e Running Jobs in the Queue e Displaying the Results e Comparing Samples Generated with the Automatic ReSpect Algorithm e Displaying a ReSpect Deconvolution Report e Loading Saved Results Automatic ReSpect deconvolution automates the process of generating chromatograms detecting chromatographic peaks extracting averaged mass spectra deconvolving unresolved isotopic peptides or proteins and generating a component list all in a single step It can optionally generate a report containing the deconvolved proteins and their mass spectrometric evidence The results of an automatic deconvolution are the same as those of a manual deconvolution You can save m
110. advances the application to the Process and Review page Reset Method Thermo Scientific Returns the parameters in the Chromatogram Parameters pane to the settings that you first chose when you loaded the raw data file Protein Deconvolution User Guide 47 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Chromatogram Page Shortcut Menus When you right click in the Chromatogram or Source Spectrum pane on the Chromatogram page a shortcut menu appears that contains the commands listed in Table 8 Table 8 Chromatogram page shortcut menu Parameter Description Mode Chromatogram pane Determines whether dragging the cursor zooms or selects a only range of scans to average e Default Averaging Averages the spectra for all the scans in the region that you drag the cursor over in the Chromatogram pane and displays them in the Source Spectrum pane e Auto Zooming Enlarges the area that you drag the cursor over in the Chromatogram pane without changing the view displayed in the Source Spectrum pane Reset Scale Restores the original chromatogram that first appeared in the Chromatogram pane Copy Bitmap Copies the view in the Chromatogram pane to the Clipboard Zoom Out Shrinks the view in the Chromatogram pane by a factor of 2 Zoom In Enlarges the view in the Chromatogram pane two times Chromatogram Pane Header The header in the Chroma
111. ae SCIENTIFIC IgG_source_cid raw _ Method Selection Run Queue Parameters Chromatogram Sample Comparison __ Reporting Deconvolve the spectrum Process Save Result As Reset Method Save as Reference gt Main Parameters ReSpect Results Source Spectrum Chromatogram Y Saved Xtract Results 2761 2696 E i mpa 2977 7551 NL 24968 x 2 ellez gg A ines highlighting all o charge states 1 3 4 5 6 4 ne Tin corresponding to the va selected average mass i 151814 672 p i 80 3 808 ao Line highlighting the i 2 40 selected average mass AA 51560 344 149595 56 Saved ReSpect Results 20 30895 424 37290 301 al 60244000 722 9359 sasag 99 100997 500 149595 DefaultMethodReSpect_1089_1094 g L 18527 4483 po da 1 r 1 r 1 1 20000 40000 60000 80000 100000 120000 140000 160000 DefaultMethodReSpect_1129_131 Mass DefaultMethodReSpect_36_57_10_ DefaultMethodReSpect_68_356_1 3 No AverageMass Intensity NEES OF Mass Std Dev PPM Std Dev Bese nacional DefaultMethodReSpect_844_874_1 Chame States Abundance Abundance 155532 625 16 119 130 00 h 3 717 95 p 3 304 3 630 152471375 23 144 214 00 y 656 70 3 304 3 630 DefaultMethodReSpect_900_942_1 100997 500 6 094 863 00 x 50 817 17 L 3 304 3 630 DefaultMethodReSpect_ 942_1020 152762 625 18 604 504 00 947 95 3 304 3 630 37290 301 7 475 119 50 x i 114 524 37 A 3 304 3 630 155469 000 7 232 607 00 K 3 654 33 3
112. am Parameters Section Source Chromatogram Section 200 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Source Spectrum Section Main Parameters ReSpect Section Advanced Parameters ReSpect Section Deconvolved Spectrum Section Mirror Plot Section ReSpect Masses Table Section Source Spectrum Evidence Section Figure 95 Partial view of the Reporting page for ReSpect deconvolution Protein Deconvolution SCIENTIFIC Created 10 17 2013 5 40 32 PM Sample Information Original File Name C XCALIBUR Intact IgG_source_cid raw Instrument Method _ C Xcalibur Intact mab_SS_1 meth Vial CStk1 01 23 Injection Volume uL 40 0 Sample Volume uL 0 ISTD Amount 0 Dil Factor Chromatogram Parameters Use Restricted Time False Time Range 0 004648833 6 2300942 Rel Intensity Threshold 1 Chromatogram m z Range 1000 4000 Use Auto Spectrual Averging False Source Chromatogram IgG_source_cid ProteinDeconvolution NL 3 10E9 F RT 3 41 S 1171 m ProteinDeconvolution Report Displaying a ReSpect Deconvolution Report Manual ReSpect isotopically Unresolved Help IgG_source_cid raw Method Selection Run Queue Parameters Chromatogram Process and Review Sample Comparison Note The Protein Deconvolution application does not support copying and pas
113. ameter defaults 3 Optional Adjust the view in the Chromatogram pane e To enlarge the view to see more detail do one of the following Right click and choose Zoom In from the shortcut menu to zoom the entire chromatogram 152 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Right click and choose Mode gt Auto Zooming from the shortcut menu if it is not already selected and drag the red cross shaped cursor over the peak or peaks of interest to form a box as shown in Figure 69 Figure 69 Enlarging a peak by drawing a box around it Chromatogram IgG_source_cid NL 3 10E9 F 100 8 s RT 0 17 RES S 38 SH Relative Intensity RT 0 36 S 172 ne 083 1 11 1 22 1 37 146 163 180 205 229 2 43 f oT TT 05 1 0 15 20 25 RT 3 03 Re i 916 3 0 RT min Draw a box around the peak of interest Right click and choose Mode gt Auto Zooming from the shortcut menu if it is not already selected Keeping the left mouse button pressed draw a line beneath the baseline of the peaks of interest as shown in Figure 70 Figure 70 Enlarging a peak by drawing a line beneath its baseline Chromatogram lgG_source_cid NL 3 10E9 F S 1171 100 2 8 e Relative Intensity RT 3 31 tT 0 17 ao Sy 98 S 1093 A RT 0 36 S 172 RT 292 RT 3 03 il
114. ameters Xtract pane to original settings 22 returning parameters on Parameters page to original settings 36 149 returning parameters on Process and Review page to original settings 65 179 returning parameters on Reporting Parameters pane to original settings 25 133 returning parameters on Sample Comparison page to original settings 85 200 Reset Scale command ReSpect 154 157 162 180 200 Xtract 41 44 48 65 85 Resolution at 400 m z parameter 20 27 131 145 ReSpect algorithm adjusting deconvolution results 175 automatic deconvolution 215 calculating a protein quality score 4 126 136 173 174 178 calculating an extracted ion chromatogram 126 136 centroid data 155 180 183 comparing samples changing a reference spectrum in a method 196 comparing a source spectrum to a reference spectrum 190 deleting a reference spectrum from a library 80 195 deleting a reference spectrum from a method 196 displaying parameter settings of reference spectrum 70 185 194 in saved ReSpect results 197 mirror plot 185 purpose 4 185 saving deconvolved spectrum as a reference spectrum 186 saving reference spectrum 185 creating a method 123 deconvolving a spectrum 163 deleting deconvolution results 184 description 3 displaying deconvolution results 52 165 166 226 displaying reports 200 exporting deconvolution results 182 loading saved deconvolution results 212 manual deconvolution 117 Thermo Scientific most important para
115. an use this method to analyze very large non covalent complexes and antibody drug conjugates that are held together by non covalent bonding Protein Quality Score The ReSpect algorithm calculates a protein quality score for each component in the results and displays it in the results table so that you can determine whether each component is valid or spurious because of noise harmonics or other factors For detailed information on this feature see Calculating a Protein Quality Score on page 173 Manual and Automatic Deconvolution You can run the Protein Deconvolution application in manual or automatic mode e In manual mode you generate the chromatogram select the chromatographic peaks deconvolve the spectra with the ReSpect or Xtract algorithm and report the results separately one step at a time e In automatic mode the Protein Deconvolution application detects chromatographic peaks extracts averaged mass spectra deconvolves isotopically unresolved or resolved peptides or proteins and generates a component list all in a single step without any intervention You can place up to 1000 samples in the run queue for automatic processing You might want to run the Protein Deconvolution application manually when something about your workflow changes and you want to determine the optimal settings for selecting the chromatographic peaks deconvolving the spectrum or both For example you might introduce a new protein a new chrom
116. ance RT Range and Apex RT They do not include the data in the columns that appear when you click the symbol to the left of the No column In the SaveAs dialog box browse to or type the name of the file to store the results in Click Save The Protein Deconvolution application stores the data shown in the Results table in an Excel file called raw_file_name x s If you do not specify a directory it places this file by default in the raw data file directory shown on the Method Selection page When you select a result and then choose the Export Top Level or Export All command the resulting Excel file reflects the columns and order of the currently visible table To export a chromatogram source spectrum or deconvolved spectrum to a third party software file Right click in one of the following panes and choose Copy Bitmap e Chromatogram pane on the Chromatogram page e Source Spectrum pane on the Process and Review page e Deconvolved Spectrum pane on the Process and Review page Open a third party graphics software file and paste the copied picture into it To export the deconvolved spectrum mass and intensity data to an Excel or CSV file Right click in the Deconvolved Spectrum pane of the Process and Review page and choose Copy Data Open an Excel or a CSV file 3 Right click in the application and choose Paste 68 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically
117. and Review page to deconvolve the spectrum view the quality scores in the Score column of the Results table 2 Optional Sort the scores from high to low by clicking the down arrow next to the Score header or sort the scores low to high by clicking the up arrow Specifying the Output Mass Range The Output Mass Range parameter restricts the allowable mass range for the charge state deconvolution It produces the best fit possible to the observed set of peaks in a spectrum using only components that fall within the specified mass range But in ReSpect processing results for a restricted output mass range are not a simple subset of runs for a larger output mass range The results for two adjacent mass ranges such as 35 000 40 000 and 40 000 45 000 need not always form proper subsets of the results for the combined mass range in this case 35 000 45 000 Your choice of parameter values affects the components that the algorithm detects in subsets of the output mass range If you set the Output Mass Range parameter to values that do not span the expected range of component masses the ReSpect algorithm adds superfluous low amplitude components with physically implausible masses to its results list to fit the peaks that would have been associated with the missing masses These components receive low protein scores and disappear when you run the ReSpect algorithm with the full output mass range If you run the algorithm with an inappropriate output
118. and a method i Experiment Types Load Result File Results Raw irectory Result Ni Descripti pil C Program Files Protein Deconvolutio needed anh del ReSpect3537 RSP Select Result Files DefaultMethodReSpe RSP_AUTO IgG_source_cid SQUTE DefaultMethodReSpe RSP_AUTO IgG_source_cid qb SQLITE DefaultMethodReSpe RSP_AUTO DefaultMethodReSpe RSP_AUTO DefaultMethodReSpe RSP_AUTO DefaultMethodReSpe RSP_AUTO DefaultMethodReSpe RSP_AUTO DefaultMethodReSpe RSP_AUTO Manual Xtract Isotopically Resolved Manual ReSpect Isotopically Unresolved Auto Xtract Isotopically Resolved Auto ReSpect Isotopically Unresolved Load Results 5 In the Results pane select the results that you want and click Load Result The application transfers you to the Process and Review page which displays the requested saved results in its Source Spectrum Chromatogram and Deconvolved Spectrum panes and in the Results table The file name of the requested results appears in the Saved ReSpect Results pane 1 To load saved results from the Process and Review page 1 In the Method Selection page load a raw data file and select a method For instructions see Setting Up a Manual ReSpect Protein Deconvolution on page 118 2 Click the Process and Review tab 3 In the Saved Xtract Results pane or the Saved ReSpect Results pane select the results that you want to load and click Lo
119. and the methods created in previous releases of the software use this formula Nucleotide Uses an elemental formula typical for nucleotides to generate the isotope table You can save the isotope table as part of the method If you want to return the parameters in the Main Parameters Xtract pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change the parameters in this pane but do not apply them and then click another tab the message box shown in Figure 12 appears Click Yes to apply the parameter changes or No to restore the parameter defaults Figure 12 Reminder to apply parameters Protein Deconvolution Pg You have made changes to the parameters Do you want to apply the A changes Yes No 22 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method 3 Optional If you are an experienced user change the appropriate parameters in the Advanced Parameters Xtract pane e Fit Factor Measures the quality of the match between a measured isotope pattern and an averagine distribution of the same mass as a percentage Enter a value between 0 and 100 percent 0 requires a low fit only 100 means that the measured isotope profile is identical to the theoretical averagine isotope distribution
120. anges as a different method Return to the Chromatogram or the Process and Review page Note You cannot overwrite a default method If you change the parameters in the default method use the Save Method As command on the Parameters page to save the changed method to a new name e Ifthe existing default methods do not contain the appropriate extraction parameters or if there are no existing methods click Create Method to create a new method The Protein Deconvolution application automatically transfers you to the Parameters Pp y y page Follow the instructions in Creating a ReSpect Method on page 123 to set the parameters on the Parameters page e Ifyou want to study proteins on the Exactive Plus EMR mass spectrometer under native or non denaturing conditions use the ExampleMethodNativeMS method This method is a read only method available only with the ReSpect algorithm It enables automated analysis of Native MS data that is directly infused into the mass spectrometer Unlike standard intact protein data Native MS data might contain detectable protein complexes with multiple proteins embedded in them The required m z range to detect these complexes is 1000 to 10 000 or even 20000 m z Table 23 shows the differences in parameter settings between the default ReSpect method and the ExampleMethodNativeMS method Table 23 Differences in parameter settings between default and ExampleMethodNativeMS methods Parameter D
121. ant displayed and click Open Report or From any other page click the Reporting tab The Protein Deconvolution application transfers you to the Reporting page which displays the report or reports 114 Protein Deconvolution User Guide Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Loading Saved Xtract Results Loading Saved Xtract Results You can reload the results of a previous deconvolution For instructions see Loading Saved Xtract Results on page 98 Thermo Scientific Protein Deconvolution User Guide 115 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Thermo Scientific This chapter explains how to manually deconvolve isotopically unresolved mass spectra with the ReSpect algorithm For information about automatically deconvolving these spectra with the ReSpect algorithm see Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm on page 215 Contents e Setting Up a Manual ReSpect Protein Deconvolution e Creating a ReSpect Method e Selecting the Spectrum to Deconvolve e Deconvolving the Spectrum e Displaying the Results e Saving the Results of a Deconvolution e Exporting the Results of a Deconvolution e Deleting the Results of a Deconvolution e Comparing Samples e Displaying a ReSpect Deconvolution Report e Loading Saved ReSpect Results When you gen
122. are wana des 49 Deconvolving the Spectrum ois osteo s oaelo seo mies tee eek 49 Displaying the Results 5 ap delece oar Seas lagd ins cc renere 51 Displaying the Results on the Process and Review Page 52 Selecting a Reference Mass to Calculate Mass Differences 60 Adjusting the Deconvolution Results 0000s cee eee eee eee 61 Process and Review Page Parameters for the Xtract Algorithm 61 Process and Review Page Menu Bar Commands 00000 65 Process and Review Page Shortcut Menus 2 0 00 c eee ee eee 65 Saving the Results of a Deconvolution 000 0 c cece eee eee 66 Exporting the Results of a Deconvolution vec sie ie es te wd a er ee ee hones 68 Deleting the Results of a Deconvolution 2 0 0 0 eee eee eee 69 Comparing Samples sureci asacbtaneyh is Karsesded ayer alee winded ded heen eg haved 70 Comparing Samples in Saved Xtract Results 2 0 0 cee eee eee ee 82 Sample Comparison Page Parameters for the Xtract Algorithm 82 Sample Comparison Page Menu Bar Commands 4 84 Mirror Plot Shortcut Ment 60 2554 aieuas Sek wees Dek urran rr 85 Displaying an Xtract Deconvolution Report 0 00 0 eee eee eee 85 Reporting Page Toolbatyei cin tnchs ei eekors wali cele aaa 88 Sample Information Section acs adda arerelac bNaredihie tpcieredaraye Aliya daneyed bed 89 Chromatogtam Parameters Section 4h wed ch eek ees Rae di
123. ass in daltons to use in calculating the peak model This parameter is critical but does not have to be exact a value within 5 to 10 percent of the actual target is sufficient for best performance For samples where the range of masses is broad choose a mass somewhere in the middle of the range For example if the IgG light 20 kDa heavy chains 50 kDa and intact antibody are found in the same sample choose 75 kDa as the target mass The default for the default method is 150000 Da Charge State Range Sets the allowable range for the number of charge states that must appear for a component to be recognized The ReSpect algorithm rejects potential components with fewer than the minimum or greater than the maximum number of charge states The default range for the default method is 10 through 100 The default range for the ExampleMethodNativeMS method is 5 through 100 Protein Deconvolution User Guide 127 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method 128 Choice of Peak Model Specifies the appropriate peak model for the data The expected peak shapes for nucleotides are different from those for proteins and peptides because of their different isotopic composition Default Intact Protein Specifies peak model shapes that are appropriate for use with the intact protein data produced by Orbitrap MS instruments Nucleotide Specifies peak model shapes th
124. at are appropriate for use with the nucleotide data produced by Orbitrap MS instruments If you want to return the parameters in the Main Parameters ReSpect pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you make changes to the parameters on this pane but do not apply them and then click another tab the message box shown in Figure 66 appears Click Yes to apply the parameter changes or No to restore the parameter defaults Figure 66 Reminder to apply parameters Protein Deconvolution xX i You have made changes to the parameters Do you want to apply the changes Yes No 3 Optional If you are an experienced user change the appropriate parameters in the Advanced Parameters ReSpect pane Protein Deconvolution User Guide Minimum Peak Significance Specifies a significance level in standard deviations that determines whether the ReSpect algorithm discards a peak as a noise feature or retains it as a legitimate peak The ReSpect algorithm retains peaks equal to or greater than this selected significance level The higher the significance level the more stringent this filtering is The default is 1 Noise Rejection Removes noise and irrelevant features from the list of peaks This parameter is important in the ReSpect algorithm because it controls how plausible a peak must be before the algorithm uses it in a deconvolution Reducin
125. ata file containing chromatographic peaks and spectral peaks the Protein Deconvolution application takes the resolution from the following sources When you perform a manual deconvolution create a method load a raw data file and do not change the resolution the application obtains the resolution from the raw data file When you perform a manual deconvolution create a method load a raw data file and change the resolution the application uses the changed resolution for the current deconvolution However when you reload the method and perform another deconvolution it uses the resolution from the raw data file When you perform an automatic deconvolution the application obtains the resolution from the instrument When you load a data file containing only spectral peaks that is an exported spectrum file in qb raw file format the Protein Deconvolution application takes the resolution from the following sources When you perform a manual deconvolution create a method and load a raw data file the application obtains the resolution from the method When you perform an automatic deconvolution the application obtains the resolution from the saved method When you load the raw data file without loading a method the default resolution is always 15 000 The Resolution at 400 m z parameter is not needed if the ReSpect algorithm deconvolves FTMS Orbitrap or Exactive data because the data contains instrum
126. ated data For more information see To save a deconvolved spectrum as a reference spectrum on page 186 Protein Deconvolution User Guide 179 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Process and Review Page Shortcut Menus When you right click in the Source Spectrum Chromatogram or Deconvolved Spectrum panes or on the Results table on the Process and Review page a shortcut menu appears that contains the commands listed in Table 34 Table 34 Process and Review page shortcut menu Parameter Reset Scale Description Restores the original chromatogram that first appeared in the Source Spectrum or Chromatogram pane Copy Bitmap Copies the view in the Source Spectrum or Chromatogram pane to the Clipboard Copy Data Deconvolved Spectrum pane only Copies mass data x axis and intensity data y axis from the Deconvolved Spectrum pane to the Clipboard so that you can paste it into an Excel spreadsheet or another application For ReSpect deconvolutions the saved data consists of a centroid spectrum followed by the associated profile spectrum Zoom Out Shrinks the view in the Source Spectrum or Chromatogram pane by a factor of 2 Zoom In Enlarges the view in the Source Spectrum or Chromatogram pane two times Set As Reference Component Results table only Resets the value in the Delta Mass Column of the Results table
127. ates EET 8 QUtpUtS aie ba eds a eae hs MA dae ee Mag eee ne da eee ieee eas 8 Starting the Protein Deconvolution Application 000 0 e eee eee 8 Specifying the Default Raw Data File Directory 44 001 ccuencea Sewn cess 9 Adjusting the Pane Se Kunee ee ene ee een ere aah nee eae ERE Raabe 10 Exiting the Protein Deconvolution Application 0 0 eee ee 11 Chapter2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm 0 22 cece eee eee eee 13 Setting Up a Manual Xtract Protein Deconvolution 000 14 Method Selection Page Parameters si oi os cv saeciowsceaaces snedaes 16 Creating an Xtract MenhGd ite cen noee see eens Reena ceeds nb on obs 18 Parameters Page Parameters for the Xtract Algorithm 27 Menu Bar Commands on the Parameters Page 000 e eee 36 Thermo Scientific Protein Deconvolution User Guide iii Contents iv Chapter 3 Protein Deconvolution User Guide Selecting the Spectrum to Deconyolye sic a aati ates dae eee kee eee 37 Chromatogram Page Parameters for the Xtract Algorithm 44 Chromatogram Page Menu Bar Commands 00 0c eee 47 Chromatogram Page Shortcut Menus 0 00 0 c eee eee ee eee 48 Chromatogram Pane Header 0p 2s cis ad ae ted awhek Ge wee kw ees 48 Source Spectrum Pane Shortcut Menu wc jee ta eth ohana eee 48 Source Spectrum Pane Header on hota wder oes venooe w
128. ath and name of the SQLite file containing the saved results or click the Browse button to browse to the location of the file 4 In the Select Result Files area of the Load Result File pane select the name of the SQLite file containing the results and click Load The Protein Deconvolution application displays the results found in the Results pane as shown in Figure 54 98 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Loading Saved Xtract Results Figure 54 Saved results in the Results pane Protein Deconvolution Lod Ress Help 3 S C RN Fic Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 SQLITE Method Selection D4 Run Queue Parameters Chromatogram Process and Review Sample Comparison Reporti og p p poruag gt Select an experiment type a data file and a method Experiment Types Load Result File Results irectory Result N Descripti emesis C Program Files Protein Deconvolutio m rahi A Manual Xtract Isotopically Resolved gt DefaultMethodXtract Select Result Files DefaultMethodXtract Manual ReSpect Isotopically Unresolved 33_lowCESQUTE DefaukMethodXtract Opmol_michrom_protein_microtrap_1imin_OT_60K_1 SQUITE DefaultMethodXtract Opmol_michrom_protein_microtrap_11imin_OT_60K_1 qb SQLITE ITE DefaultMethodXtract Auto Xtract Isotopically Resolved hpLSQUTE DefaultMethodXtract Auto ReSpect
129. ation Parameters pane Implements the parameter settings that you selected in the Reporting Parameters pane This button is only available if you changed any parameter settings in the Reporting Parameters pane Displays parameters that control automatic deconvolution You cannot set the parameters in this pane unless you select Auto Xtract Isotopically Resolved in the Experiment Types pane Save to PDF Thermo Scientific Determines whether to save the automatically generated deconvolution report to a PDF file e Default Selected Saves the automatically generated deconvolution report to a PDF file e Cleared Does not save the automatically generated deconvolution report to a PDF file Protein Deconvolution User Guide 35 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 10 of 10 Parameter Description Auto Print Determines whether to automatically print the generated deconvolution report When you select this option the Select a Printer box becomes available so that you can select a printer e Selected Automatically prints the generated deconvolution report e Default Cleared Does not automatically print the generated deconvolution report Concatenate All Reports Determines whether to combine all reports for all deconvolved spectra from the same raw data file e Default
130. atographic setup or different mass spectrometry settings Once you determine the optimal settings you can save the new method and use the automatic processing from then on for subsequent analyses for that type of sample Sample Comparison To measure consistency among batches of proteins you can compare the deconvolved spectra from two raw data files or even two different portions of the same deconvolved spectrum You designate one spectrum as a reference spectrum and the other as a source spectrum For example the first batch of proteins can serve as the reference spectrum that you can compare subsequent batches to The Protein Deconvolution application displays a mirror plot of the two spectra which you can enlarge to see whether the structures and the relative abundance of 4 Protein Deconvolution User Guide Thermo Scientific 1 Introduction Workflow masses in the two spectra are divergent or the same Major differences in these areas between the spectra can indicate that target protein sequences have been modified by post translational modifications such as phosphorylation or glycosylation You can then use top down proteomics techniques or peptide mapping to determine the exact cause of these changes This ability to compare spectra is particularly important in determining how closely a biosimilar recombinant monoclonal antibody imitates an innovator recombinant monoclonal antibody When used with peptide mapping and glycosylation profili
131. autions and special notices CAUTION Highlights hazards to humans property or the environment Each CAUTION notice is accompanied by an appropriate CAUTION symbol IMPORTANT Highlights information necessary to prevent damage to software loss of data or invalid test results or might contain information that is critical for optimal performance of the system Note Highlights information of general interest Tip Highlights helpful information that can make a task easier Thermo Scientific Protein Deconvolution User Guide ix Preface Contacting Us Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need For Thermo Scientific products Access by phone fax email or website Technical Support U S Phone 1 800 532 4752 Fax 1 561 688 8736 Email us techsupport analyze thermofisher com Web for product support technical documentation and knowledge bases www thermoscientific com support Customer Service Sales and service User Documentation x Protein Deconvolution User Guide U S Phone 1 800 532 4752 Fax 1 561 688 8731 Email us customer support analyze thermofisher com Web for product information www thermoscientific com Ic ms Web for customizing your service request 1 From any Products amp Services web page click Contact Us 2 In the Contact Us box complete the information requested scroll to the botto
132. ays each set of Xtract results that you saved from the same raw data e Saved ReSpect Results Displays each set of ReSpect results that you saved from the same raw data e Source Spectrum Displays the spectrum that you selected in the Source Spectrum pane of the Chromatogram page 164 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results e Chromatogram Displays the chromatogram that you selected in the Chromatogram pane of the Chromatogram page When you make a change to the chromatogram on the Chromatogram page the Protein Deconvolution application automatically updates the chromatogram on the Process and Review page e Deconvolved Spectrum Displays the deconvolved spectrum resulting from applying the ReSpect algorithm Results Displays the masses and intensities of the peaks that the ReSpect algorithm detected during the deconvolution along with their quality scores 2 Optional Adjust any parameters in the Main Parameters ReSpect pane For information on these parameters see able 27 on page 135 3 Click Process in the menu bar For information on the parameters in the panes of the Process and Review page see Table 32 on page 176 Displaying the Results When the Protein Deconvolution application finishes processing it displays the deconvolved spectrum in the Deconvolved Spectrum pane of the Process and Re
133. ble When the Protein Deconvolution application finishes processing all jobs the Chromatogram Process and Review Sample Comparison and Reporting tabs are not immediately available until you click Open Result When the Protein Deconvolution application finishes analyzing a data file the Run Queue page displays the following changes e The Number of Chromatographic Peaks column displays the number of detected chromatographic peaks 220 Protein Deconvolution User Guide Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue The Number of Components Detected column displays the number of components detected from the deconvolution of the averaged spectra from all found chromatographic peaks e The Completion Time column displays the date and time that the deconvolution finished for a given raw data file e The Status column changes to Completed The column displays Completed Report Not Concatenated when the size of the generated report is too large Figure 109 shows these columns Figure 109 Run Queue page after the application finishes processing a job Thermo Protei n Deconvol ution Auto ReSpect Isotopically Unresolved Help SCIENTIFIC C Method Selection f EEA C Parameters J chromatogram A Process and Review J Sample Comparison J Reportin gt Process methods defined in the work queue Queue Manipulation Set Priority O
134. ble 13 Menu bar commands on the Sample Comparison page for Xtract deconvolution Parameter Description Save Method Saves the reference spectrum to the method However you cannot use the Save Method command to save a reference spectrum to the default method you must use Save Method As Save Method As Opens the Save dialog box so that you can save the reference spectrum to a new method Reset from Method Replaces the displayed reference spectrum from the library with the reference spectrum in the method if a reference spectrum is stored in the method Save Result As Opens the SaveAs dialog box so that you can save the selected Mirror Plot Shortcut Menu reference spectrum to the current results By right clicking in the Mirror Plot pane on the Sample Comparison page you can access a shortcut menu with the commands listed in Table 14 Table 14 Mirror Plot pane shortcut menu commands Parameter Reset Scale Description Restores the original plot that first appeared in the Mirror Plot pane Copy Bitmap Copies the view in the Mirror Plot pane to the Clipboard Zoom Out Shrinks the plot in the Mirror Plot pane by a factor of two Zoom In Enlarges the plot in the Mirror Plot pane two times Displaying an Xtract Deconvolution Report Thermo Scientific When you click Process on the Process and Review page the Protein Deconvolution application generates a report displaying several aspects of the deconvolution so tha
135. box 41 Saves x Name Descriptions Cancel 3 In the Name box of the dialog box type the name of the reference spectrum 4 In the Descriptions box type a brief description of the reference spectrum 5 Click Save 6 Optional Click the Sample Comparison tab Figure 90 shows the Sample Comparison page The reference spectrum that you just saved appears in the Reference Spectrum Library section of the page The Mirror Plot pane displays the reference spectrum in the positive direction as the source spectrum When you select it as the reference spectrum it appears in the Mirror Plot pane in the negative direction 188 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 90 Sample Comparison page showing the reference spectrum displayed as the source spectrum MO Protein Deconvolution DetauitMethodReSpect Maa apai taarea hie a SCIENTIFIC IgG source cid raw Method Selection Run Queue Parameters Chromatogram __ Process and Review Reporting i Select a reference spectrum for sample comparison Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions Reference Spectrum Library Most Abundant Experiment Select Mass ain Creation Time Descriptions Reference Spectra Raw Data File Scan Ra
136. box opens 4 Specify the path and name of a PDF file to store the reports in and click Save The Protein Deconvolution application saves the report in a file called raw_file_name pdf If you do not specify a directory it places the file by default in the raw data directory shown on the Method Selection page If you generate a PDF file immediately after you deconvolve a spectrum the file remains in the directory until you manually remove it However if you generate a PDF file from previously saved results the file remains in the directory only until you exit the application To print a report 1 Move the cursor near the bottom of the screen 2 Click the Print File icon B on the Reporting page toolbar shown in Figure 43 on page 88 3 In the Print dialog box set the appropriate printing parameters and click OK 202 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Reporting Page Toolbar For information about the icons on the Reporting page toolbar see Reporting Page Toolbar on page 88 Sample Information Section The Sample Information section of the report shown in Figure 96 displays information about the sample where the spectrum was taken Figure 96 Sample Information section for ReSpect deconvolution Sample Information Original File Name C XCALIBUR Intact IgG_source_c
137. ce Spectrum Library section of the page The Mirror Plot pane displays the reference spectrum in the positive direction as the source spectrum When you select it as the reference spectrum it appears in the Mirror Plot pane in the negative direction Protein Deconvolution User Guide 73 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 37 Sample Comparison page showing the reference spectrum saved as the source spectrum m i H Manual Xtract Isotopically Resolved FMO Protein Deconvolution DetauttMethodxtract anuai Xtact Qsotapicaly Resolved o gip he CE NOT LFS PSA_240 raw C Method Selection J RunQueve C Parameters C Chromatogram 7 Process and Review D Reporti E Ea E tog ing compare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions Reference Spectrum Library Most Abundant Mass 27756 757 Number of Peaks Raw Data File Reference Spectra Scan Range RT Range Creation Time Descriptions Xtract7383 C Program Files Protein Deconvolution source 75 90 7 27 840 01 02 2014 15 53 47 Mirror Plot Source Data File C Program Files Protein Deconvolution source files XtrachPSA_240 raw NL 6 71E 004 w 27756 7570 80 Reference 26266 2459 ba 1933
138. created a new method return to the Methods pane and select the method 218 Protein Deconvolution User Guide Thermo Scientific 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up an Automatic ReSpect Protein Deconvolution 8 In the Load Raw Data File pane select the raw data file that contains the spectral data for your sample a In the Raw Data Directory box type the path of the raw data file or click the Browse button to browse to the directory containing the file For instructions on setting up a default raw data file directory see Specifying the Default Raw Data File Directory on page 9 b Inthe Select Raw Data Files area click the name of the raw data file Although you can load a raw data file with a maximum size of 34 GB processing and reporting issues might result when you load files this large Thermo Fisher Scientific recommends that you work with files up to 2 GB If you want to run a batch of data files usually of the same sample with a given method do the following e To select contiguous file names click the name of the first raw data file then hold down the SHIFT key and click the last file name that you want to select e To select noncontiguous file names click the name of the first raw data file then hold down the CTRL key and click each separate file name c Click Add to Queue The application transfers you to the Run Queue page sho
139. ctrum to use to compare to the source spectrum Delete Deletes the selected reference spectrum Add to Method Adds the selected reference spectrum to the method that you used to generate the reference spectrum Show Details Opens a Reference Spectrum Information box see Figure 41 on page 80 displaying the deconvolution parameters used to produce the reference spectrum that you selected in the reference spectrum library Mirror Plot Pane Source Data File Displays the mirror plot of the selected reference spectrum and the source spectrum Displays the path and name of the raw data file used to produce the source spectrum Reference Data File From Displays the path and name of the raw data file used to produce the reference spectrum Specifies the origin of the selected reference spectrum e Library Originates from the reference spectrum library e Method Originates from the method Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis 84 Protein Deconvolution User Guide Displays the mass of the ions formed from molecules Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Sample Comparison Page Menu Bar Commands Table 13 describes the commands on the menu bar of the Sample Comparison page Ta
140. d peak The Xtract algorithm ignores peaks below this threshold Any spectral peak must be x times the intensity of the calculated noise for that spectrum before the Xtract algorithm considers it Range 0 no maximum The recommended value is 2 to 7 Default 3 Protein Deconvolution User Guide 27 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 2 of 10 Parameter Rel Abundance Threshold Description Specifies a threshold below which the Protein Deconvolution application filters out data for data reporting This option sets a relative threshold as a percentage of the most abundant component in the spectrum The most abundant peak in the deconvolved spectrum has a relative abundance of 100 percent and all other peaks are calculated relative to that one In the Results pane on the Process and Review page the application shows only those components that are greater than or equal to this relative abundance threshold in the deconvolved spectrum For example if the highest peak has an absolute abundance of 1000 the relative abundance is 1 percent and no peaks below an absolute abundance of 10 will appear in the deconvolved spectrum Range 0 100 Zero 0 displays all results and 100 displays only the most abundant component Default 0 Negative Charge 28 Protein Deconvolution
141. d Results Raw Data Directory CAProgram Files Protein Deconvolutior Description Select Raw Data Files a Specifying the Default Raw Data File Directory You can specify the default directory where you want to store your raw data files 1M To specify the default directory for your raw data files 1 Click the Global Settings icon pe in the upper right corner of the Protein Deconvolution window to activate the Global Settings dialog box shown in Figure 5 Thermo Scientific Protein Deconvolution User Guide 9 1 Introduction Adjusting the Pane Size Figure 5 Global Settings dialog box LL Global Settings Coe ka Global Configuration General Application Configuration Global Settings Deecioy Setting General Parameters Raw Data Directory CAXcalibur data mm 2 In the Raw Data Directory box click the Browse button to browse to the appropriate directory 3 Click Apply Adjusting the Pane Size You cannot collapse the panes of the pages of the Protein Deconvolution window except for the following panes e Main Parameters Xtract or Main Parameters ReSpect on the Process and Review page e Saved Xtract Results or Saved ReSpect Results on the Process and Review page e Main Parameters Xtract or Main Parameters ReSpect on the Sample Comparison page e Source Spectrum pane on the Sample Comparison page e Deconvolved Spectrum pane
142. d intact antibody are found in the same sample choose 75 kDa as the target mass Default 150000 Da Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 5 of 15 Parameter Charge State Range Description Sets the allowable range for the number of charge states that must appear for a component to be recognized The ReSpect algorithm rejects potential components with fewer than the minimum or greater than the maximum number of charge states Default range for the default method 10 through 100 Default range for the ExampleMethodNativeMS method 5 through 100 Choice of Peak Model Specifies the appropriate peak model for the data The expected peak shapes for nucleotides are different from those for proteins and peptides because of their different isotopic composition e Default Intact Protein Specifies peak model widths that are appropriate for use with the intact protein data produced by Orbitrap MS instruments e Nucleotide Specifies peak model widths that are appropriate for use with the nucleotide data produced by Orbitrap MS instruments Advanced Parameters ReSpect pane Displays parameters that only infrequently need changing Only experienced users should change these parameters Peak Filter Parameters Displays parameters that control how pote
143. data or where it is not possible to accurately model the peak profile Default 3 Use when a high quality deconvolution is required and there is only a small change in peak width across the region to be processed Protein Deconvolution User Guide 129 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method 4 Use when extreme deconvolutions are required This value is only relevant for good signal to noise data where there is only a very small change in peak width across the region to be processed The designed model must also be a good fit to the peak profiles You can use only integer values Decreasing the value for this parameter reduces the computation time Starting at 0 the computation time roughly doubles for each integer increase e Noise Compensation Determines whether the ReSpect algorithm improves signal detection where the noise level varies across the data Default Selected Improves signal detection where the noise level varies across the data Cleared Does not improve signal detection where the noise level varies across the data e Minimum Adjacent Charges Specifies the minimum number of peaks in a row that must appear at the low and the upper end of the input spectrum if the ReSpect algorithm is to recognize a component as real The ReSpect algorithm rejects potential components with fewer than this number of adjacent peaks The mini
144. deconvolution results 69 description 1 2 displaying reports 86 displaying results 112 exporting results of deconvolution 68 loading saved deconvolution results 98 manual deconvolution 13 Thermo Scientific Index Z printing report 88 profile data 43 profile spectra 42 purpose 1 2 results of deconvolution 51 saving a report in PDF file 88 saving results of deconvolution 66 selecting a reference mass 60 selecting a spectrum to deconvolve 37 setting up manual deconvolution 14 types of spectra to use 13 Xtract Masses Table report section 96 Z Zoom In command ReSpect 152 157 162 180 200 Xtract 40 43 48 65 85 Zoom Out command ReSpect 154 157 162 180 200 Xtract 41 44 48 65 85 Thermo Scientific Protein Deconvolution User Guide 239
145. del Parameters JSST NnneS vi Number of Peak Models 1 Baseline Correction Resolution at 400 m z 12374 Peak Width i Left Right Peak Shape Left 2 Right 2 Feature Width 0 Reporting Parameters Deconvolved Spectrum Sample Information Components Table Chromatogram with Parameters Component Detail Tables Source Spectrum Deconvolution Parameters Component Source of Evidence Plots Automation Parameters only apply to autor Save to PDF Concatenate All Reports El ussjo sjprtsv1 SJTECHPUBS PHASER5500DN Select a Printer Auto Print 124 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method 2 Optional Change the appropriate parameters in the Main Parameters ReSpect pane e Negative Charge Indicates whether the data was acquired in positive charge mode or negative charge mode during the ESI process You might need to use this option when processing compounds that contain nucleotides like those found in DNA and RNA Deprotonation of nucleotides which are acidic occurs when the compound is dissolved in a basic solution and negative voltage is applied to produce negatively charged ions Selected The data was acquired in negative charge mode Default Cleared The data
146. e Abundance 100 0000 71 6096 24 0255 Fractional Abundance 14 0691 10 0748 3 3802 6 788 8 260 6 788 8 260 6 788 8 260 3 Select the appropriate reference spectrum from the Reference Spectrum Library by double clicking the spectrum or selecting the spectrum and then clicking Select The Mirror Plot now displays a mirror plot with the reference spectrum in the negative direction and the source spectrum in the positive direction as shown in Figure 39 Text at the top of the pane identifies the name of the source data file and text at the bottom of the pane identifies the name of the reference data file 76 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 39 Mirror plot showing the source spectrum and the reference spectrum Manual Xtract isotopically Resolved EA Thermo Protein Deconvolution netauitMethodxtract Help X SCIENTIFIC PSA_240_highplraw _ Method Selection Run Queue Parameters Chromatogram Process and Review M EOI A Reporting i Select a reference spectrum for sample comparison Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions Y Reference Spectrum Library Most Abundant Experiment Mass Type Xtract7383 C Program Files Protein Deconvolution source 7
147. e C Program Files Protein Deconvolution source files Xtract PSA_240_highpl raw NL 7 85E 005 28412 9569 100 28121 8589 50 2 28453 9527 A 27498 5165 En 27716 5895 27790 6337 27862 6864 27976 7711 28105 8257 ia eee 28200 9039 28370 9318 L 28513 0207 E a o Pty TT ee a g i 28414 9952 28472 0486 27511263 277147390 7796 7844 27855 8096 L 28058 8692 28266 9389 28307 9693 3 50 27976 8125 28162 9090 Aoi 27756 7570 27934 8388 28121 8884 Mass Reference Data File C Program Files Protein Deconvolution source files Xtract PSA_240 raw From Library NL 6 71E 004 lt m In this example the base protein amino acid sequence is the same in the two samples but the pattern of glycosylation is different Many of the masses in the two samples are the same but their relative abundances are noticeably different For example the most abundant peak in the source spectrum plot is 28 412 but the most abundant peak in the reference spectrum plot is 27 756 5 Optional In the Reference Spectrum Library pane sort the data in each column by clicking in the column header and then clicking the up arrow E to sort the data from low to high or clicking the down arrow l to sort the data from high to low 6 Optional Save the results See Saving the Results of a Deconvolution on page 66 78 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra w
148. e Results on page 51 e Default Selected Displays the information that appears in the Results pane on the Process and Review page e Cleared Does not display the information that appears in the Results pane on the Process and Review page Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 9 of 10 Parameter Component Detail Tables Description Determines whether to display the Monoisotopic Mass table for each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 96 e Selected Displays the Monoisotopic Mass table in the Source Spectrum Evidence section of the report e Default Cleared Does not display the Monoisotopic Mass table in the Source Spectrum Evidence section of the report Component Source of Evidence Plots Determines whether to display the spectrum of each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 96 e Selected Displays the spectrum of each component in the Source Spectrum Evidence section of the report e Default Cleared Does not display the spectrum of each component in the Source Spectrum Evidence section of the report Apply Autom
149. e 90 Source Chromatogram Sectiotivs c 0 44 50htaead oes ead ee AS eS Be 91 Source Spectrum Secon ept i Epe E cee eee ees 92 Main Parameters Xtract Section 0 cece eee eee 93 Advanced Parameters Xtract Section 0 cece ee eee eee 93 Deconyolved Spectrum Section s es aa datas kate aa beet ete oa 94 Mirror Plot Section o an 0 tiene those Passe acata Bieta 8 ean BA a Searles 95 Xtrac Masses Table Section i 4 ween ils tees welds Geil Mm e ded a ek 96 Source Spectrum Evidence Section oso 4 aes aes aces Aree is Aes ces 96 Loading Saved tract Results o nuuanu 98 Automatically Deconvolving isotopically Resolved Mass Spectra with the Xtract Algorithm 0 ccc cece eee e eee eeeaneeee 101 Setting Up an Automatic Xtract Protein Deconvolution 101 Running Jobs in the Quetiet st pana hse hares tee ewe Mee ee a adtels 105 Run Queue Page Parameters for Automatic Xtract Deconvolution 109 Comparing Samples Generated with the Automatic Xtract Algorithm 111 Displaying the Results when haves ieee eae Se Pea Rss 112 Result Names gas ac cirpant Raines Cale ees hee ai ee RE aes 113 Thermo Scientific Thermo Scientific Chapter 4 Contents Displaying an Xtract Deconvolution Report 0 00 0 c cee eee eee 114 Loading Saved Xtract Results onnu nuunuu 115 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm ccc ssiencawte tes tinted
150. e C e Video card and monitor capable of 1280 x 1024 resolution e NTFS format Software e Adobe Reader 10 e Microsoft Windows 7 Professional with Service Pack 1 32 bit or 64 bit e NET Framework 4 0 Extended e Thermo Scientific software Protein Deconvolution Foundation Xcalibur 3 0 3 0 3 0 A DVD drive is not required if you install the software from the Internet It is required only if you install the software from a DVD License Activation and Deactivation Use the Thermo Scientific Product Licensing wizard to activate or deactivate the license for the Protein Deconvolution application To activate the license you must have an activation code from Thermo Fisher Scientific You must deactivate the license before you transfer it to another computer To start the license activation or deactivation process 1 Open the Protein Deconvolution application 2 Choose File gt About Protein Deconvolution to display the Product Licensing wizard viii Protein Deconvolution User Guide Thermo Scientific Preface Cautions and Special Notices 3 Click Activate Deactivate to start the activation or deactivation process as applicable Cautions and Special Notices Make sure you follow the cautions and special notices presented in this guide Cautions and special notices appear in boxes those concerning safety or possible system damage also have corresponding caution symbols This guide uses the following types of c
151. e F The scan filter used during the LC MS run for example FTMS p ESI Full ms 1000 00 4000 00 The scan filter indicates the type of mass analyzer used to acquire the data in the raw data file and the ionization technique used If this field is blank no scan filter was used Deconvolving the Spectrum Reaching the Process and Review page shown in Figure 76 means that you have already selected the chromatogram and source spectrum on the Chromatogram page or that the Protein Deconvolution application has transferred you because the raw data file contains a spectrum only You can zoom in and out of the chromatogram and source spectrum views but you cannot change them on the Process and Review page You must manually navigate back to the Chromatogram pane to change these views Use the Process and Review page to deconvolve the selected spectrum and to view the resulting data to ensure that the results make sense You can also export the data to a Microsoft Excel file for use in other applications To deconvolve the spectrum 1 If you are not already on the Process and Review page click the Process and Review tab Figure 76 shows the initial Process and Review page Thermo Scientific Protein Deconvolution User Guide 163 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Deconvolving the Spectrum Figure 76 Initial Process and Review page for ReSpect deconvolution Protein Deconvolution Monzi Ds
152. e ReSpect Algorithm Its When you select some components in the Results table and then choose the Export Top Level or Export All command the resulting Excel file reflects the columns that you chose and the order in which you arranged them However the columns and the order are not reflected in the generated reports When you close and then reopen the Protein Deconvolution application it does not retain the columns and order that you chose However when you simply leave an experiment and return to it the application retains this information 3 Click the column header to sort the data in each column of the peak table from lowest to highest or highest to lowest For example click the Number of Charge States column header The Protein Deconvolution application which initially displays the number of charge states in this column in order from lowest to highest now displays the number of charge states from highest to lowest Click again to display the numbers from lowest to highest 4 Click the plus sign to the far left of a row in the peak table As shown in Figure 79 five new columns appear Charge State Measured Average m z Measured Average Mass Delta Mass Da and Delta Mass PPM These values represent the charge state peaks that constitute the peaks shown in the five output columns Figure 79 Hierarchical table in the Results pane for ReSpect deconvolution SCIENTIFIC _ Method Selection Deconvoive the spectr
153. e Set Priority gt priority_level where priority_level can be one of the following e Low Processes the job after jobs that are assigned a Normal or High priority level e Default Normal Processes the job before jobs that are assigned a Low priority level but after jobs that are assigned a High priority level e High Processes the job before jobs that are assigned a Low or Normal priority level High priority jobs are highlighted in orange If you assign the same priority level to multiple jobs the Protein Deconvolution application prioritizes the jobs by the date and time that you submitted them to the queue Thermo Scientific Protein Deconvolution User Guide 107 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Running Jobs in the Queue To temporarily stop the processing in the run queue 1 Click Pause The Pause button is only available when the queue contains two or more jobs and the Status column shows Processing as shown in Figure 58 You cannot pause only a single job running in the queue The pause affects the next sample to be processed not the sample currently being processed Figure 58 Pausing an Xtract job i i Auto Xtract Isotopically Resolved O Protein Deconvolution ia Om SCIENTIFIC __ Method Selection OE C Parameters Chromatogram Process and Review J Sample Comparison Reporting o Process methods defined in the work queue Queue Manipulati
154. e Source Spectrum pane by doing one of the following For a single scan In the Chromatogram pane place the red cross shaped cursor on the chromatogram to select a single scan and to display the associated mass spectrum at that time point as shown in Figure 18 on page 42 You can use the left and right arrow keys to move to the previous or next time point in the chromatogram The spectrum window automatically updates or For multiple scans Select a region of the chromatogram to display an averaged spectrum for all the scans in the selected region in the Source Spectrum pane i Right click and choose Mode gt Averaging if it is not already selected ii Drag the red cross shaped cursor across the area of interest as shown in Figure 72 The horizontal line of this cursor aids in assessing peak height The Protein Deconvolution application calculates an average spectrum for this interval The averaging method is better suited to complex data than the single scan method Averaging spectra produces higher signal to noise ratios so higher quality spectra are highly recommended for optimal deconvolution results Tip Optional You can perform step on page 149 through step 4 on page 154 in Qual Browser in the Xcalibur data system and then right click and choose Export gt Write to RAW File so that you can import the file into the Protein Deconvolution application The spectrum appears in the Source Spectrum pane of the Ch
155. e Spectrum pane becomes available after you click Add to Method in the Reference Spectrum Library pane In either case compare these parameters to the parameters that you set to generate the spectrum for the current experiment to ensure that the two spectra are comparable 194 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 94 Reference Spectrum Information box Thermo Protein Deconvolution detauitmetnoarespect ll SCIENTIFIC IgG_source_cid raw o Method Selection Run Queue o Parameters go Chromatogram o Process and Review v Sample Comparison Reporting o Compare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Method Reference Spectrum Reference Spectrum Reference Data File Ea v Reference Spectrum Library Most Abundant Experiment Number of Mass Type Peaks C Program Files Protein Deconvolution source 1161 1246 3 39 3 50 151814 828 RSP 80 02 10 2014 14 09 51 Reference Spectra Raw Data File Creation Time Descriptions ReSpect3435 Scan Range RT Range Reference Spectrum Information Reference Source Spectrum Information Raw Data File IgG_source_cid raw Experiment Type RSP Scan Range 1161 1246 RT Range 3 39 3 50 Number of Peaks 80 Most Abundant Mass 151 814 828 Deconvolution Parameters
156. e does not display peak labels such as the retention time and scan number nor the header information for the source spectrum and chromatogram However it still displays the intensity of the most abundant peak in the entire LC MS run for the chromatogram e Deconvolved Spectrum pane When you click an isotopic cluster the Deconvolved Spectrum pane displays the deconvolved spectrum of the selected isotopic cluster as shown in Figure 32 Figure 32 Deconvolved Spectrum pane showing the deconvolved spectrum of an isotopic cluster Deconvolved Spectrum 16941 0113 100 q gt o o S 6 amp 6 6 Relative intensity N 16568 8381 1563 2961 T T T T T T T T T T T T T T T T T T T T T 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 Mass o When you click an individual charge state of a selected isotopic cluster the deconvolved mass of the specific component in the Deconvolved Spectrum pane changes It shows the mass for that single charge instead of the mass of the top level deconvolved component Selecting a Reference Mass to Calculate Mass Differences A reference mass is usually the mass of the most abundant peak in the results The Protein Deconvolution application compares the mass of all other peaks in the data set to the reference mass and places these differences in the Delta Mass column of the Results table on the Process and Review page The delta mass is useful in revealing the structure of
157. e instrument method are correct but when the amount of internal standard actually in one or more samples is different than the amount specified in the instrument method This correction eliminates the necessity of remaking any samples to the internal standard concentrations or amounts specified in the instrument method and rerunning the samples Dil Factor Specifies the dilution factor that was used to prepare the sample Chromatogram Parameters Section The Chromatogram Parameters section shown in Figure 97 displays the settings that you chose in the Chromatogram Parameters pane of the Chromatogram page For more information on these parameters see Table 29 on page 158 Figure 97 Chromatogram Parameters section for ReSpect deconvolution Chromatogram Parameters Use Restricted Time False Time Range 0 004648833 6 2300942 Rel Intensity Threshold 1 Type TIC Chromatogram m z Range 1000 4000 204 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Source Chromatogram Section The Source Chromatogram section shown in Figure 98 displays the chromatogram contained in the raw data file It is the same chromatogram that appears in the Chromatogram pane of the Chromatogram page Figure 98 Source Chromatogram section for ReSpect deconvolution Source Chromatogram IgG_source_cid ProteinD
158. e intensity that is not part of the useful signals e Compute the noise compensation which can improve the separation of noise from signals in subsequent processing e Compute noise estimates for the noise compensated data The Protein Deconvolution application applies these parameters before performing any other processing operations It must perform these functions on raw unfiltered data Peak Width Specifies the half height width of a typical peak in data intervals This value does not need to be exact The default of 0 causes the ReSpect algorithm to calculate a peak width automatically Default 0 Feature Width Specifies how wide the feature of the baseline should be in data points The default of 0 causes the ReSpect algorithm to calculate a feature width automatically Default 0 Degree of Fit Specifies the offset by which to lower or raise the baseline height This adjustment is non linear so that its effect is proportional to the underlying noise amplitude Set Degree of Fit to 0 for automatic computation or to 1 for manual computation Range 0 0 2 0 Default 0 Values less than 1 lower the computed baseline and values greater than 1 raise it 142 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 9 of 15 Parameter D
159. e name of the first job hold down the CTRL key and click each separate job On the Run Queue menu bar choose Queue Manipulation gt Remove Selected The confirmation box shown in Figure 60 appears Figure 60 Message box confirming the removal of completed jobs Protein Deconvolution X This action will remove the items from the queue permanently Do you want to continue Click Yes To remove all jobs from the queue On the Run Queue menu bar choose Queue Manipulation gt Remove All You can only remove all jobs when their status is Queued or Completed In the confirmation box shown in Figure 60 click Yes To remove all completed jobs from the queue On the Run Queue menu bar choose Queue Manipulation gt Remove Completed In the confirmation box shown in Figure 60 click Yes Run Queue Page Parameters for Automatic Xtract Deconvolution Table 22 describes the parameters available on the Run Queue page for an automatic Xtract deconvolution Table 22 Run Queue page parameters for automatic Xtract deconvolution Sheet 1 of 3 Parameter Description Menu bar commands Queue Manipulation Contains commands that cancel or remove selected deconvolution jobs from the queue Thermo Scientific Remove Selected Deletes the selected job or jobs from the queue Protein Deconvolution User Guide 109 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algo
160. e results but you cannot save it to the method You cannot delete the original reference spectrum that was saved in the results You cannot save the mirror plot that was generated with the new reference spectrum but you can copy its contents to the Clipboard to use in third party applications such as PowerPoint To view the original mirror plot that was saved with the results you must reload the results The Protein Deconvolution application does not update the report to be consistent with the contents of the modified mirror plot Sample Comparison Page Parameters for the ReSpect Algorithm Thermo Scientific Table 35 lists the parameters that are available on the Sample Comparison page for ReSpect deconvolution Table 35 Sample Comparison page parameters for ReSpect deconvolution Sheet 1 of 3 Parameter Description Method Reference Spectrum Displays information about the reference spectrum in the pane currently loaded method Reference Spectrum Displays the name of the reference spectrum in the currently loaded method as you specified it in the SaveAs dialog box Protein Deconvolution User Guide 197 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Table 35 Sample Comparison page parameters for ReSpect deconvolution Sheet 2 of 3 Parameter Descriptions Description Displays the description of the reference spectrum in the currently loaded method as you spec
161. e spectrum This parameter is particularly useful with spectra where the noise level varies significantly across the spectrum especially for highly complex spectra for 150 kDa antibodies This noise is not a product of the instrument but is instead due to the high complexity of the sample Peak Width Specifies the half height width of a typical peak in data intervals This value does not need to be exact The default is 0 which causes the ReSpect algorithm to calculate a peak width automatically Feature Width Specifies how wide the feature of the baseline should be in data points The default is 0 which causes the ReSpect algorithm to calculate a feature width automatically Degree of Fit Specifies the offset by which to lower or raise the baseline height This adjustment is non linear so that its effect is proportional to the underlying noise amplitude Set Degree of Fit to 0 for automatic computation or to 1 for manual computation Values less than 1 lower the computed baseline and values greater than 1 raise it The range is 0 0 2 0 The default is 0 Number of Iterations Specifies how far the deconvolution is to proceed 0 Use when a crude deconvolution is sufficient Use only for data with a large variation in peak width or where speed is important 1 Use where there is a substantial change in peak width across the data to be processed 2 Use if there is a significant change in peak width across the
162. e top level deconvolved component In a spectrum deconvolved with the ReSpect algorithm the area of a peak is proportional to the intensity of the associated component charge peak state so lower quality results are associated with wider peaks If two components have equal intensities the component with the lower quality results has a wider peak Selecting a Reference Mass to Calculate Mass Differences A reference mass is usually the mass of the most abundant peak in the results The Protein Deconvolution application compares the mass of all other peaks in the data set to the reference mass and places these differences in the Delta Mass column of the Results table on the Process and Review page The delta mass is useful in revealing the structure of the target component However you can select the mass of another component in the table to use as the reference mass The default delta mass of this reference component is 0 The application then compares the mass of other peaks in the data set to this default 172 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results When you load multiple results concurrently the application calculates the delta masses only for components from the same deconvolved spectrum If you export the results to the Excel application see Exporting the Results of a Deconvolution on page 182 the Excel fi
163. e used to produce the reference spectrum Specifies the origin of the selected reference spectrum e Library Originates from the reference spectrum library e Method Originates from the method Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis Displays the mass of the ions formed from molecules Sample Comparison Page Menu Bar Commands Thermo Scientific Table 36 describes the commands on the menu bar of the Sample Comparison page Table 36 Menu bar commands on the Sample Comparison page for ReSpect deconvolution Sheet Parameter Description Save Method Saves the reference spectrum to the method However you cannot use the Save Method command to save a reference spectrum to the default method you must use Save Method As Save Method As Opens the Save dialog box so that you can save the reference spectrum to a new method Protein Deconvolution User Guide 199 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Table 36 Menu bar commands on the Sample Comparison page for ReSpect deconvolution Sheet Parameter Description Reset from Method Replaces the displayed reference spectrum from the library with the reference spectrum in the method if a reference spectrum is stored in the method Save Result As Opens the SaveA
164. econvolution NL 3 10E9 RT 3 41 S 1171 Relative Intensity Table 39 lists the parameters in the Source Chromatogram section Table 39 Source Chromatogram section parameters for ReSpect deconvolution Parameter Description Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity RT min axis Displays the retention time of the spectrum which is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Thermo Scientific Protein Deconvolution User Guide 205 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Source Spectrum Section The Source Spectrum section shown in Figure 99 displays the spectrum that you selected in the Source Spectrum pane of the Chromatogram page Figure 99 Source Spectrum section for ReSpect deconvolution IgG_source_cid 1134 1260 RT 3 36 3 52 AV 126 F 2761 2701 100 Relative Intensity S 3535 4388 10 1659 4856 1 1383 1411 Last ANNIN VV VN Wy lg i 3981 4358 1000 1500 2000 2500 3000 3500 4000 m z Table 40 lists the parameters in the Source Spectrum section Table 40 Source Spectrum section parameters for ReSpect deconvolution Parameter Description Relative Intensity y axis Displays the ratio of the in
165. econvolution application 8 Instrument Method column 90 203 intact proteins 128 139 Intensity column 177 180 182 Isotope Table parameter 22 31 isotope tables 22 isotopic clusters 23 32 51 63 isotopically resolved mass spectra 1 2 13 101 isotopically unresolved mass spectra 2 3 117 215 ISTD Amount column 90 204 J job queue assigning priority levels to jobs 107 221 maximum number of samples processed 105 219 removing a single job from 109 removing all completed jobs from 109 223 removing all jobs from 109 223 Thermo Scientific Index F removing single job from 223 running jobs in 105 220 stopping job processing 108 222 L Left Right Peak Shape parameter 132 146 license activation or deactivation viii transfer viii Load button 17 123 Load Method button 16 18 120 123 Load Raw Data File pane parameters on 17 123 purpose 14 102 119 216 Load Result button 62 176 loading previous results 17 122 Low command 107 110 221 224 m z Range parameter ReSpect 121 126 137 218 Xtract 21 29 m z x axis label Chromatogram page 47 161 Source Spectrum Evidence section of report 97 212 Source Spectrum pane of the Process and Review page 62 176 Source Spectrum section of report 92 206 Main Parameters ReSpect pane parameters on 125 135 176 purpose 123 164 resizing 10 Main Parameters ReSpect report section 132 147 207 Main Parameters Xtract pane parameters on 19 27 62 purpose 18 5
166. ectra generated by the Xtract algorithm or you can compare two spectra generated by the ReSpect algorithm You can compare a spectrum generated by an automatic workflow to a spectrum generated by a manual workflow However you cannot compare any spectrum generated by an Xtract algorithm to any spectrum generated by a ReSpect algorithm For convenience you can save a reference spectrum to a method for future use For manual workflows saving the reference spectrum to a method is optional to automatically produce a mirror plot for automated workflows it is required Note You can still load the results of an automated workflow and compare samples in the Load Previous Results workflow However if you modify the parameters of the original sample comparison you cannot include the resulting mirror plot in the report The Protein Deconvolution application saves a deconvolved spectrum as a reference spectrum in a library whether you deconvolved a spectrum by using an Xtract or ReSpect workflow or loaded previously saved results You can generate an unlimited number of reference spectra The application saves the following information in the database so that if necessary you can reconstruct how you created the reference spectrum e The absolute path and name of the original raw data file used to produce the reference spectrum e The algorithm used to generate the result manual Xtract manual ReSpect automatic Xtract or automatic ReSpect
167. ed Parameters ReSpect pane parameters 128 139 purpose 123 Advanced Parameters ReSpect report section 132 147 207 Advanced Parameters Xtract pane parameters on 23 31 purpose 18 Advanced Parameters Xtract report section 24 93 Apex RT column 64 66 68 178 180 182 Auto Print parameter 36 104 148 218 Auto Zooming mode 40 48 153 162 automatic protein deconvolution methodology 4 peak detection and spectral peak modeling 5 automatic ReSpect deconvolution description 17 122 215 displaying deconvolution results 226 displaying results of deconvolution 228 loading saved results 229 methodology 4 peak detection and spectral peak modeling 5 running jobs in queue 220 saving reference spectrum 70 185 setting up 216 automatic Xtract deconvolution comparing samples 111 226 description 17 101 122 displaying results 112 114 loading saved results 115 methodology 4 peak detection and spectral peak modeling 5 Thermo Scientific running jobs in queue 105 setting up 101 Automation Parameters pane parameters on 35 104 148 218 purpose 18 124 Average Charge column 63 66 68 Average Mass column 169 177 180 182 averagine isotope distribution 23 31 64 Averaging mode 41 48 154 162 averaging multiple scans 41 154 base peak chromatogram 39 45 151 159 baseline correction 142 baseline subtraction 3 BPC chromatogram See base peak chromatogram C Calculate XIC parameter ReSpect 126 136 171 Xtract 21 29
168. efault method etapa Whe method Output Mass Range 10 000 160 000 10 000 1 000 000 Mass Tolerance 20 ppm 30 ppm Minimum Adjacent 6 10 4 10 Charges Charge State Range 10 100 5 100 M z Range 1000 4000 1000 15 000 To delete a method from the Methods pane In the Methods pane of the Method Selection window select the method that you want to delete Press the DELETE key Click Yes in the confirmation box as shown in Figure 64 Protein Deconvolution User Guide 121 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up a Manual ReSpect Protein Deconvolution Figure 64 Method deletion confirmation box Protein Deconvolution x Selected method will be deleted permanently Do you want to continue Method Selection Page Parameters The Method Selection page consists of the Experiment Types Load Raw Data File and Methods panes The parameters in these panes are the same for Xtract and ReSpect deconvolutions Experiment Types Pane Parameters Table 24 lists the parameters in the Experiment Types pane of the Method Selection page Table 24 Experiment Types pane parameters Parameter Description Manual Xtract Isotopically Deconvolves an isotopically resolved mass spectrum with the Resolved Xtract algorithm Manual ReSpect Deconvolves an isotopically unresolved mass spectrum with Isotopically Unresolved the ReSpect algorithm Auto Xtract
169. eight Displays the amount of a component in the sample Sample Volume uL Displays the volume of a component in the sample ISTD Amount Specifies the correction for the internal standard amount If the value in this box is not 0 000 the value is used in an algorithm to correct for a case when any internal standard amounts specified in the active instrument method are correct but when the amount of internal standard actually in one or more samples is different than the amount specified in the instrument method This correction eliminates the necessity of remaking any samples to the internal standard concentrations or amounts specified in the instrument method and rerunning the samples Dil Factor Specifies the dilution factor that was used to prepare the sample Chromatogram Parameters Section The Chromatogram Parameters section shown in Figure 45 displays the settings that you chose in the Chromatogram Parameters pane of the Chromatogram page For information on these parameters see Table 6 on page 44 Figure 45 Chromatogram Parameters section for Xtract deconvolution Chromatogram Parameters Use Restricted Time False Time Range 0 00585 10 9839066666667 Rel Intensity Threshold 1 Type TIC Chromatogram m z Range 300 2000 90 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Source Ch
170. election page If you generate a PDF file immediately after you deconvolve a spectrum the file remains in the directory until you manually remove it However if you generate a PDF file from previously saved results the file remains in the directory only until you exit the application To print a report Move the cursor near the bottom of the screen Click the Print File icon on the Reporting page toolbar shown in Figure 43 In the Print dialog box set the appropriate printing parameters and click OK Reporting Page Toolbar You can activate the Reporting page toolbar shown in Figure 43 by hovering the cursor near the bottom of the screen Figure 43 Reporting page toolbar 88 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report This toolbar contains the following icons Table 15 Icons on the Reporting page toolbar Opens the Save a Copy dialog box so that you can save the report in a PDF file Opens the Print dialog box so that you can print the reports Displays the previous page Displays the next page Displays the current page followed by the total number of pages in the 248 report To move to a different page double click the current page type the new page number and press ENTER _ Enlarges the view Shrinks the view Ac
171. end on the data in the raw data file This parameter is only available when you select the Use Restricted Time parameter Rel Intensity Threshold 158 Protein Deconvolution User Guide Sets a lower intensity threshold for peaks in the chromatogram as a percentage The Protein Deconvolution application ignores peaks with relative intensities below this threshold Range 0 100 Default 1 This parameter is different from the Relative Abundance parameter on the Parameters page which sets a lower intensity for signals in the spectrum vot in the chromatogram Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Table 29 Chromatogram page parameters for ReSpect deconvolution Sheet 2 of 4 Parameter Description Types Determines the type of chromatogram displayed in the Chromatogram pane e Default TIC Displays a total ion current chromatogram which shows the summed intensity across the entire range of masses being detected at every point in the analysis The range is typically several hundred mass to charge units or more In complex samples the TIC chromatogram often provides limited information as multiple analytes elute simultaneously obscuring individual species A TIC in combination with a narrow m z range is effectively an extracted ion chromatogram XIC e BPC Displays a base peak chromatogram which shows o
172. ent information in the spectrum You must set this parameter for all other spectrum types and for exported spectrum files in qb raw file format which lack instrument information If you change the resolution the application applies the changed resolution to the currently loaded data file However the next time that you use the method with a raw data file it applies the instrument resolution to the method The range of values for the Resolution at 400 m z parameter is 6000 to 240 000 The default is not a fixed number but varies from raw data file to raw data file In cases where the mass spectrometer measured the resolution in the raw data file at an m z value other than 400 the ReSpect algorithm scales it as follows to account for the variation in instrument resolution versus m z M measured 400 R R converted measured Thermo Scientific Protein Deconvolution User Guide 131 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method 132 where R converted iS the resolution to be converted Ryreasured is the resolution at the measured mass to charge ratio M measured iS the measured mass to charge ratio other than 400 Left Right Peak Shape Control the sharpness of a peak The default for both the left and the right peak shape is 2 If you want to return the parameters in the Advanced Parameters ReSpect pane to the settings tha
173. ent Source of Evidence Plots Determines whether to display the spectrum of each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 96 Selected Displays the spectrum of each component in the Source Spectrum Evidence section of the report Default Cleared Does not display the spectrum of each component in the Source Spectrum Evidence section of the report If you want to return the parameters in the Reporting Parameters pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any parameters in this pane but do not click Apply and then click another tab the message box shown in Figure 12 on page 22 appears Click Yes to apply the parameter changes or No to restore the parameter defaults 5 Save the method and give it a name a Click Save Method or Save Method As The Save Method command saves current parameter values to the existing method overwriting any previous values The Save Method As command saves parameter values to a new method Protein Deconvolution User Guide 25 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method b 26 Protein Deconvolution User Guide In the Method Name box of the Save or SaveAs dialog box type a name for the method The name cannot contain sp
174. eport to a PDF file e Auto Print Automatically prints the automatically generated deconvolution report contained in the PDF file When you select this option the Select a Printer option becomes available so that you can select the printer e Concatenate All Reports Combines all reports for all deconvolved spectra from the same raw data file into a single PDF file e Select a Printer Displays a list of available printers that can print the deconvolution report PDF file This option becomes available when you select the Auto Print option In the Automation Parameters pane click Apply 6 Click the Method Selection tab again 7 If you made changes to a method or created a new method return to the Methods pane and select the method 8 In the Load Raw Data File pane select the raw data file that contains the spectral data for your sample a Protein Deconvolution User Guide In the Raw Data Directory box type the path of the raw data file or click the Browse button to browse to the directory containing the file For instructions on setting up a default raw data file directory see Specifying the Default Raw Data File Directory on page 9 In the Select Raw Data Files area click the name of the raw data file Although you can load a raw data file with a maximum size of 34 GB processing and reporting issues might result when you load files this large Thermo Fisher Scientific recommends that you work with
175. er settings in the Main Parameters Xtract pane Advanced Parameters Displays parameters that only infrequently need changing Xtract pane Only experienced users should change these parameters Fit Factor Measures the quality of the match between a measured isotope pattern and an averagine distribution of the same mass Enter a value between 0 and 100 percent e 0 requires a low fit only e 100 means that the measured isotope profile is identical to the theoretical averagine isotope distribution Range 0 100 Default 80 percent Thermo Scientific Protein Deconvolution User Guide 31 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 6 of 10 Parameter Remainder Threshold Consider Overlaps Description Specifies the height of the smaller overlapping isotopic cluster as a percentage with respect to the height of the most abundant isotopic cluster when the Xtract algorithm attempts to resolve overlapping isotopic clusters For example if one isotopic cluster in a spectrum has an abundance of 100 and you set the Remainder Threshold parameter to 30 the Xtract algorithm ignores any overlapping clusters with an abundance less than 30 Range 0 100 percent Default 25 percent Determines whether the Xtract algorithm is more tolerant of errors when the spectrum intensity i
176. erate a deconvolved spectrum from an isotopically unresolved intact protein mass spectrum the source MS spectrum can be a single spectrum from an LC MS data file an averaged spectrum from an LC MS data file or a single spectrum from a raw data file containing only that spectrum The ReSpect algorithm transforms this source spectrum into a mass spectrum and displays it in a new pane labeled with mass units rather than with the mass to charge ratio on the x axis For information on the ReSpect algorithm see ReSpect Algorithm on page 3 Protein Deconvolution User Guide 117 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up a Manual ReSpect Protein Deconvolution The ReSpect algorithm deconvolves spectra by following this general procedure 1 It generates a peak model using parameters provided by the method These parameters include a right and left hand width and a shape that describe the width of a peak and its deviation from a Gaussian The Protein Deconvolution application calculates a shape for a target protein using the target mass in the ReSpect method and the instrument resolution along with the right and left peak shape parameters from the method The ReSpect algorithm then processes this shape It deconvolves the spectrum using this peak model to detect a list of peaks and their associated parameters such as m z value intensity and an uncertainty in m z It filters this peak
177. erest Source Spectrum IgG_source_cid 1059 1331 RT 3 26 3 61 AV 272 FTMS p ESI Full ms 1000 00 4000 00 2761 2685 100 90 80 70 2667 2533 2452 2543 2339 1691 3231 1617 Relative Intensity a 2141 5468 3374 6304 1508 7419 1659 4849 1947 3273 3619 4613 1106 7029 1276 6958 T T T T r T T T 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 mz F 3785 9021 3931 3879 T T T i T 3200 3400 3600 3800 4000 Draw a line under the baseline of the peaks of interest 156 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Right click and choose Zoom In to enlarge the view of the entire spectrum Figure 75 gives an example of the enlarged peaks Figure 75 Enlarged peaks in the Source Spectrum pane for ReSpect deconvolution Source Spectrum IgG_source_cid 1059 1331_RT 3 26 3 61 AV 272 F FTMS p ESI Full ms 1000 00 4000 00 2761 2685 2812 3428 2865 4293 28093741 2871 5502 2874 4718 2806 4102 2775 5003 F 2826 8292 2859 4731 2880 1818 2856 7191 Relative Intensity 2734 7586 2753 0285 27871884 2803 8734 2894 4574 TAE r e e Deane n e eee e ee e ines r e ee e eT 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850 2860 2870 2880 2890 2900 miz e To shrink the view of the entire spectru
178. ers ReSpect Negative Charge E m z Range Min 1000 Max 4000 a Charge Carrier H 1 00727663 Ouiput Mex Ranae Min Max aa 2H 2 013553 ms 2 a 160009 Na 22 9892213 Mass Tolerance 30 Custom Target Mass 150000 Da Rel Abundance Threshold 0 Calculate XIC v Quality Score Threshold 0 Charge State Range 10 E to 100 ga Choice of Peak Model Intact Protein v Saved Xtract Results 2761 2707 2618 5392 1659 4859 2169 7448 3535 4397 RT min Deconvolved Spectrum 400 151814 828 co So Y Saved ReSpect Results a So gt A S E v El N 77289 125 50734 332 64862 207 100999 578 142108 328 Number of Relative Fractional Mas J PPI Dev i SS Charge States asaka MOm Dena Bie Abundance Abundance 151814 828 612 809 920 00 48 19 0421 3 394 3 502 34 151975 641 396 609 632 00 47 12 3240 3 394 3 502 340 7 gt 2 Click Save As Reference in the upper right corner of the Protein Deconvolution window Note The Save As Reference command is unavailable if a deconvolved spectrum is not visible in the Deconvolved Spectrum pane or if you have not yet deconvolved the spectrum The SaveAs dialog box appears as shown in Figure 89 Thermo Scientific Protein Deconvolution User Guide 187 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Figure 89 SaveAs dialog
179. ers you to the Parameters page The Parameters page features four panes containing parameters that control the deconvolution e Main Parameters Xtract Displays basic parameters that might change often These parameters also appear on the Process and Review page e Advanced Parameters Xtract Displays parameters that change infrequently Only experienced users should change these parameters e Reporting Parameters Displays parameters that determine what information appears on the Reporting page after the Protein Deconvolution application generates the deconvolution reports e Automation Parameters Displays parameters that control the reporting for automatic deconvolution For information on automatic Xtract protein deconvolution see Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm on page 101 For detailed descriptions of the parameters on these four panes see Table 4 on page 27 To create an Xtract method 1 Click the Parameters tab if it is not already selected The default settings for the Xtract algorithm automatically populate the parameter boxes on the Parameters page as shown in Figure 11 18 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Figure 11 Parameters page for Xtract deconvolution J Protein Deconvolution Manua Maroc fotop
180. escription Deconvolution Displays parameters that control the operation of the Parameters deconvolution itself Number of Iterations Specifies how far the deconvolution is to proceed e 0 Use when a crude deconvolution is sufficient Use only for data with a large variation in peak width or where speed is important 1 Use where there is a substantial change in peak width across the data to be processed e 2 Use if there is a significant change in peak width across the data or where it is not possible to accurately model the peak profile e Default 3 Use when a high quality deconvolution is required and there is only a small change in peak width across the region to be processed e 4 Use when extreme deconvolutions are required This value is only relevant for good signal to noise data where there is only a very small change in peak width across the region to be processed The designed model must also be a good fit to the peak profiles You can use only integer values Decreasing the value for this parameter reduces the computation time Starting at 0 the computation time roughly doubles for each integer increase Noise Compensation Determines whether the ReSpect algorithm improves signal detection where the noise level varies across the data e Default Selected Improves signal detection where the noise level varies across the data e Cleared Does not improve signal detection where the noise level varies acr
181. esults Lz Results are not saved Do you want to exit without saving Ce 3 Click Yes to discard the results or No to save them The state of the run queue is retained when you close the Protein Deconvolution application even if analyses remain in the queue Thermo Scientific Protein Deconvolution User Guide 11 Manually Deconvolving lsotopically Resolved Mass Spectra with the Xtract Algorithm This chapter explains how to manually deconvolve isotopically resolved mass spectra with the Xtract algorithm For information about automatically deconvolving these spectra with the Xtract algorithm see Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm on page 101 Contents e Setting Up a Manual Xtract Protein Deconvolution e Creating an Xtract Method e Selecting the Spectrum to Deconvolve e Deconvolving the Spectrum e Displaying the Results e Saving the Results of a Deconvolution e Exporting the Results of a Deconvolution e Deleting the Results of a Deconvolution e Comparing Samples e Displaying an Xtract Deconvolution Report e Loading Saved Xtract Results When you generate a deconvolved spectrum from an isotopically resolved protein mass spectrum the source MS spectrum can be a single spectrum from an LC MS data file an averaged spectrum from an LC MS data file or a single spectrum from a raw data file containing only that spectrum The Xtract algorithm transforms this so
182. ethod m z Range Specifies the portion of the input spectrum that the Xtract algorithm processes e Min Specifies the lowest end of the input spectrum e Max Specifies the highest end of the input spectrum For example if the total mass range of the spectrum is mass 100 to 2000 a setting of 300 to 500 for the m z Range parameter means that the Xtract algorithm processes only peaks with masses between 300 and 500 m z Range 300 25 000 Default minimum 600 Default maximum 2000 Protein Deconvolution User Guide 29 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 4 of 10 Parameter Description Charge Carrier Specifies the adduct ions used during ESI processing Adduct ions bring the charge to the molecule and this charge converts it to an ion e Default H 1 00727663 Specifies that the adduct was hydrogen e K 38 9631585 Specifies that the adduct was potassium e Na 22 9892213 Specifies that the adduct was sodium e Custom Specifies that the adduct was a charge carrier other than hydrogen potassium or sodium When you select this option a box opens so that you can type the mass of the custom charge carrier Note In negative mode these adduct ions correspond to deduct rather than adduct masses Min Num Detected Charge Specifies the minimum number of c
183. ethod select the Manual ReSpect Isotopically Unresolved experiment type and follow the instructions in Creating a ReSpect Method on page 123 to set the parameters on the Parameters page Before you leave the Parameters page follow the instructions from step 5 Note If you select a ReSpect method that you used in Protein Deconvolution 1 0 but that is not the default method the chromatogram parameters might not be set correctly In this case a warning message informs you that the method might have incorrect chromatogram settings and suggests that you evaluate these settings in a manual workflow before running the automated workflow Thermo Scientific Protein Deconvolution User Guide 217 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up an Automatic ReSpect Protein Deconvolution e Ifyou want to study proteins using the Exactive Plus EMR mass spectrometer under native or non denaturing conditions use the ExampleMethodNativeMS method This method is a read only method available only with the ReSpect algorithm It enables automated analysis of Native MS data that is directly infused into the mass spectrometer Unlike standard intact protein data Native MS data contains detectable protein complexes with multiple proteins embedded in them The required m z range to detect these complexes is 1000 to 5000 or even 20000 m z Table 44 shows the differences in parameter settings b
184. etween the default ReSpect method and the ExampleMethodNativeMS method Table 44 Differences in parameter settings between the default and the ExampleMethodNativeMS methods ExampleMethodNativeMS Parameter Default method method Output Mass Range 10 000 160 000 10 000 1 000 000 Mass Tolerance 20 ppm 30 ppm Minimum Adjacent 6 10 4 10 Charges Charge State Range 10 100 5 100 M z range 1000 4000 1000 15 000 5 On the Parameters page set the parameters that control the presentation of the output a Click the Parameters tab Figure 65 on page 124 shows the Parameters page for ReSpect deconvolution b In the Automation Parameters pane of the parameters page set the following options e Save to PDF Saves the automatically generated deconvolution report to a PDF file e Auto Print Automatically prints the automatically generated deconvolution report contained in the PDF file When you select this option the Select a Printer option becomes available so that you can select the printer e Concatenate All Reports Combines all reports for all deconvolved spectra from the same raw data file into a single PDF file e Select a Printer Displays a list of available printers that can print the deconvolution report PDF file This option becomes available when you select the Auto Print option c Inthe Automation Parameters pane click Apply 6 Click the Method Selection tab again 7 If you made changes to a method or
185. evious steps You can process up to 1000 samples 10 If you want to run the job or jobs start the jobs by following the instructions in the next section Running Jobs in the Queue Running Jobs in the Queue By default the Protein Deconvolution application processes multiple jobs in the queue in the order that you submitted them If you want to change the processing order you can assign each deconvolution job a priority level For instructions see To assign a priority level to jobs in the queue on page 107 While the Protein Deconvolution application is analyzing data files in the run queue you can still use the manual workflow to create new methods If you start a job and then realize that you need to use another method change parameters or change the job s priority you can pause the run queue The Protein Deconvolution application completes the analysis of the current job and then pauses the run queue until you click Run again To pause a job see To temporarily stop the processing in the run queue on page 108 When you close the Protein Deconvolution application while the application is analyzing data files and restart the application later the application automatically opens the run queue and displays the same information that it did when you closed it even if analyses are still in the queue The samples remain queued but the application does not automatically start to process them Thermo Scientific Pro
186. files up to 2 GB When you want to run a batch of data files usually of the same sample with a given method do the following e To select contiguous file names click the name of the first raw data file hold down the SHIFT key and click the last file name that you want to select e To select noncontiguous file names click the name of the first raw data file hold down the CTRL key and click each separate file name Click Add to Queue The application transfers you to the Run Queue page shown in Figure 56 The data file or files that you selected appear on the Run Queue page with a status of Queued Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Running Jobs in the Queue Figure 56 Run Queue page a a no Protein Deconvolution Thermo Protein Deconvolution amtii a me 1 SCLENTLELC Method Selection M ED O Parameters E chromatogram E Process and Review E Sampie Comparison Reporting o Process methods defined in the work queue Queue Manipulation Set Priority Open Result Open Report Number of Chromatographic Components Completion Time Peaks Experiment Submit Time Method Name Raw Data File Type 1 Normal 10 14 2013 3 29 13 PM DefaultMethodXtract C Program Files Protein Deconvolution source files XT_AUTO Queued 9 Optional If you want to add another job or jobs to the run queue return to the Method Selection page and repeat the pr
187. form an automatic deconvolution the application obtains the resolution from the instrument When you load a data file containing only spectral peaks that is an exported spectrum file in qb raw file format the Protein Deconvolution application takes the resolution from the following sources e When you perform a manual deconvolution create a method and load a raw data file the application obtains the resolution from the method e When you perform an automatic deconvolution the application obtains the resolution from the saved method e When you load the raw data file without loading a method the default resolution is always 15 000 The Resolution at 400 m z parameter is not needed if the ReSpect algorithm deconvolves FTMS Orbitrap or Exactive data because the data contains instrument information in the spectrum You must set this parameter for all other spectrum types and for exported spectrum files in qb raw file format which lack instrument information If you change the resolution the application applies the changed resolution to the currently loaded data file However the next time that you use the method with a raw data file it applies the instrument resolution to the method Protein Deconvolution User Guide 145 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 12 of 15
188. g this confidence level increases the number of peaks but allows more noise Tests suggest that a confidence level of 95 percent works well in most cases and that you can relax it to 68 percent if necessary A confidence level of 99 percent is rarely required A confidence level of 50 percent or less might produce an unacceptable number of false positives No Noise Rejection Retains all peaks and features 50 Confidence Rejects all features up to a significance corresponding to 0 7 standard deviations 68 Confidence Rejects all features up to a significance corresponding to 1 standard deviation Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Default 95 Confidence Rejects all features up to a significance corresponding to 2 standard deviations 99 Confidence Rejects all features up to a significance corresponding to 3 standard deviations Use Relative Intensities Determines whether the Protein Deconvolution application calculates the intensity of each peak relative to the noise level of the spectrum in the vicinity of the peak For more information on this parameter see Table 27 on page 135 Default Selected Calculates the intensity of each peak relative to the noise level of the spectrum in the vicinity of the peak Cleared Calculates global noise that is the noise across the entir
189. ge You can select these parameters before or after you click Process in the Process and Review page 1 Click the Parameters tab and go to the Reporting Parameters pane of the Parameters page 2 To display the Monoisotopic Mass table for each component as shown in Figure 53 on page 97 select the Component Detail Tables check box and click Apply Thermo Scientific Protein Deconvolution User Guide 87 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report To display the spectrum of each component as shown in Figure 53 on page 97 select the Component Source of Evidence Plots check box and click Apply Ifyou select these parameters after you generate the report click the Report tab to see the Source Spectrum Evidence section To save the report in a PDF file Move the cursor near the bottom of the screen The Reporting page toolbar shown in Figure 43 appears Click the Show Acrobat icon ys The Adobe Acrobat application toolbar appears at the top of the screen On the Adobe toolbar click the Save File icon The Save a Copy dialog box opens Specify the path and name of a PDF file to store the reports in and click Save The Protein Deconvolution application saves the report in a file called raw_file_name pdf If you do not specify a directory it places the file by default in the raw data directory shown on the Method S
190. gram After your job has finished processing click Open Result on the Run Queue page Click the Chromatogram tab From the Windows Start menu choose All Programs gt Accessories gt Snipping Tool Drag the cursor that appears around the area of the chromatogram that you want to capture Right click in the Snipping Tool and choose Copy Open a third party software file and paste the copied picture into it The Protein Deconvolution application creates averaged spectra from the start scan to the end scan for each chromatographic peak It saves each deconvolution result from a given chromatographic peak The name of each deconvolution result is as follows method_name_start_scan_end_scan_date_time_experiment_type where Method_name is the name of the method used for automated deconvolution Start_scan and end_scan are the scan numbers for the averaged chromatographic peak Date and time are generated at the time the run is started for the given data file Experiment_type is the type of algorithm used to deconvolve the averaged spectrum either XT_AUTO for automatic Xtract or RSP_AUTO for automatic ReSpect This designation only appears when you use the automatic Xtract or ReSpect algorithm to deconvolve a spectrum Protein Deconvolution User Guide 113 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Figure 61 gives an example of a result name
191. gram and Deconvolved Spectrum panes e Click the number of a charge state peak in the No column in the Results table or e Click the sign to the left of a charge state peak and then click the number of one of its charge states in the Charge State column When you click on a charge state peak or one of its charge states in the results table you see the following changes in the panes of the Process and Review page e Source Spectrum pane When you click a charge state peak the Source Spectrum pane overlays onto the spectrum blue lines representing the calculated monoisotopic m z values for each charge state in the charge state peak as shown in Figure 82 170 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results Figure 82 Source Spectrum pane showing the calculated monoisotopic m z values for each charge state in a charge state peak Source Spectrum 848 5576 100 942 7305 737 9650 50 653 0065 696 2056 1918 3199 500 1000 1500 2000 When you click an individual charge state of the selected charge state peak the Source Spectrum pane displays a blue line representing the calculated monoisotopic m z value for the charge state as shown in Figure 83 Figure 83 Source Spectrum pane showing the calculated monoisotopic m z value for an individual charge state
192. h the Xtract Algorithm Displaying an Xtract Deconvolution Report Figure 53 Source Spectrum Evidence section for Xtract deconvolution Monoisotopic Mass 16940 9831 BUR RE wR Calculated Monoisotopic Charge Monoisotopic Mass for this Mostabund m z Normalized Fit Left Fit Right m z Charge Intensity F758 16940 3937 998 1291 73809 4153 81 10 0 00 0 00 2 1741 169410013 942 7312 140337 8561 92 20 0 00 0 00 992 6390 16940 9876 893 1665 127290 5611 93 20 8 30 15 50 948 0574 16940 9903 848 5591 141482 8145 94 40 0 00 0 00 907 7217 16940 9657 808 1973 293916805 87 10 0 00 0 00 771 0529 16940 9921 771 5068 77086 5009 870 0 00 0 00 737 5726 16940 9820 738 0073 70953 7535 8280 0 00 0 00 706 8524 169409924 707 2982 57434 0825 80 40 0 00 0 00 678 6474 16940 9748 679 0471 345 9472 90 40 0 00 0 00 52 5843 16540 9686 652 9688 20853 9762 86 90 0 00 0 00 628 4518 16941 0059 628 8240 13975 7511 86 60 0 00 0 00 06 0081 16940 9890 606 4016 36967739 84 10 0 00 0 00 Source Spectrum Evidence Myoglobin_30pmol_michrom_proteim_microtrap_1imin_OT_60K_1 166 RT 3 65 NL 4 19E5 Thermo Scientific F FIMS p ESI Full ms 300 00 2000 00 nas 892 2206 at a0 i O08 0697 70 2 2 vt E i 1060 3777 au 028 98 i J 1013912844 1211 8618 1413 6667 1696 0089 1382 3551 400 600 800 1000 1200 1400 1600 1800 2000 miz Table 21 lists the parameters in the Source Spectrum Evidence section Table 21 Source Spectrum Evidence section paramete
193. harge deconvolution to convert the remaining peaks from a mass to charge spectrum to a mass spectrum The ReSpect algorithm uses peak spacing patterns which are indicative of mass to determine what the average mass should be The ReSpect algorithm can accommodate both low charge state spectra and data with a low signal to noise ratio so it does not require high quality data to produce meaningful results You can use it to confirm molecular masses of proteins Figure 2 shows an isotopically unresolved mass spectrum Figure 2 Isotopically unresolved mass spectrum IgG_source_cid 1123 1331 RT 3 35 3 61 AV 208 F FTMS p ESI Full ms 1000 00 4000 00 2761 2698 1005 2711 9761 2920 4838 a 2667 2545 a 2977 7553 r 3037 3632 eo 2574 1155 407 2449 6080 si 2339 1702 204 10 J 3785 9041 3931 3901 0 A ant on oon eT ee 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 miz 3231 1633 Relative Intensity 3374 6321 1508 7424 1659 4855 1843 7270 3619 4633 1106 7032 1276 6962 Thermo Scientific Protein Deconvolution User Guide 3 1 Introduction Features ExampleMethodNativeMS Method Included in the ReSpect algorithm is the ExampleMethodNativeMS method which is suitable for use in the structural analysis of proteins that is for measuring and studying intact proteins and protein complexes in their active form under native or non denaturing conditions You c
194. harge states required to produce a component No components with less than this minimum number appear in the deconvolved spectrum This parameter must be an integer greater than or equal to 1 Range 1 no maximum Default 3 30 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 5 of 10 Parameter Description Isotope Table Specifies the type of isotope table to use Isotope tables simulate the distribution of isotopic peaks in m z for different choices of the monoisotopic mass The Xtract algorithm chooses the monoisotopic mass with the best fit between the theoretical and the observed isotope distribution To generate an isotope table the Protein Deconvolution application uses a chemical formula to describe the type of molecule You can choose one of the following formulas e Default Protein Uses an averagine formula to generate the isotope table The default method and the methods created in previous releases of the software use this formula e Nucleotide Uses an elemental formula typical for nucleotides to generate the isotope table You can save the isotope table as part of the method Apply Implements the parameter settings that you selected in the Main Parameters Xtract pane This button is only available if you changed any paramet
195. he chromatographic peak and deconvolves an isotopically unresolved mass spectrum with the ReSpect algorithm Load Results Loads the saved results of a previous deconvolution Load Raw Data File Pane Parameters Table 2 lists the parameters in the Load Raw Data File pane of the Method Selection page Table 2 Load Raw Data File pane parameters Parameter Raw Data Directory Description Specifies the directory where the raw data file containing the spectrum to deconvolve is located Select Raw Data Files Specifies the name of the raw data file containing the spectrum to deconvolve Load Thermo Scientific Loads the specified raw data file Protein Deconvolution User Guide 17 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Methods Pane Parameters Table 3 lists the parameters in the Methods pane of the Method Selection page Table 3 Methods pane parameters Parameter Description Name Specifies the name of the method to use in the deconvolution Description Briefly describes the method to use in the deconvolution Create Method Activates the Parameters page so that you can specify the parameters for a new method Load Method Loads the specified existing method Creating an Xtract Method When you click Create Method in the Methods pane of the Method Selection page the Protein Deconvolution application automatically transf
196. he library Reference Spectra Raw Data File Displays the names of the reference spectra available in the reference spectrum library Displays the name of the original raw data file that produced the selected reference spectrum Scan Range RT Range Displays the scan range that the selected reference spectrum was derived from Displays the retention time range that the selected reference spectrum was derived from Most Abundant Mass Displays the mass of the most abundant component in the selected reference spectrum Experiment Type Displays the algorithm used to generate the selected reference spectrum e XT Indicates the manual Xtract algorithm e RSP Indicates the manual ReSpect algorithm e XT_AUTO Indicates the automatic Xtract algorithm e XT_RSP Indicates the automatic ReSpect algorithm Number of Peaks Creation Time Displays the number of peaks in the selected reference spectrum Displays the date and time that the selected reference spectrum was generated Protein Deconvolution User Guide 83 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Table 12 Sample Comparison page parameters for Xtract deconvolution Sheet 3 of 3 Parameter Description Descriptions Displays the description of the selected reference spectrum as you specified it in the SaveAs dialog box Select Selects the highlighted reference spe
197. heads ceanae oe 117 Setting Up a Manual ReSpect Protein Deconvolution 118 Method Selection Page Parameters ios iach tae Ae oes 122 Creating a ReSpect Method wicca ant aete se eb t ee emt aut mea 123 Parameters Page Parameters for the ReSpect Algorithm 135 Menu Bar Commands on the Parameters Page 0 00 ee eee 149 Selecting the Spectrum to Deconyolve 4 00 wee adele ded aie lea a 149 Obtaining the Best Results with the ReSpect Algorithm 157 Chromatogram Page Parameters for the ReSpect Algorithm 158 Chromatogram Page Menu Bar Commands 000 000005 161 Chromatogram Pane Shortcut Menus oiscnin tiene tates se 162 Chromatogram Pane Headet sx a ioc Yap wacenssw k oneness ed raed 162 Source Spectrum Pane Shortcut Met o 6 sag ea be caked tel wae hie oN 163 Source Spectrum Pane Header i t c65 sot cares emt ares ee ee 163 Deconvolving the Spectrum ic t s a cc x Lutestrothed Lecjabbnteie decieg eeld baeradanayralinye 163 Displaying the ResultSinkan dew ers ee ton toa a ake bee 165 Displaying the Results on the Process and Review Page 166 Selecting a Reference Mass to Calculate Mass Differences 172 Calculating a Protein Quality Scote so cscc ies a eae eat ee es 173 Specifying the Output Mass Range ssuuuuuusuuunnne eerren 175 Adjusting the Deconvolution Results 000 00 ee eee ee eee 175 Process and Review Page Parameters f
198. hich is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Deconvolved Spectrum pane Displays the deconvolved spectrum Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis Results table Protein Deconvolution User Guide Displays the mass of the ions formed from molecules Displays the masses and intensities of the isotopic clusters that the Xtract algorithm detected during the deconvolution Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Table 9 Process and Review page information for Xtract deconvolution Sheet 2 of 3 Parameter Description Monoisotopic Mass Displays a weighted average of the monoisotopic masses of each charge state i X Monoisotopic Mass of This Charge x Charge Normalized Intensity Monoisotopic Mass Sum Intensity where is the sequential order of the charge in the Charge column Sum Intensity Displays the sum of the intensities of the isotopic clusters in a charge state Number of Charge States Displays the number of detected isotopic clusters for a given deconvolved mass Average Charge Displays the average of the charge numbers in the Charge column Delt
199. his section see Chromatogram Parameters Section on page 205 e Default Selected Displays the Chromatogram Parameters section of the report e Cleared Does not display the Chromatogram Parameters section of the report Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 13 of 15 Parameter Source Spectrum Description Determines whether to display the Source Spectrum section of the report For information on this section see Source Spectrum Section on page 206 e Default Selected Displays the Source Spectrum section of the report e Cleared Does not display the Source Spectrum section of the report Deconvolution Parameters Determines whether to display the Main Parameters ReSpect and Advanced Parameters ReSpect section of the report For information on this section see Main Parameters ReSpect Section on page 207 and Advanced Parameters ReSpect Section on page 207 e Default Selected Displays the Main Parameters ReSpect and Advanced Parameters ReSpect sections of the report e Cleared Does not display the Main Parameters ReSpect and Advanced Parameters ReSpect sections of the report Deconvolved Spectrum Determines whether to display the Deconvolved Spectrum section of the report F
200. hm redraws the BPC with the most intense peak within the selected m z range rather than the whole spectrum In both cases when you select the m z range around the protein signals of interest any peaks for the background components generally disappear from the chromatogram and the only peak left is for the target proteins The default values for both limits depend on the data in the raw data file The default is the entire chromatogram for the given spectrum Use Auto Spectral Averaging Determines whether the Protein Deconvolution application ignores all chromatographic peak picking when averaging spectra Selected Creates the averaged spectrum from all spectra in the specified retention time range so it creates only one deconvolution result from an automated run By default the Protein Deconvolution application selects the full retention time range of a given data set so it produces the averaged spectrum for the full run Default Cleared Averages and deconvolves all spectra in the run This parameter is only available in the Auto Xtract experiment type Protein Deconvolution User Guide 39 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve If you want to return the parameters in the Chromatogram Parameters pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any para
201. hod and give it a name a Click Save Method or Save Method As The Save Method command saves current parameter values to the existing method overwriting any previous values The Save Method As command saves parameter values to a new method Protein Deconvolution User Guide 133 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method b 134 Protein Deconvolution User Guide In the Method Name box of the Save or SaveAs dialog box type a name for the method The name cannot contain any spaces or non alphanumeric characters It can include underscores In the Description box briefly describe the method For example you might want to describe the sample and the proteins analyzed Figure 67 shows a completed SaveAs dialog box Figure 67 Completed SaveAs dialog box 41L SaveAs x Method Name test2 at Descriptions IgG intact Save Cancel Click Save Note The Protein Deconvolution application saves all methods that you create in the database in C ProgramData ThermoScientific ProteinDeconvolution methods sqlite You cannot save individual methods to a directory that you choose The next time that you access the Method Selection page and click Manual ReSpect Isotopically Unresolved you will see the name of the method that you saved in the Methods pane The application transfers you to the Chromatogram page so that you can selec
202. ia Roke Help X Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 RAW _ Method Selection Run Queue Chromatogram Process and Review Sample Comparison Reporting i Specify the adduct ions used during ESI processing Save Method Save Method As Reset Method Main Parameters Xtract Output Mass M OMH m z Range Min 600 Max 2000 SCIENTIFI Apply Resolution at 400 m z 60000 Charge Carrier H 100727663 D K 38 9631585 S N Threshold 3 D Na 22 9892213 Rel Abundance Threshold 0 Custom Negative Charge Min Num Detected Charge 3 Calculate XIC 7 Isotope Table _ Protein Advanced Parameters Xtract Fit Factor Charge Range Remainder Threshold Minimum Intensity Consider Overlaps Expected Intensity Error Reporting Parameters Sample Information T Deconvolved Spectrum v Apply 7 Chromatogram With Parameters v Components Table v Source Spectrum v Component Detail Tables F Deconvolution Parameters Ej Component Source Of Evidence Plots Automation Parameters Only apply to automated workflow Save to PDF v Concatenate All Reports v Apply Auto Print Select a Printer _ ussjo sjprtsv1 SJTECHPUBS PHASER5SOODN Note The Xtract default parameter settings provide a good balance between sensitivity and report size by detecting all of
203. ically deconvolve isotopically resolved mass spectra with the Xtract algorithm For information about manually deconvolving these spectra with the Xtract algorithm see Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm on page 13 Contents e Setting Up an Automatic Xtract Protein Deconvolution e Running Jobs in the Queue e Displaying the Results e Comparing Samples Generated with the Automatic Xtract Algorithm e Displaying an Xtract Deconvolution Report e Loading Saved Xtract Results Automatic Xtract deconvolution automates the process of generating chromatograms detecting chromatographic peaks extracting averaged mass spectra deconvolving resolved isotopic peptides or proteins and generating a component list all in a single step It can optionally generate a report containing the deconvolved proteins and their mass spectrometric evidence The results of an automatic deconvolution are the same as those of a manual deconvolution Setting Up an Automatic Xtract Protein Deconvolution In automatic protein deconvolution you add files to a run queue and then deconvolve the queued files First you select the Xtract deconvolution algorithm a method and a raw data file Protein Deconvolution User Guide 101 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up an Automatic Xtract Protein Deconvolution To set up an automatic protein deconvolutio
204. ication expands the range to 15 000 to 160 000 m z in the mirror plot 4 Optional Right click in the Mirror Plot pane and choose Zoom In to enlarge the plot as shown in Figure 40 or hold down the left mouse button and drag the cursor beneath the part of the spectrum that you are interested in Thermo Scientific Protein Deconvolution User Guide 71 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples The zoom setting in the mirror plot remains the same until you change the deconvolution mass range or load a previous result This behavior is the same as that of the deconvolved spectrum in the Deconvolved Spectrum of the Process and Review page Figure 40 Enlarged mirror plot Protein Deconvolution defauitethodxtract Kiera kaa iA E E 1 SCIENTIFIC PSA_240_highplraw v Method Selection Run Queue o Parameters v Chromatogram o Process and Review g Reporting o Compare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Show Details Descriptions Y Reference Spectrum Library Most Abundant Experiment Number of Si Select a lt a Xtract7383 C Program Files Protein Deconvolution source 75 90 7 27 840 27756 757 XT 69 01 02 2014 15 53 47 Reference Spectra Raw Data File Scan Range RT Range Mirror Plot Source Data Fil
205. id raw Instrument Method C Xcalibur Intact mab_SS_1 meth Vial CStk1 01 23 Injection Volume uL 40 0 Sample Volume uL 0 ISTD Amount 0 Dil Factor 1 Table 38 lists the parameters in the Sample Information section All the parameters in this section are read only Table 38 Sample Information section parameters for ReSpect deconvolution Sheet 1 of 2 Parameter Description Original File Name Displays the name of the original raw data file If you rename the raw data file the original name of the raw data file still appears on the report Instrument Method Displays the name of the instrument method file Vial Displays the position number of the sample in the autosampler Injection Volume uL Displays the injection volume of the sample to be injected in microliters Sample Weight Displays the amount of a component in the sample Sample Volume uL Displays the volume of a component in the sample Thermo Scientific Protein Deconvolution User Guide 203 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Table 38 Sample Information section parameters for ReSpect deconvolution Sheet 2 of 2 Parameter Description ISTD Amount Specifies the correction for the internal standard amount If the value in this box is not 0 000 the value is used in an algorithm to correct for a case when any internal standard amounts specified in the activ
206. ified it in the SaveAs dialog box Reference Data File Displays the name of the raw data file that produced the reference spectrum in the currently loaded method Show Details Opens a Reference Spectrum Information box see Figure 94 on page 195 displaying the deconvolution parameters used to produce the reference spectrum that you selected in the currently loaded method Remove Removes a reference spectrum from the currently loaded method Reference Spectrum Library pane Displays commands that allow you to select reference spectra to show in the mirror plot display information about specific reference spectra add reference spectra to the method and delete reference spectra from the library Reference Spectra Raw Data File Displays the names of the reference spectra available in the reference spectrum library Displays the name of the original raw data file that produced the selected reference spectrum Scan Range RT Range Displays the scan range that the selected reference spectrum was derived from Displays the retention time range that the selected reference spectrum was derived from Most Abundant Mass Displays the mass of the most abundant component in the selected reference spectrum Experiment Type Displays the algorithm used to generate the selected reference spectrum e XT Indicates the manual Xtract algorithm e RSP Indicates the manual ReSpect algorithm e XT_A
207. igher sensitivity that is with a slightly narrower width threshold to identify narrow shoulders or noise like peaks that the peak detector might have missed in the first pass This option increases sensitivity at the cost of a potential increase in the false positive rate Chromatogram m z Range Specifies the range of m z values used as input to the chromatogram The Xtract algorithm ignores the portions of the spectrum outside this range The default values for both limits depend on the data in the raw data file The default is the entire chromatogram for the given spectrum Use Auto Spectral Averaging Determines whether the Protein Deconvolution application ignores all chromatographic peak picking when averaging spectra e Selected Creates the averaged spectrum from all spectra in the specified retention time range so it creates only one deconvolution result from an automated run e Default Cleared Averages and deconvolves all spectra in the run This parameter is only available in the Auto Xtract experiment type Apply Implements the parameter settings that you selected This button is only available if you changed any parameter settings in the Chromatogram Parameters pane Chromatogram pane Displays the chromatogram contained in the raw data file Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity
208. in Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Figure 42 Partial view of the Reporting page for Xtract deconvolution Protein Deconvolution defauitmethoaxtract alien nt esa Help SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_LRAW o Method Selection Run Queue o Parameters o Chromatogram o Process and Review g Sample Comparison ProteinDeconvolution Report Created 10 17 2013 4 54 31 PM Sample Information Original File Name Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 RAW Instrument Method C Xcalibur data tonya Intacts 11min_OT_60K_SID_15V meth Vial 1A5 Injection Volume uL 5 Sample Weight 0 sample Volume uL 0 ISTD Amount 0 Dil Factor 1 Chromatogram Parameters Use Restricted Time Time Range Rel Intensity Threshold Type Sensitivity Source Chromatogram Myoglobin_30pmol_michrom_protein_microtrap_1imin_OT_60K_1 ProteinDeconvolution NL 8 43E7 F 3 65 RT 3 66 S 166 Note The Protein Deconvolution application does not support copying and pasting chromatograms and source spectra from the Reporting page To display the Source Spectrum Evidence section Note The Source Spectrum Evidence Section appears in the report only if you select the Component Detail Tables and Component Source of Evidence Plots parameters in the Parameters pa
209. in the Chromatogram pane Default TIC Displays a total ion current chromatogram BPC Displays a base peak chromatogram The base peak is the largest peak in a spectrum For information on these types of chromatograms see Table 6 on page 44 Sensitivity Specifies the sensitivity with which the chromatographic peak detector identifies peaks Low Directs the chromatographic peak detector to perform one pass at the default sensitivity Default High Directs the chromatographic peak detector to perform a second pass at higher sensitivity that is with a slightly narrower width threshold to identify narrow shoulders or noise like peaks that the peak detector might have missed in the first pass This option increases sensitivity at the cost of a potential increase in the false positive rate Chromatogram m z Range Specifies the range of m z values used to create the chromatogram You can use this parameter to select a narrower range The Xtract algorithm ignores the portions of the spectrum outside this range You might want to create a narrower range because the intact proteins are usually at a higher m z value and any small molecule contaminants and background are below 600 m z Instead of creating a TIC using the full m z range the Xtract algorithm calculates a TIC by summing those protein peaks within the narrower m z range The resulting TIC is basically an extracted ion chromatogram XIC The Xtract algorit
210. iple scans The number spectra that were averaged to create the source spectrum for example 39 F The scan filter used during the LC MS run for example FTMS p ESI Full ms 300 00 2000 00 The scan filter indicates the type of mass analyzer used to acquire the data in the raw data file and the ionization technique used If this field is blank no scan filter was used Deconvolving the Spectrum Reaching the Process and Review page shown in Figure 22 means that you have already selected the chromatogram and source spectrum on the Chromatogram page or that the Protein Deconvolution application has transferred you because the raw data file contains a spectrum only You can zoom in and out of the chromatogram and source spectrum views but you cannot change them on the Process and Review page You must manually navigate back to the Chromatogram pane to change these views Use the Process and Review page to deconvolve the selected spectrum and to view the resulting data to ensure that the results make sense You can also export the data to a Microsoft Excel spreadsheet for use in other applications To deconvolve the spectrum 1 If you are not already on the Process and Review page click the Process and Review tab Figure 22 shows the initial Process and Review page Thermo Scientific Protein Deconvolution User Guide 49 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Deconvolving the Spect
211. irectory 9 starting 8 Xtract algorithm See Xtract algorithm Protein Deconvolution window 8 protein quality score 4 126 173 174 Q qb files 131 Qual Browser 6 41 154 quality score 178 Quality Score Threshold parameter 126 136 Queue Manipulation menu 109 223 224 Raw Data Directory area 17 123 Raw Data File column 83 198 Raw Data File parameter 110 225 raw data files containing one spectrum 13 117 default location 9 displaying available 14 102 119 216 loading 17 123 location 17 123 selecting 104 219 recombinant monoclonal antibodies 5 Record Number parameter 110 224 Reference Data File parameter 83 84 198 199 reference mass 60 172 Reference Spectra column 83 198 Protein Deconvolution User Guide 235 Index R reference spectrum changing in a method 196 comparing deconvolved spectra 4 70 185 190 deleting from a method 196 deleting from reference spectrum library 195 saving deconvolved spectrum as 186 saving in database 65 179 Reference Spectrum Library pane deleting reference spectrum from 80 195 parameters on 83 198 sorting data in 78 193 Reference Spectrum parameter 82 197 Rel Abundance Threshold parameter 20 28 125 136 Rel Intensity Threshold parameter 38 45 151 158 Relative Abundance column 63 66 68 178 180 182 Relative Intensity y axis label Chromatogram page 46 47 160 161 Chromatogram pane of the Process and Review page 62 176 Deconvolved Spectrum pane of the P
212. isotopic m z 4 he column 40 20 3 15635 2961 16508 8381 R E AA E ION A O T N AA 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 Mass Saved ReSpect Results Number of Charge Average Charge Delta Mass Relative Fractional States Abundance Abundance 16941 0113 315 704 75 20 19 82 0 00 100 0000 42 2091 3 303 3 969 3 671 2 16923 0410 216 752 20 20 60 17 97 68 6566 28 9794 3 303 3 969 3 653 Calculated Mass Charge Normalized F 1881 3459 16922 9635 1882 4489 706 89 9030 1693 3120 16922 9742 1694 3067 111744 9530 i 0 00 1539 4661 16922 9617 1540 3659 1 154 09 93 60 7 1411 2612 sens nese ne nse eee Selected value in 996 4807 16922 9966 997 0701 19 963 28 85 20 the Calculated 941 1766 16923 0212 941 7328 26 718 04 891 6940 16923 0370 892 2208 29 956 09 7 7 Monoisoto p icm Z 847 1596 16923 0457 847 6608 2910341 806 8667 16923 0585 807 3446 25 868 14 t 7 column 770 2367 16923 0492 770 6924 23 528 05 7367919 16923 0399 737 2274 19 153 83 Sum Intensity For more information on the columns in the Results table see Table 9 on page 62 To display the results in the Source Spectrum Chromatogram and Deconvolved Spectrum panes e Click the number of an isotopic cluster in the No column in the Results table e Click the sign to the left of an isotopic cluster and then click the number of one of its charge states in the Charge State column When you click an isotopic cluster
213. it Time Displays the time that you placed the job into the run queue Method Name Displays the method used to deconvolve the averaged spectrum Raw Data File 110 Protein Deconvolution User Guide Displays the path and name of the raw data file used to deconvolve the averaged spectrum Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Generated with the Automatic Xtract Algorithm Table 22 Run Queue page parameters for automatic Xtract deconvolution Sheet 3 of 3 Parameter Experiment Type Description Displays the type of algorithm used to deconvolve the averaged spectrum e XT_AUTO Deconvolves the spectrum with the automatic Xtract algorithm e RSP_AUTO Deconvolves the spectrum with the automatic ReSpect algorithm Number of Chromatographic Peaks Displays the number of chromatographic peaks detected in the spectrum If the Protein Deconvolution application finds no chromatographic peaks or proteins the value is 0 Number of Components Detected Displays the number of components detected from the deconvolution of each averaged spectrum from all chromatographic peaks If the Protein Deconvolution application finds no chromatographic peaks or proteins the value is 0 Completion Time Displays the date and time that the processing finished for the given raw data file Status Displays the real time stat
214. ith the Xtract Algorithm Comparing Samples If you select a method that includes a reference spectrum and select another reference spectrum from the reference spectrum library the Save Results As command saves the reference spectrum from the reference spectrum library To display the settings of the parameters used to generate the reference spectrum Select the reference spectrum in the Reference Spectrum Library pane Do one of the following e Click Show Details in the Reference Spectrum Library pane to display all the deconvolution parameters that you selected in the reference spectrum library to generate the reference spectrum not the currently loaded parameters as shown in Figure 41 or e Click Show Details in the Method Reference Spectrum pane to display all the deconvolution parameters that you selected in the method to generate the reference spectrum not the currently loaded parameters The Show Details command in the Method Reference Spectrum pane becomes available after you click Add to Method in the Reference Spectrum Library pane In either case compare these parameters to the parameters that you set to generate the spectrum for the current experiment to ensure that the two spectra are similar Protein Deconvolution User Guide 79 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 41 Reference Spectrum Information box Protein Deconvolutio
215. k Yes Thermo Scientific Protein Deconvolution User Guide 195 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples To delete a saved reference spectrum from a method Click the Sample Comparison tab if you are not already on the Sample Comparison page The Method Reference Spectrum pane displays the reference spectrum name and the name of the reference data file It also displays a description of this reference spectrum if there is one In the Method Reference Spectrum pane click Remove The deleted reference spectrum is removed from the Method Reference Spectrum pane and the mirror plot Save the method as follows e To save the method under the existing name select the method from the library and click Save Method e Ifyou used the default method save the method under another name i Click Save Method As The Save dialog box appears ii Type the name of the method in the Method Name box iii Type a brief description of the method in the Descriptions box iv Click Save To change a reference spectrum in a method Note You might want to save the method at this point otherwise you cannot apply the Remove command beyond this analysis Click the Sample Comparison tab if you are not already on the Sample Comparison page Remove the existing reference spectrum from the method See To delete a saved reference spectrum from a method
216. l hundred mass to charge units or more In complex samples the TIC chromatogram often provides limited information because multiple analytes elute simultaneously obscuring individual species A TIC in combination with a narrow m z range is effectively an extracted ion chromatogram XIC e BPC Displays a base peak chromatogram which shows only the most intense peak in each spectrum The base peak chromatogram represents the intensity of the most intense peak at every point in the analysis Base peak chromatograms for each spectrum often have a cleaner look and are therefore more informative than TIC chromatograms because the background is reduced by focusing on a single analyte at every point For intact protein spectra the TIC often looks better The BPC is usually better for smaller molecules where the entire signal exists in a single charge state Thermo Scientific Protein Deconvolution User Guide 45 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Table 6 Chromatogram page parameters for Xtract deconvolution Sheet 3 of 4 Parameter Sensitivity Description Sensitivity Specifies the sensitivity with which the chromatographic peak detector identifies peaks e Low Directs the chromatographic peak detector to perform one pass at the default sensitivity e Default High Directs the chromatographic peak detector to perform a second pass at h
217. le reflects the updated state of the reference mass When you load results that you previously saved see Loading Saved ReSpect Results on page 212 you can change the reference component but the Protein Deconvolution application does not update the reports to reflect the change to the component table because it cannot change reports that it already generated To select a reference mass 1 In the Results table select the row of the component that you want to use as the reference peak 2 Choose Set as Reference Component The Protein Deconvolution application resets the value in the Delta Mass Column to 0 for the chosen component and recalculates the delta mass value for all other components in the Results table When you have multiple results loaded concurrently and then change the reference component the application modifies only those results from the deconvolved spectrum associated with the reference component Calculating a Protein Quality Score When the Protein Deconvolution application runs the ReSpect algorithm it calculates a protein quality score for each component shown in the Results table and displays it in the Score column so that you can easily assess the quality of the deconvolved components These scores can help you determine whether each component is valid or spurious because of noise harmonics or other factors The ReSpect algorithm accepts a significant false positive rate to avoid the risk of false
218. le that contains both the parameter settings that you applied to the raw data file and the results of the deconvolution These results appear in the Results table of the Process and Review page When you want to view the results in another Protein Deconvolution session you can select Load Results on the Protein Deconvolution window to load the SQLite file and display the results of the deconvolution For more information on loading previous results refer to Loading Saved Xtract Results on page 98 or Loading Saved ReSpect Results on page 212 Starting the Protein Deconvolution Application To start the Protein Deconvolution application Choose Start gt All Programs gt Thermo Protein Deconvolution or double click the Protein Deconvolution icon ll The Protein Deconvolution window appears with the Method Selection page selected as shown in Figure 4 8 Protein Deconvolution User Guide Thermo Scientific 1 Introduction Specifying the Default Raw Data File Directory Figure 4 Method Selection page of the Protein Deconvolution window Protein Deconvolution Manai Niradi Dot icaty Besohet a ie SCIENTIFIC Method Selection gt Select an experiment type a data file and a method Experiment Types Load Raw Data File Methods Manual Xtract Isotopically Resolved gt Manual ReSpect Isotopically Unresolved Auto Xtract Isotopically Resolved Auto ReSpect Isotopically Unresolved Loa
219. lecular mass The application identifies multiple peaks in the mass spectrum associated with different charge states of the same component and displays information about the masses and abundance of that component For example peaks at 1000 m z 1111 m z and 1250 m z might be the charge states 10 9 and 8 for a protein with a mass of 10 000 Da The Protein Deconvolution application includes two independent deconvolution algorithms for mass spectral data e Xtract which deconvolves isotopically resolved mass spectra that is spectra in which it is possible to distinguish separate peaks for different isotopic compositions of the same component Protein Deconvolution User Guide 1 1 Introduction Features e ReSpect which deconvolves isotopically unresolved or unseparated mass spectra that is spectra in which it is not possible to distinguish the separate peaks for different isotopic compositions of the same component Whether mass spectra are isotopically resolved or unresolved depends not on the specific instrument model but on the resolution of the instrument the mass of the compounds involved and the details of the experiment run In addition you can use the Protein Deconvolution application to do the following e Produce more than one deconvolved spectrum for any given mass spectrum e Perform a manual or automated deconvolution e Generate a report containing the deconvolved spectrum and the mass spectrometry evidence for i
220. ll not be usable Specifies the adduct ions used during ESI processing Adduct ions bring the charge to the molecule that converts it to an ion e Default H 1 00727663 Specifies that the adduct was hydrogen e 2H 2 013553 Specifies that the adduct was deuterium e Na 22 9892213 Specifies that the adduct was sodium e Custom Specifies that the adduct was a charge carrier other than hydrogen deuterium or sodium When you select this option a box opens so that you can type the mass of the custom charge carrier Note In negative mode these adduct ions correspond to deduct rather than adduct masses Protein Deconvolution User Guide 135 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 2 of 15 Parameter Description Rel Abundance Specifies a threshold below which the Protein Deconvolution Threshold application filters out data for data reporting This option sets a relative threshold as a percentage of the most abundant component in the spectrum The most abundant peak in the deconvolved spectrum has a relative abundance of 100 percent and all other peaks are calculated relative to that one In the Results pane on the Process and Review page the application shows only those components that are greater than or equal to this relative abundance threshold in the deconvolved spect
221. llowing document as a PDF file from the data system computer Protein Deconvolution Quick Start Guide To view the product manuals From the Microsoft Windows taskbar do the following e Choose Start gt All Programs gt Thermo Protein Deconvolution gt Manuals e From the Protein Deconvolution window choose Help gt Manuals For access to the application Help follow this procedure To view application specific Help e From the application window choose Help gt Protein Deconvolution Help e If information about setting parameters is available for a specific view page or dialog box click Help or press the F1 key for information about setting parameters Thermo Scientific Protein Deconvolution User Guide vii Preface System Requirements e In applications that have a Communicator bar click the field or parameter to display definitions required actions ranges defaults and warnings For more information visit www thermoscientific com System Requirements The Protein Deconvolution application requires a license In addition ensure that the system meets these minimum requirements IMPORTANT Before you install the device driver ensure that the data system computer has a compatible version of the Thermo Foundation platform as noted in the Protein Deconvolution x x Release Notes System Minimum requirements Computer e 2 GHz processor with 2 GB RAM e DVD drive e 300 GB or greater available on driv
222. lving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report where e Method_name is the name of the method used for automated deconvolution Start_scan and end_scan are the scan numbers for the averaged chromatographic peak e Date and time are generated at the time the run is started for the given data file e Experiment_type is the type of algorithm used to deconvolve the averaged spectrum either XT_AUTO for automatic Xtract or RSP_AUTO for automatic ReSpect This designation only appears when you use the automatic Xtract or ReSpect algorithm to deconvolve a spectrum Figure 113 gives an example of a result name Figure 113 DefaultMethodReSpect_2730_2752_3_9 2012_12_40_18_PM_RSP_AUTO tt Method Start Date Time Experiment name scan type End scan All results generated from an analysis of a given raw data file are stored in the SQLite file associated with the raw data file The SQLite file and the raw data file have the same name but different suffixes For example if you analyze a file called IgG_source_cid raw the results are stored in the IgG_source_cid sqlite file All results for a given automated run on a given data file have the same date and time stamp For information on loading saved results see Loading Saved Results on page 229 Displaying a ReSpect Deconvolution Report When you click Run on the Run Queue page the Protein Deconvolution application
223. m and click Send Web for downloading documents mssupport thermo com 1 On the Terms and Conditions web page click I Agree 2 In the left pane click Customer Manuals 3 To locate the document click Search and enter your search criteria For Document Type select Manual Email to send feedback directly to Technical Publications techpubs lcms thermofisher com Web to complete a survey about this Thermo Scientific document www surveymonkey com s PQM6P62 Thermo Scientific Introduction Features Thermo Scientific This chapter describes the purpose features workflows inputs and outputs of the Protein Deconvolution application It also explains how to start and exit the application specify a default raw data file directory and adjust pane sizes Contents e Features e Workflow e Inputs and Outputs e Starting the Protein Deconvolution Application e Specifying the Default Raw Data File Directory e Adjusting the Pane Size e Exiting the Protein Deconvolution Application Electrospray ionization ESI of intact peptides and proteins produces mass spectra that contain series of multiply charged ions with associated mass to charge m z ratios The resulting spectrum is complex and difficult to interpret requiring mathematical algorithms for the analysis of the data Through a process called deconvolution the Protein Deconvolution application uses such algorithms to transform a charge state series into a mo
224. m right click and choose Zoom Out e To reset the view to the original spectrum right click and choose Reset Scale Unlike adjustments in the Chromatogram pane which you use to select a spectrum for processing adjustments in the Source Spectrum pane do not affect the spectrum that the Protein Deconvolution application deconvolves In particular they do not change the m z range that the deconvolution algorithm uses 6 When the spectrum is suitable for ReSpect processing click the Process and Review tab and follow the instructions in Deconvolving the Spectrum on page 163 Obtaining the Best Results with the ReSpect Algorithm Low outlying peaks in the source spectrum are less accurate than high peaks and fade into noise Follow these suggestions to increase the stringency of the deconvolution decrease noise and produce better results e Narrow the m z range as much as possible around the more abundant peaks in the distribution perhaps those above 10 to 20 percent relative abundance e In the Main Parameters ReSpect pane of the Process and Review page or the Parameters page adjust the values of the Output Mass Range parameter Harmonics overtones are a byproduct of the ReSpect algorithm and other deconvolution algorithms They are normal in a distribution but you can avoid them by narrowing the range to the region around the target mass e Inthe Main Parameters ReSpect pane of the Process and Review page or the Parameters
225. m Parameters pane to adjust the chromatogram displayed in the Chromatogram pane The automation algorithm uses these parameters to generate the chromatogram and select the appropriate chromatographic peaks e Use Restricted Time Determines whether the Protein Deconvolution application Protein Deconvolution User Guide zooms the part of the chromatogram that you designate with the Time Limits parameters Selected Enlarges the designated part of the chromatogram Default Cleared Displays the entire chromatogram Time Limits Specifies the beginning and the end of the chromatogram that you want to zoom The default values for both limits depend on the data in the raw data file This parameter is only available when you select the Use Restricted Time check box Rel Intensity Threshold Sets a lower intensity threshold for peaks in the chromatogram as a percentage The Protein Deconvolution application ignores peaks with relative intensities below this threshold The values are 0 to 100 inclusive The default is 1 This parameter is different from the Relative Abundance parameter on the Parameters page which sets a lower abundance threshold for signals in the spectrum not in the chromatogram Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve e Types Specifies the type of chromatogram to display
226. meters 118 Native MS method 4 obtaining best results 157 printing report 202 profile data 155 156 180 183 purpose 3 saving a report in PDF file 202 saving results of deconvolution 181 selecting a reference mass 172 selecting a spectrum to deconvolve 149 setting up manual deconvolution 118 specifying output mass range 126 157 167 175 specifying the output mass range 137 175 steps in processing 118 studying proteins under native or non denaturing conditions 121 218 types of spectra to use 117 ReSpect Masses Table report section 210 result file names 67 182 Result Method command ReSpect 227 Xtract 112 result names 113 227 results of a deconvolution 52 53 112 166 167 226 Results table columns in 54 62 168 177 displaying the deconvolution results in 8 53 165 167 purpose 51 165 results of deconvolution in 51 retention time 46 62 160 177 RT min x axis label 46 62 91 160 177 205 RT Range column exporting data in 66 68 180 182 purpose 5 64 83 178 198 Run Queue page jobs queued on 104 219 parameters on 109 224 running jobs in queue 106 220 See also job queue S S N Threshold parameter 20 27 sample comparison purpose 4 70 185 ReSpect algorithm See ReSpect algorithm comparing samples Xtract algorithm See Xtract algorithm comparing samples Sample Comparison page menu bar commands 84 199 Method Reference Spectrum pane 81 82 196 197 Mirror Plot pane 76 84 191 199 200 Thermo Scientific
227. meters in this pane but do not click Apply and then click another tab the message box shown in Figure 12 on page 22 appears Click Yes to apply the parameter changes or No to restore the parameter defaults 3 Optional Adjust the view in the Chromatogram pane Protein Deconvolution User Guide e To enlarge the view to see more detail do one of the following Right click and choose Zoom In from the shortcut menu to zoom the entire chromatogram Right click and choose Mode gt Auto Zooming from the shortcut menu if it is not already selected and drag the red cross shaped cursor over the peak or peaks of interest to form a box as shown in Figure 15 Figure 15 Enlarging a peak by drawing a box around it wromatogram soglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 NL 8 43E7 3 65_ RT 3 66 100 EARI 80 60 40 RT 0 58 RT 1 02 s 28 SH 47 R T 5 90 Sf 268 6 21 6 59 1 22 4 69 3 97 ANS 4 62 4 91 5 34 198 2 38 2 88 3 01 J 7 06 7 26 7 55 814 847 881 9 06 9 33 60 65 70 75 80 85 90 95 40 45 5 0 55 RT min 10 00 10 27 10 0 10 Draw a box around the peak of interest Right click and choose Mode gt Auto Zooming from the shortcut menu if it is not already selected Keeping the left mouse button pressed draw a line beneath the baseline of the peaks of interest as shown in Figure 16 Figure 16 Enlarging a peak by drawing a line beneath it
228. might produce an unacceptable number of false positives 140 Protein Deconvolution User Guide No Noise Rejection Retains all peaks and features 50 Confidence Rejects all features up to a significance corresponding to 0 7 standard deviations 68 Confidence Rejects all features up to a significance corresponding to 1 standard deviation Default 95 Confidence Rejects all features up to a significance corresponding to 2 standard deviations 99 Confidence Rejects all features up to a significance corresponding to 3 standard deviations Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 7 of 15 Parameter Use Relative Intensities Description Determines whether the Protein Deconvolution application calculates the intensity of each peak relative to the noise level of the spectrum in the vicinity of the peak The ReSpect algorithm performs this calculation automatically when you select the Noise Compensation option which is selected by default The relative intensity reflects the signal to noise ratio of a feature and informally attempts to capture its reliability in the deconvolution results Relative intensity is more effective than intensity if the noise level varies appreciably in different regions of the spectrum as is often the case in mass
229. mpare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions Y Reference Spectrum Library Most Abundant Experiment Mass Type 151814 828 RSP Creation Time Descriptions Reference Spectra Raw Data File Scan Range ReSpect3435 3 39 3 50 C Program Files Protein Deconvolution source 1161 1246 02 10 2014 14 09 51 Mirror Plot Source Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid raw NL 1 96E 008 151813 623 100 151976 123 151655 323 152137 823 on 152062 023 151494 623 152305 923 152431 923 152268 523 151896 223 151735 823 152481 523 152595 023 152728 123 151571 423 151615 453 151753 953 151932 653 152186 053 Relative Intensity So y 63 152755 853 Y 152097 253 y y 152589 553 152636 153 S 151495 353 152274 753 152428 353 151654 253 151975 653 152138 353 4 853 100 Mass Reference Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid raw From Library NL 6 13E 008 i B In this example comparing one portion of the deconvolved spectrum to another helps to establish that the long tail shown in the Chromatogram pane is due to structural changes in the protein and not an artifac
230. mponent in the spectrum The most abundant peak in the deconvolved spectrum has a relative abundance of 100 percent and all other peaks are calculated relative to that one In the Results pane on the Process and Review page the application shows only those components that are greater than or equal to this relative abundance threshold in the deconvolved spectrum For example if the highest peak has an absolute abundance of 1000 the relative abundance is 1 percent and no peaks below an absolute abundance of 10 will appear in the deconvolved spectrum The minimum value is 0 which displays all results and the maximum value is 100 which displays only the most abundant component The default is 0 Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Negative Charge Indicates whether the data was acquired in positive charge mode or negative charge mode during the ESI process You might want to use this option when processing compounds that contain nucleotides like those found in DNA and RNA When these compounds are acquired in negative mode the resulting mass spectra are often clearer Deprotonation of nucleotides which are acidic occurs when the compound is dissolved in a basic solution and negative voltage is applied to produce negatively charged ions Selected The data was acquired in negative charge mode Default Cleared
231. mum values for this parameter are 1 and 1 and there are no maximum values The default values for the default method are 6 and 10 The default values for the ExampleMethodNativeMS method are 4 and 10 Note The performance of the ReSpect algorithm improves as the number of charge states increases Tests suggest that this algorithm always yields reliable results if the minimum numbers of allowed charge states at the low and high end of the m z range are 6 and 10 respectively These numbers are therefore the default parameters In cases where a sample does not provide this number of charge states parameter values of 6 and 6 should still give high quality results For smaller numbers of charge states reliability is lower so you should confirm results by other means if possible e Number of Peak Models Controls the resolution of the peak modeling process by dividing the observed m z range into a uniformly spaced set of regions equal to this number The Protein Deconvolution application generates a single peak model for each of these regions on the basis of the observed m z value and instrument resolution at the midpoint of each region The default is 1 130 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method e Resolution at 400 m z Defines the resolution of the source spectrum at an m z value of 400 When you load a d
232. n Advanced Parameters ReSpect Minimum Peak Significance 1 Noise Rejection 95 Confidence Use Relative Intensities True Peak Width 0 Feature Width 0 Degree of At 0 Number of Iterations 3 Noise Compensation True Minimum Adjacent Charges 6 10 Number of Peak Models 1 Resolution at 400 m z 12374 Left Right Peak Shape 2 2 Thermo Scientific Protein Deconvolution User Guide 207 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Deconvolved Spectrum Section The Deconvolved Spectrum section shown in Figure 102 displays the same information that appears in the Deconvolved Spectrum pane of the Process and Review page Figure 102 Deconvolved Spectrum section for ReSpect deconvolution Deconvolved Spectrum 151814 375 abundance 51560 434 146557 172 100997 063 76380 492 16583 086 37972 895 20000 40000 60000 80000 100000 120000 140000 160000 Mass Table 41 lists the parameters in the Deconvolved Spectrum section Table 41 Deconvolved Spectrum section parameters for ReSpect deconvolution Parameter Description Abundance y axis Displays the relative peak abundance Mass x axis Displays the actual mass of an ion in atomic mass units 208 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Mi
233. n DetaultMethodXtract Manual ethan Aieshcs Oe SCIENTIFIC PSA_240_highplraw o Method Selection Run Queue o Parameters o Chromatogram o Process and Review 4 Sample Comparison fe Reporting o Compare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions v Reference Spectrum Library Most Abundant Mass Number of Peaks Experiment Type C Program Files Protein Deconvolution source 75 90 7 27 840 27756 757 xT 69 01 02 2014 15 53 47 Reference Spectra Raw Data File Scan Range RT Range Creation Time Descriptions Reference Spectrum Information Reference Source Spectrum Information Raw Data File PSA_240 raw Experiment Type xT Scan Range 75 90 RT Range 7 27 840 Number of Peaks 69 Most Abundant Mass 27 756 757 Deconvolution Parameters Output Mass M Fit Factor 80 Resolution at 400 m z 240000 Remainder Threshold 25 Mirror Plot S N Threshold 3 Consider Overlaps True Source Data File C Program Files Protein Deconvolution source files XtractiPSA_240_highpl raw Rel Abundance Threshold 0 Charge Range 5 50 Negative Charge False Minimum Intensity si 28412 9569 100 m z Range 600 2000 Expected Intensity Error 3 j Charge Carrier H Min Num Detected Charge 3 50 Isotope Table Protein saas
234. n User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Mirror Plot Section The Mirror Plot section shown in Figure 51 displays the mirror plot as it appears in the Mirror Plot pane of the Sample Comparison page Figure 51 Mirror Plot section for Xtract deconvolution Manual Xtract sotopically Resolved Help 4 Protein Deconvolution DefauitMethodXtract PSA_240_highpl raw S C LENT LEIC z Method Selection Run Queue a Parameters B Chromatogram o Process and Review Sample Comparison Reporting i Select a reference spectrum for sample comparison Save Method Save Method As Reset from Method Save Result As Y Method Reference Spectrum Reference Spectrum Reference Data File Descriptions Y Reference Spectrum Library Most Abundant Experiment Number of Mass Type Xtract7383 C Program Files Protein Deconvolution source 75 90 7 27 840 27756 757 xT 69 Reference Spectra Raw Data File Scan Range RT Range Creation Time Descriptions 01 02 2014 15 53 47 Y Mirror Plot Source Data File C Program Files Protein Deconvolution source files Xtract PSA_240_highpl raw NL 7 85E 005 400 28412 9569 80 60 40 20 49484 6559 0 20 k 4049394 6136 26266 2459 60 80 Ho 27756 28121 8589 27862 6864 25714 8120 Tr t 25715 8758 Relative Intensity Mass NL 6 71E
235. n ei eeren insta tive tte ee ete kl eur Ae tak 209 ReS pect Masses Table Section i ii4 5 c 50 So cacia Soe acd tele eke OS Sued 210 Source Spectrum Evidence Section tus dep w inl ajark anece alse runnner 210 Loading Saved Respect Results isa wd tun cee beehes eee eae Bee ee 212 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm ccc cece eee eee eee eee eee eens 215 Setting Up an Automatic ReSpect Protein Deconvolution 216 Running Jobs in the Quetiesee sosiaas ot das See eee eee ee 220 Run Queue Page Parameters for Automatic ReSpect Deconvolution 224 Comparing Samples Generated with the Automatic ReSpect Algorithm 226 Displaying the Results o na cy anger di At et ee alias Dae e es 226 Res l Names 4 ck ner chek tiene ENUE EEEE Gee pen eed Roe kes et 227 Displaying a ReSpect Deconvolution Report 0002 cece 228 Loading Saved Results iuta sccm dain ae parton Reh ces E easels ies 229 ndek ee ee errs 231 Thermo Scientific Preface This guide describes how to use the Thermo Protein Deconvolution 3 0 application to deconvolve mass spectra Contents e Related Documentation e System Requirements e License Activation and Deactivation e Cautions and Special Notices e Contacting Us Related Documentation The Protein Deconvolution application includes complete documentation In addition to this guide you can also access the fo
236. n of the input spectrum that the ReSpect algorithm processes Min Specifies the lowest end of the input spectrum Max Specifies the highest end of the input spectrum For example if the total mass range of the spectrum is mass 100 to 2000 a setting of 300 to 500 for the m z Range parameter means that the ReSpect algorithm processes only peaks with masses between 300 and 500 m z The valid range is 300 through 25 000 The default range for the default method is 1000 through 4000 The default range for the ExampleMethodNativeMS method is 1000 through 15 000 Exercise caution in specifying the width of the m z range Using too large an m z range might cause the ReSpect algorithm to incorporate weak noisy and poorly characterized peaks into some of its fittings with a corresponding loss in the quality of the results In general try to restrict the m z range to the more intense regions of the spectrum Output Mass Range Specifies the required output mass range Min Specifies the lowest end of the mass range Max Specifies the highest end of the mass range The default range for the default method is 10 000 160 000 The default range for the ExampleMethodNativeMS method is 10 000 1 000 000 Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method For more information on the effect of the Output Mass Range paramete
237. n with the Xtract algorithm 1 Start the Protein Deconvolution application according to the instructions in Starting the Protein Deconvolution Application on page 8 2 Click the Method Selection tab if it is not already selected The Method Selection page contains three panes as shown in Figure 55 e Experiment Types pane Displays the available deconvolution algorithms and a command that you can use to load the saved results of previous deconvolutions e Methods pane Displays the available methods e Load Raw Data File pane Displays the list of the available raw data files for the selected algorithm 3 In the Experiment Types pane click Auto Xtract Isotopically Resolved Several more tabs appear along the top of the Protein Deconvolution window 102 Protein Deconvolution User Guide Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up an Automatic Xtract Protein Deconvolution Figure 55 Initial Method Selection page Protein Deconvolution Auto Xirect isotopically Besoko a iik SCLENTLELG Method Selection o Run Queue Parameters Chromatogram B Process and Review a Sample Comparison B Reporting o Select an experiment type a data file and a method Experiment Types Methods Load Raw Data File Description Raw Data Directory C Program Files Protein Deconvolutior Manual Xtract Isotopically Resolved
238. ncatenated when the size of the generated report is too large Figure 57 shows these columns 106 Protein Deconvolution User Guide Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Running Jobs in the Queue Figure 57 Run Queue page after the application finishes processing a job Thermo protein Deconvolution Se calls SCIENTIFIC p __ Method Selection Run Queue Parameters Chromatogram S Process and Review Sample Comparison o Reporting o Process methods defined in the work queue Queue Manipulation Set Priority Open Result Open Report FER Number of Number Of Submit Time Raw Data File B lt Chromatographic Components Completion Time Mio Peaks Detected 1 Normal 10 14 2013 3 29 13 PM _DefaultMethodXtract C Program Files Protein Deconvolution source files XTAUTO 10 14 2013 3 33 39 PM Completed For information about the columns on this page see Run Queue Page Parameters for Automatic Xtract Deconvolution on page 109 3 Optional Once the status displayed in the Status column is Completed you can open the results or open a report See Displaying the Results on page 112 or Displaying an Xtract Deconvolution Report on page 114 To assign a priority level to jobs in the queue 1 Click the Run Queue tab to open the Run Queue page if it is not already opened 2 Click a job to select it 3 In the Run Queue menu bar choos
239. nfirmation box as shown in Figure 10 Figure 10 Method deletion confirmation box Protein Deconvolution X Selected method will be deleted permanently Do you want to continue 16 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Method Selection Page Parameters Setting Up a Manual Xtract Protein Deconvolution The Method Selection page consists of the Experiment Types Load Raw Data File and Methods panes The parameters in these panes are the same for Xtract and ReSpect deconvolutions Experiment Types Pane Parameters Table 1 lists the parameters in the Experiment Types pane of the Method Selection page Table 1 Experiment Types pane parameters Parameter Manual Xtract Isotopically Resolved Manual ReSpect Isotopically Unresolved Description Deconvolves an isotopically resolved mass spectrum with the Xtract algorithm Deconvolves an isotopically unresolved mass spectrum with the ReSpect algorithm Auto Xtract Isotopically Resolved Automatically detects chromatographic peaks creates an averaged spectrum from all the mass spectra within the chromatographic peak and deconvolves an isotopically resolved mass spectrum with the Xtract algorithm Auto ReSpect Isotopically Automatically detects chromatographic peaks creates an Unresolved averaged spectrum from all the mass spectra within t
240. ng spectral comparison can help you identify and quantify an intact protein mass a primary sequence and post translational modifications It also helps you visualize very small differences between the two spectra Chromatographic Peak Detection and Spectral Peak Modeling In manual deconvolution you select the spectrum to deconvolve In automatic deconvolution the Protein Deconvolution application uses the Parameterless Peak Detection PPD algorithm to analyze the raw data and to separate peaks from noise in chromatograms This algorithm does the following to locate peaks in a chromatogram e Constructs a chromatogram after applying any parameters that you set on the Chromatogram page It also creates a report e Assigns peak numbers e Generates a peak list e Determines the peak start and peak end points To locate peaks in spectra the ReSpect algorithm fits a peak model to the spectrum to locate potential peaks This peak model resembles a Gaussian distribution You can control the characteristics of this peak model by modifying the relevant parameters on the Parameters page of the Protein Deconvolution window Extracted lon Chromatogram Calculation for Deconvolved Spectra Workflow Thermo Scientific The Protein Deconvolution application calculates an extracted ion chromatogram from a selected component It displays this chromatogram in the Chromatogram pane of the Process and Review page as well as in the Chromatogram page
241. nge RT Range ReSpect3435 C Program Files Protein Deconvolution source 1161 1246 3 39 3 50 151814 828 RSP 02 10 2014 14 09 51 Delete Add to Method Show Details Source Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid raw NL 6 13E 008 ia 151814 853 80 Pe Reference 5 2 F 16585 453 50965 253 64862 253 76465 853 142108 353 149769 453 f 155397953 S pectru m a displayed as a SoA source spectrum The Sample Comparison page contains three panes e Method Reference Spectrum Displays information about the reference spectrum in the currently loaded method e Reference Spectrum Library Displays commands that allow you to select reference spectra to show in the mirror plot display information about specific reference spectra add reference spectra to the method and delete reference spectra from the library e Mirror Plot Displays the mirror plot of the selected reference spectrum and the source spectrum For detailed information on the commands and parameters on the Sample Comparison page see Table 35 on page 197 7 If you want to make this reference spectrum available in the method that you used to deconvolve the reference spectrum select the spectrum in the Reference Spectrum Library and click Add to Method in the Reference Spectrum Library pane Thermo Scientific Protein Deconvolution User Guide 189 4 Manually Deconvolving Isotopically Unresolved Mass Spectra wi
242. nly the most intense peak in each spectrum This means that the base peak chromatogram represents the intensity of the most intense peak at every point in the analysis Base peak chromatograms for each spectrum often have a cleaner look and are therefore more informative than TIC chromatograms because the background is reduced by focusing on a single analyte at every point For intact protein spectra the TIC often looks better The BPC is usually best for smaller molecules where all of the signal exists in a single charge state Sensitivity Sensitivity Specifies the sensitivity with which the chromatographic peak detector identifies peaks e Low Directs the chromatographic peak detector to perform one pass at the default sensitivity e Default High Directs the chromatographic peak detector to perform a second pass at higher sensitivity that is with a slightly narrower width threshold to identify narrow shoulders or noise like peaks that might have been missed by the first pass This option increases sensitivity at the cost of a potential increase in the false positive rate Thermo Scientific Protein Deconvolution User Guide 159 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Table 29 Chromatogram page parameters for ReSpect deconvolution Sheet 3 of 4 Parameter Chromatogram m z Range Description Specifies the range of m z values used
243. ntial peaks in the spectrum that might be associated with compounds are identified and which ones are excluded as being too small The Protein Deconvolution application applies these parameters after it applies the Baseline Correction parameters Minimum Peak Significance Thermo Scientific Specifies a significance level in standard deviations that determines whether the ReSpect algorithm discards a peak as a noise feature or retains it as a legitimate peak The ReSpect algorithm retains peaks equal to or greater than this selected significance level The higher the significance level the more stringent this filtering is Default 1 Protein Deconvolution User Guide 139 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 6 of 15 Parameter Description Noise Rejection Removes noise and irrelevant features from the list of peaks This parameter is important in the ReSpect algorithm because it controls how plausible a peak must be before the algorithm uses it in a deconvolution Reducing this confidence level increases the number of peaks but allows more noise Tests suggest that a confidence level of 95 percent works well in most cases and that you can relax it to 68 percent if necessary A confidence level of 99 percent is rarely required A confidence level of 50 percent or less
244. o Parameters Chromatogram f Process and Review E Sample Comparison E Reporting gt Select an experiment type a data file and a method Experiment Types Load Raw Data File Methods Raw Data Directory C Program Files Protein Deconvolutior i Description Manual Xtract Isotopically Resolved gt DefaultMethodXtract Default method for Xtract Select Raw Data Files HCDfraction03_lowCE raw Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60 Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60 Manual ReSpect Isotopically Unresolved Auto Xtract Isotopically Resolved Auto ReSpect Isotopically Unresolved Load Results Create Method 5 In the Methods pane specify the extraction method to use by doing one of the following e Ifone of the existing methods contains the appropriate parameters select the name of the method of interest and click Load Method Thermo Scientific Protein Deconvolution User Guide 15 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up a Manual Xtract Protein Deconvolution The Protein Deconvolution application automatically transfers you to the Chromatogram page if the raw data file contains a chromatogram or to the Process and Review page if it contains a spectrum only To use the Chromatogram page follow the instructions in Selecting the Spectrum to Deconvolve
245. o a spectrum generated by a manual workflow However you cannot compare any spectrum generated by an Xtract algorithm to any spectrum generated by a ReSpect algorithm For convenience you can save a reference spectrum to a method for future use For manual workflows saving the reference spectrum to a method is optional to automatically produce a mirror plot for automated workflows it is required Note You can still load the results of an automated workflow and compare samples in the Load Previous Results workflow However if you modify the parameters of the original sample comparison you cannot include the resulting mirror plot in the report The Protein Deconvolution application saves a deconvolved spectrum as a reference spectrum in a library whether you deconvolved a spectrum by using an Xtract or ReSpect workflow or loaded previously saved results You can generate an unlimited number of reference spectra The application saves the following information in the database so that if necessary you can reconstruct how you created the reference spectrum 70 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples The absolute path and name of the original raw data file used to produce the reference spectrum The algorithm used to generate the result manual Xtract manual ReSpect automatic Xtract or automatic
246. o perform one pass at the default sensitivity Default High Directs the chromatographic peak detector to perform a second pass at higher sensitivity that is with a slightly narrower width threshold to identify narrow shoulders or noise like peaks that the peak detector might have missed in the first pass This option increases sensitivity at the cost of a potential increase in the false positive rate Protein Deconvolution User Guide 151 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve e Chromatogram m z Range Specifies the range of m z values used to create the chromatogram You can use this parameter to select a narrower range The ReSpect algorithm ignores the portions of the spectrum outside this range You might want to create a narrower range because the intact proteins are usually at a higher m z and any small molecule contaminants and background are below 600 m z Instead of creating a TIC using the full m z range the ReSpect algorithm calculates a TIC by summing those protein peaks within the narrower m z range The resulting TIC is basically an extracted ion chromatogram XIC The ReSpect algorithm redraws the BPC with the most intense peak within the selected m z range rather than the whole spectrum In both cases any peaks for background components generally disappear from the chromatogram and the only peak left is for the target proteins
247. o the directory containing the file For instructions on setting up a default raw data file directory see Specifying the Default Raw Data File Directory on page 9 b In the Select Raw Data Files area click the name of the raw data file c Click Load If another raw data file was previously loaded the warning box shown in Figure 8 appears Figure 8 Unsaved Data dialog box Protein Deconvolution z Any unsaved data will be lost Do you want to continue d Click Yes Although you can load a raw data file with a maximum size of 34 GB processing and reporting issues might result when you load files this large Thermo Fisher Scientific recommends that you work with files up to 2 GB 14 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up a Manual Xtract Protein Deconvolution Several more tabs appear along the top of the Protein Deconvolution window and the available methods including a default method appear in the Methods pane as shown in Figure 9 Note The Run Queue tab is not available unless you select Auto Xtract or Auto ReSpect in the Experiment Types pane Figure 9 Method Selection page for manual Xtract deconvolution after the raw data file is loaded Protein Deconvolution Manual Xiract sotopicaBy Resolved Help 4 SCENT LF LC Myoglobin_30pmol_michrom_protein_microtrap_1imin_OT_60K_LRAW Run Queue f
248. olution application deletes all the results for the corresponding raw data file 184 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples Note After you delete the SQLite file the run still appears in the run queue in the Auto ReSpect workflow Use the Queue Manipulation gt Remove Selected command to remove it from the queue Comparing Samples As noted in Sample Comparison on page 4 you can compare the deconvolved spectra from two different samples or you can compare two different averaged spectra from the same LC MS run You designate one spectrum as the reference spectrum and the other as the source spectrum When you compare the source spectrum to a reference spectrum the Protein Deconvolution application displays a mirror plot of the two spectra see Figure 92 on page 192 The reference spectrum is the plot in the negative direction and the current spectrum is the plot in the positive direction You can use this plot to see whether the structures and the relative abundance of masses in the two spectra are divergent or the same Divergence can indicate that the target protein sequences have been modified by post translational modifications such as phosphorylation or glycosylation You can then use top down proteomics techniques or peptide mapping to determine the exact cause of these changes You can compare two sp
249. on 108 222 PDF files saving report in automated ReSpect 218 saving report in automated Xtract 104 saving report in ReSpect 8 35 148 202 saving report in Xtract 8 85 88 peak deconvolution 3 peak detection 4 5 peak models 127 128 130 138 139 144 Peak Width parameter 129 142 peptide mapping 5 70 185 phosphorylation 5 70 185 potassium 21 30 PPM Std Dev column 177 180 182 Print dialog box 88 89 202 Print File icon 88 89 202 printing deconvolution report 36 88 148 202 Priority parameter 110 224 Process and Review page Deconvolved Spectrum pane 51 165 deconvolving a spectrum 49 163 displaying deconvolution results 52 112 166 226 Main Parameters ReSpect pane 164 Main Parameters Xtract pane 50 menu bar commands 65 179 parameters on 61 176 Results table 51 165 Thermo Scientific Index P retention of layout dimensions 52 165 Saved ReSpect Results pane 50 164 Saved Xtract Results pane 50 164 shortcut menus 65 180 Source Spectrum pane 51 164 165 Process command 51 65 179 profile data 43 156 180 183 Protein Deconvolution application adjusting pane size 10 automatic mode 4 displaying deconvolution results 226 displaying results 112 114 226 displaying results of deconvolution 228 exiting 11 features inputs 8 loading saved results 115 229 manual mode 4 manual workflow 5 outputs 8 peak detection and modeling 5 ReSpect algorithm See ReSpect algorithm specifying default raw data file d
250. on Briefly describes the method to use in the deconvolution Create Method Activates the Parameters page so that you can specify the parameters for a new method Load Method Loads the specified existing method Creating a ReSpect Method When you click Create Method in the Methods pane of the Method Selection page the Protein Deconvolution application automatically transfers you to the Parameters page The Parameters page features four panes containing parameters that control the deconvolution e Main Parameters ReSpect Displays basic parameters that might change often These parameters also appear on the Process and Review page e Advanced Parameters ReSpect Displays parameters that change infrequently Only experienced users should change these parameters e Reporting Parameters Displays parameters that determine what information appears on the Reporting page after the Protein Deconvolution application generates the deconvolution reports Thermo Scientific Protein Deconvolution User Guide 123 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method e Automation Parameters Displays parameters that control the reporting for automatic deconvolution For information on automatic ReSpect protein deconvolution see Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm on page 215 For detailed descriptions of the parameters
251. on Library See CDL Component Source of Evidence Plots parameter 25 35 133 148 Components Table parameter 25 34 133 147 Concatenate All Reports parameter 36 104 149 218 Consider Overlaps parameter 23 32 contacting us x Copy Bitmap command ReSpect 162 180 200 Xtract 48 65 85 Copy Data command 65 180 Create Method button 16 18 121 123 Creation Time column 83 198 CSV files 68 182 183 deconvolution 1 deconvolution algorithms 1 Deconvolution Parameters parameter 24 34 132 147 deconvolution report displaying 114 228 printing 36 148 deconvolution result names 113 227 deconvolution results 51 112 165 226 232 Protein Deconvolution User Guide Deconvolved Spectrum pane deconvolved spectrum in 51 165 displaying results in 58 60 170 172 exporting the source spectrum 68 183 highlighting calculated mass 169 parameters on 62 177 purpose 51 165 Deconvolved Spectrum parameter 25 34 133 147 Deconvolved Spectrum report section description 94 displaying 25 34 133 147 208 Degree of Fit parameter 129 142 deisotoping 2 Delete command 84 199 deleting deconvolution results 69 184 Delta Mass column 66 68 177 180 182 Delta Mass Da column 168 179 Delta Mass parameter 63 Delta Mass PPM column 168 179 Description column 17 123 Descriptions column 84 199 Descriptions parameter 83 198 deuterium 125 135 device drivers viii Dil Factor column 90 204 documentation accessing vii additi
252. on Set Priority Open Result Open Report Number of Number Of Record Experiment i Chromatographic Components Completion Time Submit Time Method Name Raw Data File Bihis Type Peaks Detected Normal 10 14 2013 3 42 28 PM DefaultMethodXtract C Program Files Protein Deconvolution source files XTAUTO Processing Normal 10 14 2013 3 42 32 PM _ _ DefaultMethodXtract C Program Files Protein Deconvolution source files XTAUTO Queued Pause A confirmation box similar to that shown in Figure 59 appears Figure 59 Confirmation box N Analyzing C Program Files Protein Deconvolution source y files Xtract Myoglobin_30pmol_michrom_protein_microtrap_11min_OT _60K_1 RAW Queue will pause after the analysis is completed 2 Click OK After the current analysis finishes the rest of the jobs remain in the run queue in the Queued state 3 Click Run to have the Protein Deconvolution application process the remaining jobs 108 Protein Deconvolution User Guide Thermo Scientific 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Running Jobs in the Queue To remove a selected job or jobs from the queue Select the job or jobs to remove from the queue Remove multiple jobs by doing the following e To select contiguous files names click the name of the first job hold down the SHIFT key and click the last job that you want to select e To select noncontiguous names click th
253. on according to the instructions in Starting the Protein Deconvolution Application on page 8 118 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up a Manual ReSpect Protein Deconvolution 2 Click the Method Selection tab if it is not already selected The Method Selection page contains three panes e Experiment Types pane Displays the available workflows and a command for loading the saved results of previous deconvolutions Load Raw Data File pane Displays the list of the available raw data files for the selected algorithm e Methods pane Displays the available methods 3 In the Experiment Types pane click Manual ReSpect Isotopically Unresolved 4 In the Load Raw Data File pane select the raw data file that contains the spectral data for your sample a In the Raw Data Directory box type the path of the raw data file or click the Browse button to browse to the directory containing the file For instructions on setting up a default raw data file directory see Specifying the Default Raw Data File Directory on page 9 b Inthe Select Raw Data Files box click the name of the raw data file c Click Load If another raw data file was previously loaded the message box shown in Figure 62 appears Figure 62 Unsaved Data dialog box Protein Deconvolution Ex Any unsaved data will be lost Do you
254. onal vii E ESI 1 21 125 Exactive mass spectrometers 8 20 27 131 145 Exactive Plus EMR mass spectrometer 121 218 ExampleMethodNativeMS method Charge State Range parameter 127 139 m z Range parameter 126 137 Mass Tolerance parameter 127 138 Minimum Adjacent Charges parameter 130 144 Output Mass Range parameter 126 137 purpose 121 218 Use Auto Spectral Averaging parameter 152 160 Excel files 68 182 183 exiting Protein Deconvolution application 11 Expected Intensity Error parameter 24 33 Experiment Type column 83 198 Experiment Type parameter 111 225 Experiment Types pane parameters on 17 122 purpose 14 102 119 216 Export All command 54 66 68 168 180 182 Export Top Level command 54 66 68 168 180 182 Thermo Scientific exported spectrum files in qb raw file format 20 27 131 145 extracted ion chromatogram 5 126 136 178 F Feature Width parameter 129 142 Fit column 64 Fit Right column 64 Fit column 57 Fit Factor parameter 23 31 Fit column 54 Fit Left column 54 64 Fit Right column 54 Fractional Abundance column 63 66 68 178 180 182 From parameter 84 199 FTMS mass spectrometers 8 20 27 131 145 G Gaussian distribution 5 118 Global Settings dialog box 9 Global Settings icon 9 glycosylation 5 70 78 185 H harmonics 4 157 173 174 High command 107 110 222 224 hydrogen 21 30 125 135 Injection Volume uL column 90 203 inputs to Protein D
255. onvolve centroid spectra and profile spectra The Protein Deconvolution application automatically chooses the appropriate type of spectrum The Source Spectrum pane displays profile information if it is available and centroid information if it is not 42 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve e Centroid data represents mass spectral peaks in terms of two parameters the centroid the weighted center of mass and the relative intensity the normalized area of the peak The data is displayed as a bar graph of relative intensity versus m z e Profile data represents the entire spectrum as a succession of points in m z and relative intensity The data is displayed as a line graph of relative intensity versus m z The source spectrum also appears in the Process and Review pane for deconvolution 5 Optional Adjust the view in the Source Spectrum pane if necessary To enlarge the view to see more detail do one of the following Drag the red cross shaped cursor over the peak or peaks of interest to form a box as shown in Figure 19 Figure 19 Enlarging an area by drawing a box around the peaks of interest Source Spectrum Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 148 178 RT 3 27 3 88 AV 30 F FTMS p ESI Full ms 300 00 2000 00 e7 391 2844 Relative Intensity di 0
256. or a CSV file Right click in the application and choose Paste The Protein Deconvolution application exports mass x axis and intensity y axis data from the Deconvolved Spectrum pane to the Excel or the CSV file For ReSpect deconvolutions the saved data consists of a centroid spectrum followed by the associated profile spectrum Figure 87 gives an example of the mass and intensity data in an Excel file Protein Deconvolution User Guide 183 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Deleting the Results of a Deconvolution Figure 87 Mass and intensity data from the deconvolved spectrum in an Excel file Qa kae EE Home Insert Page Layout Formulas B Become jua Aa a iil Clipboard Font m Mass Intensity 3373 199 5517 949 5612 34 5612 627 5612 986 5616 34 5616 669 5622 005 5622 322 5622 644 5626 5626 329 5626 664 5627 673 5630 658 5631 989 5632 318 5635 34 5635 657 5652 329 5660 986 Deleting the Results of a Deconvolution You can manually delete the results of deconvolution outside of the Protein Deconvolution application To delete the results 1 Navigate to the directory containing the raw data files 2 Select the SQLite file or files containing the results that you want to delete The SQLite file has the same name as the original raw data file 3 Right click and choose Delete The Protein Deconv
257. or example if you set this parameter range from 1 through 5 the Xtract algorithm considers only charge states 1 through 5 for deconvolution It ignores charge states 6 and higher The default range is 5 through 50 Minimum Intensity Specifies a minimum intensity threshold to filter out possible background noise even when you set the S N Threshold parameter to zero The minimum value is 0 and there is no maximum The default value is 1 Protein Deconvolution User Guide 23 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method e Expected Intensity Error Specifies the permissible percentage of error allowed in calculating the ratio of the most abundant isotope to the next isotope higher in mass in the isotope series The default is 3 If you want to return the parameters in the Advanced Parameters Xtract pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any parameters in this pane but do not click Apply and then click another tab the message box shown in Figure 12 on page 22 appears Click Yes to apply the parameter changes or No to restore the parameter defaults 4 Optional In the Reporting Parameters pane select the parts of the generated deconvolution report that you want to display e Sample Information Determines whether to display the Sample Information section of the report
258. or information on this section see Deconvolved Spectrum Section on page 208 e Default Selected Displays the Deconvolved Spectrum section of the report e Cleared Does not display the Deconvolved Spectrum section of the report Components Table Determines whether to display in the report the information that appears in the Results table on the Process and Review page For information on this section see Displaying the Results on page 165 e Default Selected Displays the information that appears in the Results pane on the Process and Review page e Cleared Does not display the information that appears in the Results pane on the Process and Review page Protein Deconvolution User Guide 147 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 14 of 15 Parameter Component Detail Tables Description Determines whether to display the Average Mass table for each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 210 e Selected Displays the Average Mass table in the Source Spectrum Evidence section of the report e Default Cleared Does not display the Average Mass table in the Source Spectrum Evidence section of the report Component Source of Evidence Plots
259. or one of its charge states in the Results table you see the following changes in the panes of the Process and Review page e Source Spectrum pane When you click an isotopic cluster the Source Spectrum pane overlays onto the spectrum blue lines representing the calculated monoisotopic m z values for each charge state in the isotopic cluster as shown in Figure 29 58 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 29 Source Spectrum pane showing the calculated monoisotopic m z values for each charge state in an isotopic cluster Source Spectrum 848 5571 1004391 2844 When you click an individual charge state of the selected isotopic cluster the Source Spectrum pane displays a blue line representing the calculated monoisotopic m z value for the charge state as shown in Figure 30 Figure 30 Source Spectrum pane showing the calculated monoisotopic m z value for an individual charge state in an isotopic cluster Source Spectrum 848 5571 1004391 2844 50 1060 3778 1304 9267 1696 0063 1918 3183 500 1000 1500 2000 e Chromatogram pane When you click an isotopic cluster in the Results table and you selected the Calculate XIC check box on the Parameters page the Chromatogram pane displays the chromatogram overlaid onto the isotopic cluster s extracted ion chromatogram
260. or the ReSpect Algorithm 176 Process and Review Page Menu Bar Commands 0000 179 Process and Review Page Shortcut Menus 0000s eee eee 180 Saving the Results of a Deconvolution i094 cesewes ceased vgn vanced 181 Exporting the Results of a Deconvolution 0 0 0 e eee ee eee 182 Deleting the Results of a Deconvolution 0 0 0 0 cece 184 Comparing Samples oerset wage pias Glee aes A Ea ty oleae 185 Comparing Samples in Saved ReSpect Results 00000000 197 Sample Comparison Page Parameters for the ReSpect Algorithm 197 Sample Comparison Page Menu Bar Commands 04 199 Mirror Plot Shortcut Menwins spec dente road eee td dso Raden 200 Protein Deconvolution User Guide v Contents vi Chapter 5 Protein Deconvolution User Guide Displaying a ReSpect Deconvolution Report 0 00 cece eee eee 200 Reporting Page Toolbar tcctunhioustuntaawavtades Mratdivauncs Siac 203 Sample Information Section uss tia ca ae ews ae pw AOE 203 Chromatogram Parameters Section ac datos a 0N cates as eee dee 204 Source Chromatogram Sectioning i sa veh ee eee a ae nw heh wp ee eek wes 205 Source Spectrum Section 6 eee cece eens 206 Main Parameters ReSpect Sections Acs enna wecae errereen nuna 207 Advanced Parameters ReSpect Section 0 1 0 cece eee eee eee 207 Deconivolved Spectrum Sectioni o 005 i cie cede ieee eee ae ees 208 Mirror Plot Sectio
261. ore than one deconvolved spectrum for any given source spectrum For example if the sample contains more than one protein you might want to perform two deconvolutions with two different mass ranges 22 000 24 000 for immunoglobulin G IgG light chain and 50 000 52 000 for IgG heavy chain Thermo Scientific Protein Deconvolution User Guide 215 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up an Automatic ReSpect Protein Deconvolution Setting Up an Automatic ReSpect Protein Deconvolution In automatic protein deconvolution you add files to a run queue and then deconvolve the queued files First you select the ReSpect deconvolution algorithm a raw data file and a method To set up an automatic protein deconvolution with the ReSpect algorithm 1 Start the Protein Deconvolution application according to the instructions in Starting the Protein Deconvolution Application on page 8 2 Click the Method Selection tab if it is not already selected The Method Selection page contains three panes as shown in Figure 107 e Experiment Types pane Displays the available deconvolution algorithms and a command that you can use to load the saved results of previous deconvolutions e Methods pane Displays the available methods e Load Raw Data File pane Displays the list of the available raw data files for the selected algorithm 3 In the Experiment Types pane click Auto ReSpect
262. oss the data Thermo Scientific Protein Deconvolution User Guide 143 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 10 of 15 Parameter Description Minimum Adjacent Specifies the minimum number of peaks in a row that must Charges appear at the lower and the upper end of the spectrum if the ReSpect algorithm is to recognize a component as real The ReSpect algorithm rejects potential components with fewer than this number of adjacent peaks Minimum values 1 and 1 Maximum values None Default values for the default method 6 and 10 Default values for the ExampleMethodNativeMS method 4 and 10 Note The performance of the ReSpect algorithm improves as the number of charge states increases Tests suggest that this algorithm always yields reliable results if the minimum number of allowed charge states at the low and high end of the m z range is 6 and 10 respectively These numbers are therefore the default parameters In cases where a sample does not provide this number of charge states parameter values of 6 and 6 should still give high quality results For smaller numbers of charge states reliability is lower so you should confirm the results by other means if possible Peak Model Displays parameters that place restrictions on the width and shape Parameters that a peak must have to be as
263. page 37 through step 4 in Qual Browser in the Xcalibur data system and then right click and choose Export gt Write to RAW File so that you can import the file into the Protein Deconvolution application Thermo Scientific Protein Deconvolution User Guide 41 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve The spectrum appears in the Source Spectrum pane of the Chromatogram page as shown in Figure 18 Figure 18 Spectrum in the Source Spectrum pane of the Chromatogram page for Xtract deconvolution a a telae i i Manual Xtract Isotopicaliy Resolved Thermo Protein Deconvolution Detauitmethodxtract poca Help 3 SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_1imin_OT_60K_LRAW o Method Selection v F v Parameters v Chromatogram k Process and Review Sample Comparison B Reporting gt Select the chromatogram to deconvolve Save Method Save Method As Reset Method Chromatogram Parameters Use Restricted Time E Types mc m Ages Time Limits 0 01 to 10 98 Sensitivity High 1 Rel Intensity Threshold 1 Chromatogram m z Range 600 0000 to 2000 0000 Chromatogram Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 NL 8 43E7 F 3 65 RT 3 66 1005 166 Relative Intensity RT 0 58 RT 1 02 S 28 Sit AT RT 5 90 491 5 34 S 268 621 6 59 7 06 7 26 7 55 8 14 _122 169 198 238 298 3 01 8 47 8 81
264. page displays four results unless one of the averaged spectra from a chromatographic peak fails to produce a deconvolved spectrum For more information on this page see Displaying the Results on page 51 To adjust the size of the panes on this page see Adjusting the Pane Size on page 10 The Save Method Save Method As and Result Method commands are not available when you access the Chromatogram page and the Process Save Result As and Reset Method commands are not available when you access the Process and Review page from the Open Result command on the Run Queue page 112 Protein Deconvolution User Guide Thermo Scientific Result Names Thermo Scientific A N ON 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results You cannot open the results if the Protein Deconvolution application has not yet analyzed the raw data file or is in the process of analyzing it Opening the results does not stop the application from analyzing subsequent data sets To adjust the chromatogram Click the Method Selection tab Click Manual Xtract Isotopically Resolved and reload the raw data file by following the instructions in Creating an Xtract Method on page 18 Adjust the chromatogram by following the instructions in Selecting the Spectrum to Deconvolve on page 37 Resubmit the job to the job queue for automatic processing To copy a chromato
265. pect ha aretha a i IgG_source_cid raw _ Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting i Source Chromatogram Process Save ResultAs Reset Method Save as Reference SCIENTIFIC Y Main Parameters ReSpect Negative Charge F m z Range Min 1000 Max 4000 Apply Charge Carrier H 1 00727663 Output Mass Range Min 1000 Max 160000 2H 2 013553 jsa z T Na 22 9892213 Mass Tolerance 30 Custom Target Mass 150000 Da Rel Abundance Threshold 0 5 ra Charge State Range 10 to 100 Calculate XIC v A iy Choice of Peak Model Quality Score Threshold 0 Results Source Spectrum Y Saved Xtract Results 400 2761 2696 3535 4380 RT min Deconvolved Spectrum Saved ReSpect Results DefaultMethodReSpect_1089_1 DefaultMethodReSpect_1129_1 DefaultMethodReSpect_36_57_ DefaultMethodReSpect_68_356 DefaultMethodReSpect_844_87 DefaultMethodReSpect_893_90 DefaultMethndReSnect 900 94 4 Load Result The Process and Review page displays the following panes Note To size these panes vertically see Adjusting the Pane Size on page 10 e Main Parameters ReSpect Displays the same parameters as those in the Main Parameters ReSpect pane on the Parameters page so that you can adjust them without returning to the earlier page e Saved Xtract Results Displ
266. pectrum pane of the Process and Review page as a set of peaks in mass and relative intensity It also displays the component list in the Results pane as a table of masses intensities charge state information and quality scores See Figure 23 on page 53 The values in the columns of the Results table represent the outputs of the deconvolution Each peak in the Results table is composed of isotopic clusters Each isotopic cluster in the original spectrum provides evidence for the peak in the deconvolved spectrum You can expand each entry in this table to display detailed information about the individual charge states that the entry contains Protein Deconvolution User Guide 51 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Note When you do any of the following the layout dimensions of the Process and Review page remain fixed e Navigate away from the Process and Review page to another page of the Protein Deconvolution application e Open results from the Run Queue e Load previous results e Switch algorithms from Xtract to ReSpect or from ReSpect to Xtract However the layout dimensions do not remain fixed when you close the application and reopen it They revert to the default dimensions See the following sections e Displaying the Results on the Process and Review Page e Selecting a Reference Mass to Calculate Mass Differences e Adjusting the Deconvolution
267. pen Result Open Report Number of Chromatographic Components Completion Time Peaks 1 Normal 10 14 2013 4 14 49PM_ _DefaultMethodReSpect C Program Files Protein Deconvolution source files RSP_AUTO 10 14 2013 4 20 23 PM Completed Experiment Submit Time Method Name Raw Data File Type For information about the columns on this page see Run Queue Page Parameters for Automatic ReSpect Deconvolution on page 224 3 Optional Once the status displayed in the Status column is Completed you can open the results or open a report See Displaying the Results on page 226 or Displaying a ReSpect Deconvolution Report on page 228 To assign a priority level to jobs in the queue 1 Click the Run Queue tab to open the Run Queue page if it is not already opened 2 Click a job to select it 3 In the Run Queue menu bar choose Set Priority gt priority_level where priority_level can be one of the following e Low Processes the job after jobs that are assigned a Normal or High priority level e Default Normal Processes the job before jobs that are assigned a Low priority level but after jobs that are assigned a High priority level Thermo Scientific Protein Deconvolution User Guide 221 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue e High Processes the job before jobs that are assigned a Low or Normal priority level High priority jobs are
268. port e Default Cleared Does not automatically print the generated deconvolution report Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Table 27 Parameters page parameters for ReSpect deconvolution Sheet 15 of 15 Parameter Description Concatenate All Determines whether to combine all reports for all deconvolved Reports spectra from the same raw data file e Default Selected Opens one report for a single raw data file e Cleared Opens a report for each chromatographic peak in the raw data file Select a Printer Displays a list of available printers that can print the deconvolution report PDF file This option becomes available when you select the Auto Print option Menu Bar Commands on the Parameters Page Table 28 describes the commands on the menu bar of the Parameters page Table 28 Menu bar commands on the Parameters page for ReSpect deconvolution Parameter Description Save Method Saves the current parameter values to the existing method overwriting any previous values Save Method As Saves the parameter values to a new method Reset Method Returns the parameters on the Parameters page to the settings that you first chose when you loaded the raw data file Selecting the Spectrum to Deconvolve When you click Save Method or Save Method As after you have set the parameters on the Parameters page
269. pplication displays the results of the deconvolution in the Deconvolved Spectrum pane and in the Results table of the Process and Review page To display the results on the Process and Review page Click the Process and Review tab if the Process and Review page is not already open The Process and Review page displays the deconvolved spectrum in the Deconvolved Spectrum pane as shown in Figure 77 Figure 77 Deconvolved spectrum on the Process and Review page for ReSpect deconvolution Protein Deconvolution east i maison eee TE SCIENTIFIC IgG_source_cid raw Chromatogram Sample Comparison _ Reporting i Deconvolution completed successfully Process Save Result As Reset Method Save as Reference Method Selection Run Queue Parameters Negative Charge a m z Range Min 1000 Max 4000 Appl Charge Carrier H 1 00727663 Output Mass Range Min Max 16000 2H 2 013553 ee ms 10000 160000 Na 22 9892213 Mass Tolerance 30 Ppm Custom Target Mass 150000 Da Rel Abundance Threshold 0 Charge State Range 10 S to 10 E Calculate XIC A Choice of Peak Model naz Quality Score Threshold o iceofPeakModel itachmicinan Results Source Spectrum Chromatogram Saved Xtract Results 2761 2696 RT 3 41 100 s 1171 2618 5381 2977 7551 100 50 RT 0 36 RT 3 31 62 2373 1261 50 391 ad 3e3 1409 1859 4854 3201 2790 3707 8040 oi 172 4147 1 37 1 71 268 S 1093 a AS 50
270. r ReSpect algorithm Evaluate raw results or deconvolution results Is component of No interest present Compare samples optional Generate report and save results Thermo Scientific Select a RAW file Single spectrum contain LC MS data or a single spectrum 1 Introduction Workflow generated in Qual Browser Protein Deconvolution User Guide 7 1 Introduction Inputs and Outputs Inputs and Outputs The Protein Deconvolution application can accept the following input and generate the following output Inputs The input file to the Protein Deconvolution application is the raw data file from a mass spectrometry experiment The raw data file can contain LC MS data or a spectrum only You can use the Protein Deconvolution application with data from Exactive Orbitrap and Fourier transform mass spectrometry FTMS series mass spectrometers Outputs As output the Protein Deconvolution application produces a deconvolved spectrum and peak information It generates the following e A report on the results of the deconvolution that appears on the Reporting page of the Protein Deconvolution window For information on the specific contents of this report see Displaying an Xtract Deconvolution Report on page 85 for the Xtract algorithm and Displaying a ReSpect Deconvolution Report on page 200 for the ReSpect algorithm You can save this report in a PDF file e An SQLite fi
271. r Guide 155 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve e Profile data represents the entire spectrum as a succession of points in m z and intensity The data is displayed as a line graph of relative intensity versus m z The source spectrum also appears in the Process and Review pane for deconvolution 5 Optional Adjust the view in the Source Spectrum pane if necessary e To enlarge the view to see more detail do one of the following Drag the red cross shaped cursor over the peak or peaks of interest to form a box as shown in Figure 73 Figure 73 Enlarging an area by drawing a box around the peaks of interest Source Spectrum IgG_source_cid 1059 1331_RT 3 26 3 61 AV 272 F FIMS p ESI Full ms 1000 00 4000 00 100 90 80 70 2977 7538 2574 1143 agg sse 2452 2543 2339 1691 Relative Intensity 30 3231 1617 2141 5468 3374 6304 1508 7419 1659 4849 1947 3273 14106 7029 1276 6958 pinag T T T T T T T T T 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 mz 3785 9021 3931 3879 T T jj 7 T 3200 3400 3600 3800 4000 Draw a box around the peaks of interest Keeping the left mouse button pressed draw a line beneath the baseline of the peaks of interest as shown in Figure 74 Figure 74 Enlarging an area by drawing a line beneath the baseline of the peaks of int
272. r on the results see Specifying the Output Mass Range on page 175 Mass Tolerance Specifies the global allowable error for the m z values of peaks in a charge state series as they appear in the input spectrum This parameter compensates for calibration errors and the effects of local noise peak overlaps and other sources of mismatches between the model and the actual peak profiles Mass Tolerance is an important parameter in the ReSpect algorithm because it controls the tolerance for peaks in m z when the algorithm uses these peaks to produce a particular deconvolved component As you increase the value of this parameter the algorithm expands the plausible set of charge state peaks but at the same time it correspondingly increases the false positive rate As described in Calculating a Protein Quality Score on page 173 the ReSpect algorithm can exclude these false positives to some degree For most purposes the Ppm setting provides better results than the Da setting A Ppm setting between 20 and 50 is a good starting point Default Ppm Specifies the mass tolerance in parts per million The default for the default method is 20 ppm charge number The default for the ExampleMethodNativeMS method is 30 ppm charge number Da Specifies the mass tolerance in daltons The default for the default method is 0 05 Da The default for the ExampleMethodNativeMS method is 0 05 Da Target Mass Specifies an expected target m
273. ract Results Displays each set of Xtract results that you saved from the same raw data e Saved ReSpect Results Displays each set of ReSpect results that you saved from the same raw data 50 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results e Source Spectrum Displays the spectrum that you selected in the Source Spectrum pane of the Chromatogram page e Chromatogram Displays the chromatogram that you selected in the Chromatogram pane of the Chromatogram page When you make a change to the chromatogram on the Chromatogram page the Protein Deconvolution application automatically updates the chromatogram on the Process and Review page e Deconvolved Spectrum Displays the deconvolved spectrum that results from applying the Xtract algorithm e Results Displays the masses and intensities of the peaks that the Xtract algorithm detected during the deconvolution along with their quality scores 2 Optional Adjust any parameters in the Main Parameters Xtract pane For information on these parameters see Table 4 on page 27 3 Click Process in the menu bar For information on the parameters in the panes of the Process and Review page see Table 9 on page 62 Displaying the Results Thermo Scientific When the Protein Deconvolution application finishes processing it displays the deconvolved spectrum in the Deconvolved S
274. rameter on the Parameters page The table shows all the charge states that the Protein Deconvolution application detected for that component It displays the same parameters as those displayed in the Results pane on the Process and Review page For information on these parameters see Table 32 on page 176 The graph shows the peaks in the scan that are associated with a particular component The table in Figure 105 shows only a partial list of values 210 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Figure 105 Source Spectrum Evidence section for ReSpect deconvolution Average Mass 151814 546875 Charge State Measured Average m z Measured Average Mass Delta Mass Da Delta Mass PPM 42 3615 630 0 377 2 483 151814 170 43 3531 620 151816 331 1 784 11 753 44 3451 453 151819 596 5 049 33 257 45 3374 659 151814 336 0 211 1 391 46 3301 382 151817 241 2 694 17 746 47 3231 135 151816 026 1 480 9 746 48 3163 855 151816 713 2 166 14 270 49 3099 323 151817 458 2 912 19 178 50 3037 323 151815 786 1 239 8 163 51 2977 776 151815 186 0 639 4 212 52 2920 543 151815 856 1 309 8 623 53 2865 458 151815 915 1 368 9 009 54 2812 405 151815 452 0 905 5 964 55 2761 287 151815 364 0 817 5 382 56 2711 985 151814 772 0 225 1 484 57 2664 429 151815 022 0 475 3 128 58 2618 516 151815 527 0 980 6 454 59 2574 139 151814 767 0 2
275. reases this threshold might rise The precise threshold below which you might want to ignore components depends on the quality of the spectrum the peak confidence level and the mass tolerance that the ReSpect algorithm uses Tests indicate that 20 might be a good starting point when you select a threshold e Scores between 20 and 40 generally indicate good components e Scores above 40 indicate components of very high quality The protein score calculation algorithm is powerful but it cannot overcome a poor choice of control parameters for the ReSpect algorithm In particular if you run the algorithm with an inappropriate mass or m z range too low a peak confidence level or too high a mass tolerance the results could include fits to noise that no scoring algorithm could identify To specify a minimum component quality score 1 Click the Parameters tab or the Process and Review tab 2 In the Quality Score Threshold box in the Main Parameters section specify the minimum quality score that a component must have to appear in the Results table 174 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results You can enter any floating point number The default is 0 The ReSpect algorithm discards components that do not meet this minimum score To view and sort the quality score 1 After you click Process on the Process
276. requires a very good even though not exact fit between the measured pattern and the averagine pattern A fit factor of 100 means that the observed peaks in the measured isotope profile are absolutely identical to those in a theoretical averagine distribution and that any missing peaks fall below a restrictive threshold Fit Left Displays the quality of the match between a measured isotope pattern and an averagine distribution that is one dalton smaller than the calculated monoisotopic mass Fit Right 64 Protein Deconvolution User Guide Displays the quality of the match between a measured isotope pattern and an averagine distribution that is one dalton larger than the calculated monoisotopic mass Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Process and Review Page Menu Bar Commands Table 10 describes the commands on the menu bar of the Process and Review page Table 10 Menu bar commands on the Process and Review page for Xtract deconvolution Parameter Description Process Deconvolves the spectrum and displays the results in the Results table Save Result As Saves the current deconvolution results in an SQLite file Reset Method Returns the parameters on the Process and Review page to the settings that you first chose when you loaded the raw data file Save As Reference Saves the selected deconvolved spectrum
277. rithm Running Jobs in the Queue Table 22 Run Queue page parameters for automatic Xtract deconvolution Sheet 2 of 3 Parameter Remove Completed Description Deletes all completed jobs from the queue Remove All Deletes all jobs from the queue Set Priority Specifies the order in which the Protein Deconvolution application deconvolves the spectra in the queue Low Processes the job after jobs that are assigned a Normal or High priority level Normal Default Processes the job before jobs that are assigned a Low priority level but after jobs assigned a High priority level High Processes the job before jobs that are assigned a Low or Normal priority level Open Result Transfers you to the Process and Review page after the Protein Deconvolution application finishes processing a job Open Report Transfers you to the Reporting page after the Protein Deconvolution application finishes processing a job Queue Record Number Displays a sequential number that identifies each job Priority Displays the priority level that each job in the queue has e Low Processes the job after jobs that are assigned a Normal or High priority level e Normal Processes the job before jobs that are assigned a Low priority level but after jobs that are assigned a High priority level e High Processes the job before jobs that are assigned a Low or Normal priority level Subm
278. rmation 3 Click the column header to sort the data in each column of the peak table from lowest to highest or highest to lowest For example click the Number of Charge States column header The Protein Deconvolution application which initially displays the number of charge states in this column in order from lowest to highest now displays the number of charge states from highest to lowest Click again to display the numbers from lowest to highest Click the plus sign to the far left of a row in the peak table As shown in Figure 25 eight new columns appear Charge State Calculated Monoisotopic m z Monoisotopic Mass for This Charge Mostabund m z Charge Normalized Intensity Fit Fit Left and Fit Right These values represent the isotopic clusters with different charge states from the source spectrum that were used to produce the peak in the deconvolved spectrum 54 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 25 Hierarchical table in the Results pane for an Xtract deconvolution SCIENTIFIC Protein Deconvolution defauttmethoaxtract al Casio Ceo __ Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting Myoglobin 30pmol_michrom_protein_microtrap_11min_OT_60K_ RAW Deconvoive the spectrum gt Main Parameters Xtract Results K Y
279. rmation box shown in Figure 112 click Yes To remove all completed jobs from the queue On the Run Queue menu bar choose Queue Manipulation gt Remove Completed In the confirmation box shown in Figure 112 click Yes Protein Deconvolution User Guide 223 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue Run Queue Page Parameters for Automatic ReSpect Deconvolution Table 45 describes the parameters available on the Run Queue page for an automatic ReSpect deconvolution Table 45 Run Queue page parameters for automatic ReSpect deconvolution Sheet 1 of 2 Parameter Menu bar commands Description Queue Manipulation Contains commands that cancel or remove selected deconvolution jobs from the queue Remove Selected Deletes the selected job or jobs from the queue Remove Completed Deletes all completed jobs from the queue Remove All Deletes all jobs from the queue Set Priority Specifies the order in which the Protein Deconvolution application deconvolves the spectra in the queue Low Processes the job after jobs that are assigned a Normal or High priority level Normal Default Processes the job before jobs that are assigned a Low priority level but after jobs that are assigned a High priority level High Processes the job before jobs that are assigned a Low or Normal priority level
280. rocess and Review page 62 177 Sample Comparison page 84 199 Source Chromatogram section of report 91 205 Source Spectrum Evidence section of report 97 212 Source Spectrum pane of the Process and Review page 62 176 Source Spectrum section of report 92 95 206 209 release notes viii Remainder Threshold parameter 23 32 Remove All command 109 223 224 Remove command 83 198 Remove Completed command 109 110 223 224 Remove Selected command 109 223 224 Reporting page Advanced Parameters Xtract section 93 Chromatogram Parameters section 90 204 Deconvolved Spectrum section 94 208 displaying deconvolution report 8 85 86 114 200 228 Main Parameters Xtract section 93 Mirror Plot section 95 209 ReSpect Masses Table section 210 Sample Information section 89 203 sections in 86 200 Source Chromatogram section 91 205 Source Spectrum Evidence section 96 210 Source Spectrum section 92 206 toolbar 88 Xtract Masses Table section 96 Reporting Parameters pane ReSpect 123 132 146 Xtract 18 24 33 236 Protein Deconvolution User Guide Reset Method command returning parameters on Advanced Parameters ReSpect pane to original settings 132 returning parameters on Advanced Parameters Xtract pane to original settings 24 returning parameters on Chromatogram Parameters pane to original settings 40 47 152 161 returning parameters on Main Parameters ReSpect pane to original settings 128 returning parameters on Main Par
281. romatogram Section The Source Chromatogram section shown in Figure 46 displays the chromatogram contained in the raw data file It is the same chromatogram that appears in the Chromatogram pane of the Chromatogram page Figure 46 Source Chromatogram section for Xtract deconvolution Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 ProteinDeconvolution NL 3 51E7 i RT 3 65 S 166 100 Relative Intensity RT 10 74 SH 484 RT min Table 17 lists the parameters in the Source Chromatogram section Table 17 Source Chromatogram section parameters for Xtract deconvolution Parameter Description Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity RT min x axis Displays the retention time of the spectrum which is the time after injection at which a compound elutes Retention time can also refer to the total time that the compound is retained on the chromatograph column Thermo Scientific Protein Deconvolution User Guide 91 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Source Spectrum Section The Source Spectrum section shown in Figure 47 displays the spectrum that you selected in the Source Spectrum pane of the Chromatogram page Figure 47 Source Spectrum section for Xtract deconvolution Myoglobin_30pmol_michrom_protein_microtrap_1
282. romatogram page as shown in Figure 72 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve Figure 72 Spectrum in the Source Spectrum pane of the Chromatogram page for ReSpect deconvolution LL Thermo Protein Deconvolution m i i Manual ReSpect sotopically Unresolved Thermo protein Deconvolution ual Respect Gsxtopicaty D heip 3 SCIENTIFIC IgG_source_cid raw o Method Selection J e Parameters C Process and Review Sample Comparison t Reporting Select the chromatogram to deconvolve Save Method Save Method As Reset Method Chromatogram Parameters Relative Intensity Use Restricted Time F Types mc x App a Time Limits 0 00 to 623 Sensitivity High 1 Rel Intensity Threshold 1 Chromatogram m z Range 1000 0000 to 4000 0000 fi Chromatogram 196_source_cid NL 3 10E9 RT 3 41 100 S 1171 80 5 RT 0 36 382 3 91 407 434 Riam RT 3 03 repeat 4 61 4 78 5 01 5 14 5 34 5 47 5 60 5 90 6 00 0 1 s gs1 S 918 0 67 0 83 1 11 1 22 1 37 1 46 1 63 1 80 2 05 2 29 2 43 0 5 1 0 15 20 25 3 0 35 40 45 5 0 5 5 6 0 RT min Relative Intensity Source Spectrum IgG_source_cid 1089 1348 RT 3 30 3 63 AV 259 F FTMS p ESI Full ms 1000 00 4000 00 100 2761 2696 2865 4305 90 80 70 60 50 40 30 20 10 2977 7551 3037
283. rror Plot Section The Mirror Plot section shown in Figure 103 displays the mirror plot as it appears in the Mirror Plot pane of the Sample Comparison page Figure 103 Mirror Plot section for ReSpect deconvolution Mirror Plot Source Data File IgG_source_cid raw 100 NL 2 741 80 NL 7 221 60 40 151813 6 20 50653 0 2 ii e 60662 5 100992 4 0 E g 100908 8 d 2 379537 _ 40 50605 1 60 30 100 50000 100000 150000 m z Reference Data File IgG_source_cid raw Table 42 lists the parameters in the Mirror Plot section Table 42 Mirror Plot section parameters for ReSpect deconvolution Parameter Description Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity Mass x axis Displays the mass of the ions formed from molecules Thermo Scientific Protein Deconvolution User Guide 209 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report ReSpect Masses Table Section The ReSpect Masses Table section shown in Figure 104 displays the results of the deconvolution It contains the same columns as those in the Results pane on the Process and Review page For information on the columns in this table see Table 32 on page 176 Figure 104 ReSpect Masses Table section for ReSpect deconvolution ReSpect Masses Table SACE Ea 47 1 70 0 00
284. rs for Xtract deconvolution Parameter Relative Intensity y axis Description Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules Protein Deconvolution User Guide 97 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Loading Saved Xtract Results Loading Saved Xtract Results If you saved the results of an Xtract deconvolution you can reload them at a later time You can load multiple results as long as they are from a single SQLite file You can concurrently load multiple Xtract results or multiple ReSpect results but not both types of results at the same time For information on comparing samples using saved Xtract results see Comparing Samples in Saved Xtract Results on page 82 You can use one of two ways to load the results of previous deconvolutions e Choose the Load Results command on the Method Selection page e Click the applicable results in the Saved Xtract Results pane on the Process and Review page Note When you load results generated in version 1 0 any parameters added since version 1 0 are set to 0 To load saved results from the Method Selection page 1 Click the Method Selection tab 2 In the Experiment Types pane click Load Results 3 In the Raw Data Directory box of the Load Result File pane type the p
285. rtion of the input spectrum that the ReSpect algorithm processes e Min Specifies the lowest end of the input spectrum e Max Specifies the highest end of the input spectrum For example if the total mass range of the spectrum is mass 100 to 2000 a setting of 300 to 500 for the m z Range parameter means that the Xtract algorithm processes only peaks with masses between 300 and 500 m z Range 300 25 000 Default range for the default method 1000 4000 Default range for the ExampleMethodNativeMS method 1000 15 000 Output Mass Range Specifies the required output mass range e Min Specifies the lowest end of the mass range e Max Specifies the highest end of the mass range Default range for the default method 10 000 160 000 Default range for the ExampleMethodNativeMS method 10 000 1 000 000 For more information on the Output Mass Range parameter see Specifying the Output Mass Range on page 175 Protein Deconvolution User Guide 137 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 4 of 15 Parameter Mass Tolerance Description Specifies the global allowable error for the m z values of peaks in a charge state series as they appear in the input spectrum This parameter compensates for calibration errors and the effects of local noise peak overlaps and other sources
286. rum Figure 22 Initial Process and Review page for Xtract deconvolution Protein Deconvolution DefauitMethodXtract Manual ragt Gsotopicaly Resolved Help SCIENTIFIC Myoglobin_30pmol_michrom_protein_microtrap_1imin_OT_60K_LRAW z Method Selection Run Queue z Parameters z Chromatogram Sample Comparison Reporting i Deconvolve the spectrum Process Save ResultAs Reset Method Save as Reference Y Main Parameters Xtract Output Mass M OMH m z Range Min 600 Max 2000 Apply Resolution at 400 m z 60000 Charge Carrier H 1 00727663 S N Threshold 3 K 38 9631585 Na 22 9892213 Rel Abundance Threshold 0 Custom Negative Charge Min Num Detected Charge 3 Calculate XIC Isotope Table Chromatogram Y Saved Xtract Results 893 1164 RT 5 90 1304 9242 4696 2001 1918 3288 9 S 268 6 75 7 40 8 47 8 94 9 64 RT min Y Saved ReSpect Results Average Mass Sum Intensity etot Mass Std Dev PPM Std Dev Delta Mass Hene iii RT Range Charge States Abundance Abundance The Process and Review page displays the following panes when you first access it Note To size these panes vertically see Adjusting the Pane Size on page 10 e Main Parameters Xtract Displays the same parameters as those in the Main Parameters Xtract pane on the Parameters page so that you can adjust them without returning to the earlier page e Saved Xt
287. rum For example if the highest peak has an absolute abundance of 1000 the relative abundance is 1 percent and no peaks below an absolute abundance of 10 will appear in the deconvolved spectrum Range 0 100 Zero 0 displays all results and 100 displays only the most abundant component Default 0 Calculate XIC Determines whether the Protein Deconvolution application calculates the extracted ion chromatogram from a range of deconvolved spectra This check box is not available for single spectra because there is no chromatogram e Default Selected Calculates the extracted ion chromatogram e Cleared Does not calculate the extracted ion chromatogram The application saves this setting to the method Quality Score Specifies a minimum protein quality score that components must Threshold have to be displayed in the Results table The Protein Deconvolution application discards components that have a score beneath this threshold For more information on the protein quality score see Calculating a Protein Quality Score on page 173 You can enter any floating point number Default 0 136 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 3 of 15 Parameter m z Range Description Specifies the po
288. rum is now available in the method The Protein Deconvolution application populates the fields of the Method Reference Spectrum pane The From area at the bottom of the Mirror Plot pane changes to Method Optional for manual workflows but required for automatic workflows if you want to compare the data to be analyzed to a reference spectrum Save the method containing the reference spectrum as follows e To save the method under the existing name select the reference spectrum from the library and click Save Method or e Ifyou used the default method save the method under another name i Select the reference spectrum from the library and click Save Method As The Save dialog box appears ii Type the name of the method in the Method Name box iii Type a brief description of the method in the Descriptions box iv Click Save When you use this method in the future the Protein Deconvolution application uses the saved reference spectrum as the reference spectrum in the mirror plot To compare a deconvolved source spectrum to a reference spectrum Deconvolve the source spectrum that you want to compare to the reference spectrum For best results with the automatic Xtract workflow use the method to which you saved the reference spectrum to generate the source spectrum Figure 38 shows a spectrum that was deconvolved from the chromatogram in the PSA_240_highpI raw file between approximately 6 7 and 8 2 minutes It is the
289. s and Review ff EIA Reporting compare samples for deconvolved spectra Save Method Save Method As Reset from Method Save Result As Method Reference Spectrum Reference Spectrum Reference Data File Show Bee Reference Spectrum Library Reference Spectra Raw Data File Scan Range RT Range SEASSA EET TE Descriptions Mass Type Peaks ReSpect3435 C Program Files Protein Deconvolution source 1161 1246 3 39 3 50 151814 828 02 10 2014 14 09 51 Add to Method to Method Show Details Source Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid raw NL 1 96E 008 w 151813 623 20 Source 60 2 40 152431 923 spectrum 5 149561 623 g 70 25176 523 38134723 49243 323 74697 723 101052 923 i pea p am 1 7 20416585453 50965 253 64862 253 76465 853 149765 453 155397 953 3 40 Reference 80 pe 151814 853 spectrum Mass Reference Data File C Program Files Protein Deconvolution source files ReSpect Q Exactive lgG_source_cid raw From Library NL 6 13E 008 Note If you try to compare a spectrum generated by an Xtract algorithm to a spectrum generated by a ReSpect algorithm an error message appears The mirror plot is scaled to 100 percent in both directions but the actual intensities of the spectra can be completely different You can tell the different intensities of the peaks in each spectrum by looking at the NL values see Source Spectrum Pane
290. s baseline Chromatogram Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 NL 8 43E7 F 3 65 RT 3 66 100 e s 2 s Relative Intensity RT 0 58 RT 1 02 s28 SHAT RT 5 90 19 aa 238 288 3 01 462 49 534 S 268 621 6 59 oder T TTET pT i rT TTT T 05 10 15 20 25 30 3540 45 5 0 357 4 7 06 7 26 755 8 14 8 47 881 9 06 9 33 60 65 70 75 8 0 85 90 95 10 0 55 RT min 10 00 10 27 10 58 Draw a line under the baseline of the peak of interest Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve When you zoom a region of the chromatogram or the spectrum the Protein Deconvolution application recalculates the values on the y axis so that 100 percent represents the highest abundance in the displayed region as shown in Figure 17 The abundances shown on the y axis remain at values relative to the most abundant component in the plot Drawing a box rescales the chromatogram or spectrum to the relative height of the box Figure 17 Chromatogram automatically scaled to the maximum height of the peak in the Chromatogram pane Chromatogram Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 NL 8 51E7 F RT 3 66 3 65 S 166 007 Relative Intensity Tatui Y 8 I o 4 4 J J TT T T TTT TTT Torere r Saal 3 35 3 40 5 3 60 RT min If there is
291. s dialog box so that you can save the selected reference spectrum to the current results Mirror Plot Shortcut Menu By right clicking in the Mirror Plot pane on the Sample Comparison page you can access a shortcut menu with the commands listed in Table 37 Table 37 Mirror Plot pane shortcut menu commands Parameter Description Reset Scale Restores the original plot that first appeared in the Mirror Plot pane Copy Bitmap Copies the view in the Mirror Plot pane to the Clipboard Zoom Out Shrinks the plot in the Mirror Plot pane by a factor of two Zoom In Enlarges the plot in the Mirror Plot pane two times Displaying a ReSpect Deconvolution Report When you click Process on the Process and Review page the Protein Deconvolution application generates a report displaying several aspects of the deconvolution so that you can track the progression of the data You can view this report on the Reporting page and save it as a PDF file When you load multiple results the application still generates a single report containing the concatenated results However the report can only contain results from either the Xtract or ReSpect algorithm but not both To display a report Click the Reporting tab when you have finished analyzing the data The Reporting page partially shown in Figure 95 displays a summary of all results for a given data file It contains the following sections Sample Information Section Chromatogr
292. s pane deleting results from 67 182 making visible 181 purpose 50 62 67 164 176 182 resizing 10 67 182 Protein Deconvolution User Guide 237 Index T Scan Range column 83 198 Score column 180 182 Score parameter 178 Select a Printer parameter 36 104 149 218 Select command 84 199 Select Raw Data Files area 17 123 Sensitivity parameter 39 46 151 159 Set As Reference Component command 66 180 Set Priority menu 107 110 221 224 Show Acrobat icon 88 89 202 Show Details command 79 83 84 194 198 199 signal to noise threshold 20 27 141 single scans 41 154 sodium 21 30 125 135 software requirements viii Source Chromatogram report section 91 205 Source Data File parameter 84 199 source spectrum 4 70 185 Source Spectrum Evidence report section description 96 210 displaying 87 202 displaying Average Mass table in 133 displaying Monoisotopic Mass table in 25 Source Spectrum pane adjusting the view 43 156 creating spectrum in 41 154 displaying calculated monoisotopic m z values 170 displaying results in 58 170 displaying selected spectrum 42 155 exporting the source spectrum 68 183 header 49 163 highlighting charge states 169 highlighting Measured Average m z values 169 highlighting most abundant isotope 55 parameters on 47 62 160 176 purpose 38 51 151 164 165 shortcut menu 48 163 Source Spectrum parameter 24 34 132 147 Source Spectrum report section description 92 206 displaying 24 34
293. s significantly higher than expected for the theoretical isotopic cluster e Default Selected The Xtract algorithm is more tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster Because this option can lead to increased false positives select it only in cases where you expect overlapping isotopic clusters in a data set e Cleared The Xtract algorithm is less tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster Charge Range 32 Protein Deconvolution User Guide Specifies the lowest and highest charge state to be deconvolved e Low Specifies the lowest charge state e High Specifies the highest charge state As an example if you set this parameter range from 1 through 5 the Xtract algorithm considers only charge states 1 through 5 for deconvolution It ignores charge states 6 and higher Default range 5 50 Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method Table 4 Parameters page parameters for Xtract deconvolution Sheet 7 of 10 Parameter Minimum Intensity Description Specifies a minimum intensity threshold to filter out possible background noise even when you set the S N Threshold parameter to zero Range 0 no maximum Default 1 Expected Intensity Error Specifies the
294. sociated with a compound Number of Peak Controls the resolution of the peak modeling process by dividing Models the observed m z range into a uniformly spaced set of regions equal to this number The Protein Deconvolution application generates a single peak model for each of these regions on the basis of the observed m z value and instrument resolution at the midpoint of each region Default 1 144 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Table 27 Parameters page parameters for ReSpect deconvolution Sheet 11 of 15 Parameter Resolution at 400 m z Description Defines the resolution of the source spectrum at an m z value of 400 When you load a data file containing chromatographic peaks and spectral peaks the Protein Deconvolution application takes the resolution from the following sources e When you perform a manual deconvolution create a method load a raw data file and do not change the resolution the application obtains the resolution from the raw data file e When you perform a manual deconvolution create a method load a raw data file and change the resolution the application uses the changed resolution for the current deconvolution However when you reload the method and perform another deconvolution it uses the resolution from the raw data file e When you per
295. source spectrum Protein Deconvolution User Guide 75 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples Figure 38 Chromatogram used as a source spectrum Protein Deconvolution defauitmethoaxtract Manual wirnct Osteo Deon ait SCIENTIFIC S N Threshold 3 Rel Abundance Threshold 0 Negative Charge F Calculate XIC v Saved Xtract Results Charge Carrier Min Num Detected Charge Isotope Table 1497 3218 4509 5054 1354 8189 1896 4035 1186 4677 H 1 00727663 K 38 9631585 Na 22 9892213 Custom 3 3 ae F d 3 Relative Intensity PSA_240_highpLraw a Method Selection Run Queue o Parameters g Chromatogram Sample Comparison Reporting i Deconvolution completed successfully Process Save Result As Reset Method Save as Reference Y Main Parameters Xtract Output Mass M OMH m z Range Min 600 Max 2000 Apply Resolution at 400 m z 240000 15 RT min NL 8 118 Deconvolved Spectrum 100 80 Y Saved ReSpect Results 60 2 A 5 D a 20 9484 6559 28412 9570 28121 8590 27862 6368 25714 8120 Monoisotopic Mass 28412 9570 28121 8590 28453 9528 Number of Charge Sum Intensity States Average Charge 786 138 39 0 00 562 950 50 2 Click the Sample Comparison tab Delta Mass 291 10 188 873 64 41 00 Relativ
296. splay the information that appears in the Results table on the Process and Review page Component Detail Tables Determines whether to display the Average Mass table for each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 210 Selected Displays the Average Mass table in the Source Spectrum Evidence section of the report Default Cleared Does not display the Average Mass table in the Source Spectrum Evidence section of the report Component Source of Evidence Plots Determines whether to display the spectrum of each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 210 Selected Displays the spectrum of each component in the Source Spectrum Evidence section of the report Default Cleared Does not display the spectrum of each component in the Source Spectrum Evidence section of the report If you want to return the parameters in the Reporting Parameters pane to the settings that you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any parameters in this pane but do not click Apply and then click another tab the message box shown in Figure 66 on page 128 appears Click Yes to apply the parameter changes or No to restore the parameter defaults 5 Save the met
297. sults table Adjusting the Deconvolution Results If you are not satisfied with the deconvolution results you can adjust them To adjust the Xtract deconvolution results 1 Do one of the following e Adjust the parameters in the Main Parameters Xtract pane on either the Process and Review page or the Parameters page and click Apply or e Return to the Parameters page adjust the parameters in the Advanced Parameters Xtract pane and click Apply 2 When you finish adjusting the parameters click Process on the Process and Review page again If you are satisfied with the results you can save them by using the procedure in Saving the Results of a Deconvolution on page 66 Process and Review Page Parameters for the Xtract Algorithm The Process and Review page displays parameters that you can set for the protein deconvolution the source spectrum the deconvolved spectrum and the Results table Table 9 describes the types of information available on the Process and Review page for Xtract deconvolutions Thermo Scientific Protein Deconvolution User Guide 61 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Table 9 Process and Review page information for Xtract deconvolution Sheet 1 of 3 Parameter Main Parameters Xtract pane Description The parameters in the Main Parameters Xtract pane are the same as those in the Main Parameters
298. t Save the method as follows e To save the method under the existing name select the method from the library and click Save Method e Ifyou used the default method save the method under another name i Click Save Method As The Save dialog box appears ii Type the name of the method in the Method Name box iii Type a brief description of the method in the Descriptions box iv Click Save To change a reference spectrum in a method Note You might want to save the method at this point otherwise you cannot apply the Remove command beyond this analysis Click the Sample Comparison tab if you are not already on the Sample Comparison page Remove the existing reference spectrum from the method See To delete a saved reference spectrum from a method Select the appropriate reference spectrum in the Reference Spectrum Library pane Click Add to Method Save the method as follows e To save the method under the existing name select the method from the library and click Save Method Protein Deconvolution User Guide 81 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Comparing Samples e Ifyou used the default method save the method under another name i Click Save Method As after you select a reference spectrum and add it to the reference library The Save dialog box appears ii Type the name of the method in the Method Name box iii Type
299. t e Compare spectra Xtract Algorithm The Xtract algorithm uses a fitting scheme similar to the THRASH algorithm to deconvolve and deisotope isotopically resolved mass spectra of peptides and proteins First it examines a cluster of isotopically resolved peaks and uses the peak spacing of a cluster to determine an initial estimate of the mass of the relevant component Then it fits an averagine distribution to the observed peak profile in that cluster to determine the monoisotopic mass that best reproduces that profile Finally it combines results for all observed charge states for each mass component to produce a single mass value for that component The resulting spectrum shows only the monoisotopic masses for the components that the algorithm identified When used properly the Xtract algorithm reduces spectral noise and provides a high intensity mass spectrum of monoisotopic peaks You can use the results table called the monoisotopic mass list of the deconvolved mass spectral peaks or the extracted spectra not the original MS MS spectra as the input to various search engines Figure shows an isotopically resolved mass spectrum 1 Senko M W Beu S C McLafferty EW Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions J Am Soc Mass Spectrometry 1995 Vol 6 226 233 2 Protein Deconvolution User Guide Thermo Scientific 1 Introduction Features
300. t Protein Deconvolution on page 14 2 Click the Sample Comparison tab 3 Select a reference spectrum from the Reference Spectrum Library to add to the method 4 Click Add to Method You do not need to deconvolve the data first 5 In to the Method Selection pane select the method in the Methods pane but it is not necessary to click Edit Method 6 In the Load Raw Data File pane select the raw data file and click Add to Queue Displaying the Results After the Protein Deconvolution application completes the analysis of a raw data file you can open the results of that analysis If you want to adjust the chromatogram after you view the results you must do it manually and rerun the job To display the results of a job 1 On the Run Queue page select the completed job with the results that you want displayed 2 Click Open Result The Protein Deconvolution application transfers you to the Process and Review page which displays the output spectrum in the Deconvolved Spectrum pane and the component list in the Results pane as shown in Figure 23 on page 53 It also displays the source spectrum in the Source Spectrum pane and the chromatogram in the Chromatogram pane In the Saved Xtract Results pane it displays a result for each peak shown in the Number of Chromatographic Peaks column on the Run Queue page For example if the Number of Chromatographic Peaks column displays 4 the Results pane on the Process and Review
301. t change often These parameters also appear on the Process and Review page Output Mass Resolution at 400 m z Determines whether the Xtract algorithm returns a single peak at either the monoisotopic mass or the monoisotopic MH mass for each of the detected components e Default M Specifies that the results file contains a single peak for the monoisotopic mass for each of the detected components This option generates masses without adducts e MHe Specifies that the results file contains a monoisotopic MH mass for each of the detected components This option generates masses with adducts Defines the resolution of the source spectrum at an m z value of 400 This parameter is not needed if the Xtract algorithm deconvolves FTMS Orbitrap or Exactive data because the data contains the instrument information in the spectrum You must set this parameter for all other spectrum types and for exported spectrum files in qb raw file format which lack instrument information In deriving the resolution the behavior of the manual and the automatic Xtract algorithms follows the behavior of the manual and the automatic ReSpect algorithms described for the Resolution at 400 m z parameter in Table 27 on page 135 although the resolution does not affect the results S N Threshold Thermo Scientific Specifies a signal to noise S N threshold x above which the Xtract algorithm considers a measured peak to be a real accepte
302. t of the chromatography 5 Optional In the Reference Spectrum Library pane sort the data in each column by clicking in the column header and then clicking the up row to sort the data from low to high or clicking the down arrow to sort the data from high to low 6 Optional Save the results See Saving the Results of a Deconvolution on page 181 If you select a method that includes a reference spectrum and select another reference spectrum from the reference spectrum library the Save Results As command saves the reference spectrum from the reference spectrum library Thermo Scientific Protein Deconvolution User Guide 193 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Comparing Samples To display the settings of the parameters used to generate the reference spectrum 1 Select the reference spectrum in the Reference Spectrum Library pane 2 Do one of the following e Click Show Details in the Reference Spectrum Library pane to display all the deconvolution parameters that you selected in the reference spectrum library to generate the reference spectrum vor the currently loaded parameters as shown in Figure 94 or e Click Show Details in the Method Reference Spectrum pane to display all the deconvolution parameters that you selected in the method to generate the reference spectrum not the currently loaded parameters The Show Details command in the Method Referenc
303. t the spectrum to deconvolve For information on this process see Selecting the Spectrum to Deconvolve on page 149 Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method Parameters Page Parameters for the ReSpect Algorithm Table 27 describes the parameters on the Parameters page for a ReSpect deconvolution Table 27 Parameters page parameters for ReSpect deconvolution Sheet 1 of 15 Parameter Main Parameters ReSpect pane Description Displays basic parameters that might change often These parameters also appear on the Process and Review page Negative Charge Charge Carrier Thermo Scientific Indicates whether the data was acquired in positive charge mode or negative charge mode during the ESI process You might want to use this option when processing compounds that contain nucleotides like those found in DNA and RNA When these compounds are acquired in negative mode the resulting mass spectra are often clearer Deprotonation of nucleotides which are acidic occurs when the compound is dissolved in a basic solution and negative voltage is applied to produce negatively charged ions e Selected The data was acquired in negative charge mode e Default Cleared The data was acquired in positive charge mode IMPORTANT Do not select the Negative Charge check box if your data was acquired in positive mode Results wi
304. t you can track the progression of the data You can view this report on the Reporting page and save it as a PDF file Protein Deconvolution User Guide 85 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report When you load multiple results the application still generates a single report containing the concatenated results However the report can only contain results from either the Xtract or ReSpect algorithm but not both Note The default Xtract parameter settings provide a good balance between sensitivity and report size If you adjust these parameters so that a report becomes filled with a large number of low intensity noise peaks a system without sufficient memory might hang If your system hangs restart the application and rerun it with a more restrictive set of parameters To display a report Click the Reporting tab when you have finished analyzing the data The Reporting page partially shown in Figure 42 displays a summary of all results for a given data file It contains the following sections Sample Information Section Chromatogram Parameters Section Source Chromatogram Section Source Spectrum Section Main Parameters Xtract Section Advanced Parameters Xtract Section Deconvolved Spectrum Section Mirror Plot Section Xtract Masses Table Section Source Spectrum Evidence Section 86 Prote
305. t you first chose when you loaded the raw data file click Reset Method otherwise click Apply If you change any parameters in this pane but do not click Apply and then click another tab the message box shown in Figure 66 on page 128 appears Click Yes to apply the parameter changes or No to restore the parameter defaults 4 Optional In the Reporting Parameters pane select the parts of the generated deconvolution report that you want to display Protein Deconvolution User Guide Sample Information Determines whether to display the Sample Information section of the report For information on this section see Sample Information Section on page 203 Default Selected Displays the Sample Information section of the report Cleared Does not display the Sample Information section of the report Chromatogram with Parameters Determines whether to display the Chromatogram Parameters section of the report For information on this section see Chromatogram Parameters Section on page 205 Default Selected Displays the Chromatogram Parameters section of the report Cleared Does not display the Chromatogram Parameters section of the report Source Spectrum Determines whether to display the Source Spectrum section of the report For information on this section see Source Spectrum Section on page 206 Default Selected Displays the Source Spectrum section of the report Cleared
306. ta in the columns that appear when you click the symbol to the left of the No column 3 In the Save As dialog box browse to or type the name of the file to store the results in 4 Click Save The Protein Deconvolution application stores the data shown in the Results pane in an Excel file called raw_file_name xls If you do not specify a directory the application places this file by default in the raw data directory shown on the Method Selection page When you select a result and then choose the Export Top Level or Export All command the resulting Excel file reflects the columns and order of the currently visible table 182 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Exporting the Results of a Deconvolution To export a chromatogram source spectrum or deconvolved spectrum to a third party software file Right click in one of the following panes and choose Copy Bitmap e Chromatogram pane on the Chromatogram page e Source Spectrum pane on the Process and Review page e Deconvolved Spectrum pane on the Process and Review page Open a third party graphics software file and paste the copied picture into it To export the deconvolved spectrum mass and intensity data to an Excel or a CSV file Right click in the Deconvolved Spectrum pane of the Process and Review page and choose Copy Data Open an Excel
307. tein Deconvolution User Guide 105 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Running Jobs in the Queue Torun the jobs in the queue 1 Click the Run Queue tab to open the Run Queue page if it is not already opened 2 Click Run The Run button changes to a Pause button After a few moments the status of the job changes from Queued to Processing When the status in the Status column changes to Processing the Pause button becomes unavailable unless the queue contains other jobs with a Queued status During processing only the Method Selection and the Parameters tabs are available When the Protein Deconvolution application finishes processing all jobs the Chromatogram Process and Review Sample Comparison and Reporting tabs are not immediately available until you click Open Result When the Protein Deconvolution application finishes analyzing a data file the Run Queue page displays the following changes The Number of Chromatographic Peaks column displays the number of detected chromatographic peaks The Number of Components Detected column displays the number of components detected from the deconvolution of the averaged spectra from all found chromatographic peaks e The Completion Time column displays the date and time that the deconvolution finished for a given raw data file e The Status column changes to Completed The column displays Completed Report Not Co
308. tein Deconvolution User Guide Thermo Scientific Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method e Deconvolved Spectrum Determines whether to display the Deconvolved Spectrum section of the report For information on this section see Deconvolved Spectrum Section on page 94 Default Selected Displays the Deconvolved Spectrum section of the report Cleared Does not display the Deconvolved Spectrum section of the report Components Table Determines whether to display in the report the information that appears in the Results table on the Process and Review page For information on this section see Displaying the Results on page 51 Default Selected Displays the information that appears in the Results table on the Process and Review page Cleared Does not display the information that appears in the Results table on the Process and Review page Component Detail Tables Determines whether to display the Monoisotopic Mass table for each component in the Source Spectrum Evidence section of the report For information on this section see Source Spectrum Evidence Section on page 96 Selected Displays the Monoisotopic Mass table in the Source Spectrum Evidence section of the report Default Cleared Does not display the Monoisotopic Mass table in the Source Spectrum Evidence section of the report Compon
309. tensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules This ratio is the quantity formed by dividing the mass of an ion in daltons by the number of charges carried by the ion 206 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report Main Parameters ReSpect Section The Main Parameters ReSpect section shown in Figure 100 displays the parameter settings that you selected in the Main Parameters ReSpect pane of the Parameters page for the deconvolution For information on these parameters see Table 27 on page 135 Figure 100 Main Parameters ReSpect section for ReSpect deconvolution Main Parameters ReSpect Negative Charge False Charge Camies H 100727663 Rel Abundance Threshold 36 0 m z Range 1000 4000 Output Mass Range 10000 160000 Mass Tolerance 0 05 Target Mass 150000 Charge State Range 10 100 Advanced Parameters ReSpect Section The Advanced Parameters ReSpect section shown in Figure 101 displays the parameter settings that you selected in the Advanced Parameters ReSpect pane of the Parameters page for the deconvolution For information on these parameters see Table 27 on page 135 Figure 101 Advanced Parameters ReSpect section for ReSpect deconvolutio
310. th the ReSpect Algorithm Comparing Samples You cannot add more than one reference spectrum to a method To delete the reference spectrum in a method see To delete a saved reference spectrum from a method on page 196 To change the reference spectrum in a method see To change a reference spectrum in a method on page 196 The reference spectrum is now available in the method The Protein Deconvolution application populates the fields of the Method Reference Spectrum pane The From area at the bottom of the Mirror Plot pane changes to Method 8 Optional for manual workflows but required for automatic workflows if you want to compare the data to be analyzed to a reference spectrum Save the method containing the reference spectrum as follows e To save the method under the existing name select the reference spectrum from the library and click Save Method or e Ifyou used the default method save the method under another name i Select the reference spectrum from the library and click Save Method As The Save dialog box appears ii Type the name of the method in the Method Name box iii Type a brief description of the method in the Descriptions box iv Click Save When you use this method in the future the Protein Deconvolution application uses the saved reference spectrum as the reference spectrum in the mirror plot To compare a deconvolved source spectrum to a reference spectrum 1 Deconvolve the source
311. the Saved Results panes display both types of saved results The result file names appear in alphabetical order To load these results see Loading Saved Xtract Results on page 98 To delete the results that appear in these panes see Deleting the Results of a Deconvolution on page 69 You can collapse each pane by clicking its down arrow When the list of files becomes longer than the visible pane can accommodate a vertical scroll bar appears so that you can scroll down to find a results file Protein Deconvolution User Guide 67 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Exporting the Results of a Deconvolution Exporting the Results of a Deconvolution You can export the results in the Results table to an Excel file a chromatogram source spectrum or deconvolved spectrum to a third party software file or the deconvolved spectrum mass and intensity data to an Excel or CSV file R kod To export the results in the Results table to an Excel file 1 Select a result in the Results table 2 Right click anywhere in the Results table and choose one of the following Export All to store all the results in a file Export Top Level to store just the top level results in the Results table in a file The top level results are the data in these columns Monoisotopic Mass Sum Intensity Number of Charge States Average Charge Delta Mass Relative Abundance Fractional Abund
312. the columns that you want to display in the Results table Protein Deconvolution User Guide 53 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results Figure 24 Column Chooser dialog box for the Xtract algorithm p a ii Monoisotopic Mass Sum Intensity Number of Charge States Average Charge Delta Mass Relative Abundance Fractional Abundance RT Range 7 884 8 8 ff NA wf eR Apex RT The Protein Deconvolution application instantly makes the selected columns visible and the cleared columns invisible in the Results table For a description of the columns in the Results table see Table 9 on page 62 The Column Chooser affects only the top level components that is the isotopic clusters in the Xtract algorithm and the charge peak states in the ReSpect algorithm When you select some components in the Results table and then choose the Export Top Level or Export All command the resulting Excel file reflects the columns that you chose and the order in which you arranged them However the columns and the order are not reflected in the generated reports When you close and then reopen the Protein Deconvolution application it does not retain the columns and order that you chose However when you simply leave an experiment and return to it the application retains this info
313. the significant components while excluding low intensity noise peaks that might inflate the final report to an excessive length 2 Optional Change the appropriate parameters in the Main Parameters Xtract pane e Output Mass Determines whether the Xtract algorithm returns a single peak at either the monoisotopic mass or the monoisotopic MH mass for each of the detected components Default M Specifies that the results file contains a single peak for the monoisotopic mass for each of the detected components This option generates masses without adducts Thermo Scientific Protein Deconvolution User Guide 19 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Creating an Xtract Method 20 Mhr Specifies that the results file contains a monoisotopic MH mass for each of the detected components This option generates masses with adducts e Resolution at 400 m z Defines the resolution of the source spectrum at an m z value Protein Deconvolution User Guide of 400 This parameter is not needed if the Xtract algorithm deconvolves FTMS Orbitrap or Exactive data because it reads the centroid peaks directly from the raw data You must set this parameter for all other spectrum types and for exported spectrum files in qb raw file format which lack instrument information Note An exported mass spectrum file is a mass spectrum file that you create by exporting mass spectral data from
314. the target component However you can select the mass of another component in the table to use as the reference mass for a given deconvolved spectrum The default delta mass of this reference component is 0 The application then compares the mass of other peaks in the data set to this default When you load multiple results concurrently the application calculates the delta masses only for components from the same deconvolved spectrum If you export the results to the Excel application see Exporting the Results of a Deconvolution on page 68 the Excel file reflects the updated state of the reference mass 60 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results When you load results that you previously saved see Loading Saved Xtract Results on page 98 you can change the reference component but the Protein Deconvolution application does not update the reports to reflect the change to the component table because it cannot change reports that it already generated lt To select a reference mass 1 In the Results table right click the row of the component that you want to use as the reference peak 2 Choose Set as Reference Component The Protein Deconvolution application resets the value in the Delta Mass Column to 0 for the chosen component and recalculates the delta mass value for all other components in the Re
315. thod on page 123 The Source Spectrum pane on the Process and Review page shows the same spectrum as the Source Spectrum pane on the Chromatogram page The Chromatogram pane on the Process and Review page shows the same chromatogram as the Chromatogram pane on the Chromatogram page To display the results in the Results table 1 Optional If necessary expand the Results table by following the instructions in Adjusting the Pane Size on page 10 2 Optional Select the columns that you want to display as follows a Click the Column Chooser icon Eg b In the Column Chooser dialog box shown in Figure 78 select the check boxes corresponding to the columns that you want to display in the Results table Figure 78 Column Chooser dialog box for the ReSpect algorithm Column Chooser B 78 8 NA 8 fs 8 S S NS N N The Protein Deconvolution application instantly makes the selected columns visible and the cleared columns invisible in the Results table For a description of the columns in the Results table see Table 32 on page 176 The Column Chooser affects only the top level components that is the isotopic clusters in the Xtract algorithm and the charge peak states in the ReSpect algorithm Thermo Scientific Protein Deconvolution User Guide 167 4 Manually Deco Displaying the Resu nvolving Isotopically Unresolved Mass Spectra with th
316. three panes Note To size these panes vertically see Figure 6 on page 11 e Chromatogram Parameters Contains parameters that you can use to adjust the view in the Chromatogram pane e Chromatogram Displays a chromatogram of the data in the raw data file A chromatogram view shows the intensities of one or more masses as a function of time By default the Chromatogram pane displays a total ion current TIC chromatogram as shown in Figure 68 The application s internal peak selection mechanism selects the peaks in the mass spectrum associated with different charge states of the same component The chromatogram is fully magnified You can use the zooming and averaging functions in this pane to generate a spectrum for instructions see step 3 on page 152 150 Protein Deconvolution User Guide Thermo Scientific Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Selecting the Spectrum to Deconvolve e Source Spectrum Displays the spectrum to deconvolve either single scan or averaged that you selected in the Chromatogram pane The mass spectrum in this pane is empty until you select a region in the chromatogram 2 Optional Use the parameters in the Chromatogram Parameters pane to adjust the chromatogram displayed in the Chromatogram pane The automation algorithm uses these parameters to generate the chromatogram and select the appropriate chromatographic peaks e Use
317. ting chromatograms and source spectra from the Reporting tab Thermo Scientific Protein Deconvolution User Guide 201 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying a ReSpect Deconvolution Report C To display the Source Spectrum Evidence section Note The Source Spectrum Evidence Section appears in the report only if you select the Component Detail Tables and Component Source of Evidence Plots parameters in the Parameters page You can select these parameters before or after you click Process in the Process and Review page 1 Click the Parameters tab and go to the Reporting Parameters pane of the Parameters page 2 To display the Average Mass table for each component as shown in Figure 105 on page 211 select the Component Detail Tables check box and click Apply 3 To display the spectrum of each component as shown in Figure 105 on page 211 select the Component Source of Evidence Plots check box and click Apply 4 If you select these parameters after you generate the report click the Report tab to see the Source Spectrum Evidence section To save the report in a PDF file 1 Move the cursor near the bottom of the screen The Reporting page toolbar shown in Figure 43 on page 88 appears 2 Click the Show Acrobat icon ys The Adobe Acrobat toolbar appears at the top of the screen 3 On the Adobe toolbar click the Save File icon The Save a Copy dialog
318. tion application zooms the part of the chromatogram that you define with the Time Limits parameters e Selected Zooms the specified part of the chromatogram e Default Cleared Displays the entire chromatogram Time Limits Specifies the beginning and the end of the chromatogram that you want to zoom The default values for both limits depend on the data in the raw data file This parameter is only available when you select the Use Restricted Time check box 44 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Selecting the Spectrum to Deconvolve Table 6 Chromatogram page parameters for Xtract deconvolution Sheet 2 of 4 Parameter Description Rel Intensity Threshold Sets a lower intensity threshold for peaks in the chromatogram as a percentage The Protein Deconvolution application ignores peaks with relative intensities below this threshold Range 0 100 Default 1 This parameter is different from the Relative Abundance parameter on the Parameters page which sets a lower intensity for signals in the spectrum vot in the chromatogram Types Determines the type of chromatogram displayed in the Chromatogram pane e Default TIC Displays a total ion current chromatogram which shows the summed intensity across the entire range of masses being detected at every point in the analysis The range is typically severa
319. tion application deletes all the results for the corresponding raw data file Note After you delete the SQLite file the run still appears in the run queue in the Auto Xtract workflow Use the Queue Manipulation gt Remove Selected command to remove it from the queue Comparing Samples As noted in Sample Comparison on page 4 you can compare the deconvolved spectra from two different samples or you can compare two different averaged spectra from the same LC MS run You designate one spectrum as the reference spectrum and the other as the source spectrum When you compare the source spectrum to a reference spectrum the Protein Deconvolution application displays a mirror plot of the two spectra see Figure 39 on page 77 The reference spectrum is the plot in the negative direction and the current spectrum is the plot in the positive direction You can use this plot to see whether the structures and the relative abundance of masses in the two spectra are divergent or the same Divergence can indicate that the target protein sequences have been modified by post translational modifications such as phosphorylation or glycosylation You can then use top down proteomics techniques or peptide mapping to determine the exact cause of these changes You can compare two spectra generated by the Xtract algorithm or you can compare two spectra generated by the ReSpect algorithm You can compare a spectrum generated by an automatic workflow t
320. tions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history Revision A March 2014 Software version Thermo Protein Deconvolution 3 0 0 and later Thermo Foundation 3 0 and later Microsoft Windows 7 Professional 32 bit Service Pack 1 For Research Use Only Not for use in diagnostic procedures Contents PICTACGs ciate i aa i nota sar a E a A SAE E vii Related Documentation a6 ce ccc sd adh rnvnier ior ertes tenaceennew nes vii Syst in Requirements se ous esis vs ees cera see ela Ee ted weg eee eek lew viii License Activation and Deactivation i 6 ce cbs vcas ceeo eee cee es viii Cautions and Special Notices i442 eek kad epee ed pee eee Or eN ix Contacting Usi 05 bs0 ceed cadiegae Ghd eaa bas ae Gee eee whe naa 5 x Chapter1 Introduction 0 0 0 0 0 1 Features cies tei ha aoa ae heal nai Wate eat aah A 1 XiractAlgorithni ear a a a PPO area re 2 Respect Algorithm oii eiie ker cebohiveri eid ckekowee sin ies 3 Manual and Automatic Deconvolution o o de ened shana eee Kune se 4 Sample Comparison sssi entres erta ia E dee meee eee eee 4 Chromatographic Peak Detection and Spectral Peak Modeling 5 Extracted lon Chromatogram Calculation for Deconvolved Spectra 5 Workflow anise KL eee ies Rete Lae ph os Kee 5 Inp ts and OUP eke pee eee heeded epee es aeaea SEE EEE pes 8 VOUS sevee E he eee Seances E E E ed Mods soins Meaein gs h
321. tivates an Adobe Acrobat application toolbar so that you can perform the functions available in an Acrobat file Sample Information Section The Sample Information section of the report shown in Figure 44 displays information about the sample from which the spectrum was taken Figure 44 Sample Information pane for Xtract deconvolution Sample Information Original File Name Myoglobin_30pmol_michrom_protein_microtrap_11min_OT_60K_1 RAW Instrument Method C Xcalibur data tonya Intacts 11min_OT_60K_SID_15V meth Vial 1A5 Injection Volume uL 5 Sample Weight 0 Sample Volume uL 0 ISTD Amount 0 Dil Factor 1 Thermo Scientific Protein Deconvolution User Guide 89 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying an Xtract Deconvolution Report Table 16 lists the parameters in the Sample Information section All the parameters in this section are read only Table 16 Sample Information section parameters for Xtract deconvolution Parameter Description Original File Name Displays the name of the original raw data file If you rename the raw data file the original name of the raw data file still appears on the report Instrument Method Displays the name of the instrument method file Vial Displays the position number of the sample in the autosampler Injection Volume uL Displays the injection volume of the sample to be injected in microliters Sample W
322. to the reference spectrum database along with the associated data For more information see To save a deconvolved spectrum as a reference spectrum on page 71 Process and Review Page Shortcut Menus When you right click in the Source Spectrum Chromatogram or Deconvolved Spectrum panes or on the Results table on the Process and Review page a shortcut menu appears that contains the commands listed in Table 11 Table 11 Process and Review page shortcut menu Sheet 1 of 2 Parameter Reset Scale Description Restores the original chromatogram that first appeared in the Source Spectrum or Chromatogram pane Copy Bitmap Copies the view in the Source Spectrum or Chromatogram pane to the Clipboard Copy Data Deconvolved Spectrum pane only Copies mass data x axis and intensity data y axis from the Deconvolved Spectrum pane to the Clipboard so that you can paste it into an Excel spreadsheet or another application For Xtract deconvolutions the saved data consists of a centroid spectrum Zoom Out Shrinks the view in the Source Spectrum or Chromatogram pane by a factor of 2 Zoom In Enlarges the view in the Source Spectrum or Chromatogram Thermo Scientific pane two times Protein Deconvolution User Guide 65 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Saving the Results of a Deconvolution Table 11 Process and Review page shortcut menu Sheet 2 of 2
323. togram pane displays the following information e The name of the raw data file for example Myoglobin_30pmol_microm_protein_microtrap_11min_OT_60K_1 e NL The intensity of the most abundant peak in the entire LC MS run for example 8 51E7 F The scan filter used during the LC MS run The scan filter indicates the type of mass analyzer used to acquire the data in the raw data file and the ionization technique used If this field is blank no scan filter was used Source Spectrum Pane Shortcut Menu When you right click in the Source Spectrum pane a shortcut menu appears that contains all of the commands in Table 8 except Mode but they apply to the Source Spectrum pane rather than the Chromatogram pane For information on these commands see Table 8 48 Protein Deconvolution User Guide Thermo Scientific 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Deconvolving the Spectrum Source Spectrum Pane Header The header in the Source Spectrum pane displays the following information e Name of the raw data file for example Myoglobin_30pmol_microm_protein_microtrap_11min_OT_60K_1 e Scan number or range of scan numbers for example 149 187 e RT Retention time which is the time in the mass chromatogram when any particular precursor ion is observed for example 3 30 4 08 e NL single scans The intensity of the most abundant peak in the entire LC MS run for example 3 83E5 e AV mult
324. trum gt Main Parameters Xtract Source Spectrum v Saved Xtract Results Method Selection Run Queue Parameters Chromatogram Sample Comparison Reporting Process Save ResultAs Reset Method Save as Reference Nago S838 Relative Intensity o 50 536 1664 o 500 Deconvolved Spectrum 1 6000 7000 Saved ReSpect Results Chromatogram im 391 2844 893 1164 Eroa 10604415 oan 100 RT 0 58 ETA Spectral line 304 1696 2001 1918 3288 Pa r 01 S 268 6 75 7 40 8 47 8 94 9 64 corresponding to 1000 1500 2000 1 2 3 4 5 6 7 8 9 10 me RT min a selected value in the Mostabund 00 16941 0113 m z column 16568 8381 15635 2961 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 Mass 4 15 16 TEn Monoisotopic he Mass te T 16941 0113 X a ri 16923 0410 i a 3 16922 8878 4 14 98 18 12 10 5966 44727 13 1302 7694 16922 9025 1303 5380 3 762 86 96 00 0 00 0 00 14 1209 7864 16922 8779 1210 4987 0 00 0 00 gt 15 1129 2011 16922 8745 6 Ee Selected value in 16 1058 6890 16922 8981 1059 3154 402 0 00 7 10 4 16905 0254 31 121 70 12 21 16 35 9 8578 4 1609 3 303 3 969 3 641 th e M osta bu nd g 5 16568 8381 26 967 16 3 6055 3 303 3 969 3 619 m z column L Number of Charge Relative Fractional p Sum Intensity RT Range Apex RT Average Char Delta Mass States cabana Abundance Abundance 315 704 75 20 19 82 0 00 100 0000 42 2091 3 303 3 969 3 671
325. ult As Reset Method Save as Reference Results Source Spectrum Chromatogram mE 893 1164 RT 3 66 104 7 mis S 166 50 1060 4415 0 RT 5 90 536 1664 i 1304 9242 1696 2001 1918 3288 g S 268 6 75 7 40 8 47 8 94 9 64 500 1000 1500 2000 5 miz RT min Deconvolved Spectrum 16941 0113 100 a Spectral line 4 x corresponding to i assas 2951 16508281 a selected value 6000 7000 2000 9000 10000 11000 12000 13000 14000 15000 16000 17000 nia in the aan Monoisotopic Results Deta Ma Relative actoral r Rang gt Mass for This a 1 16941 0113 315 704 75 20 19 82 0 00 100 0000 422091 3 303 3 969 3 671 Ch arge column 2 16923 0410 216 752 20 16 20 60 1797 68 6566 28 9794 3 303 3 969 3 653 i a 3 16922 8878 33 454 02 4 14 98 18 12 10 5966 44727 3 303 3 969 3 658 a 4 16905 0254 31 121 70 12 2116 35 99 9 8578 41609 3303 3969 3 641 a 5 16568 8381 26 967 16 14 20 34 372 17 8 5419 3 6055 3 303 3 969 3 619 ee 4 1184 4963 16568 7910 1185 2076 451 95 83 10 0 00 0 00 15 1105 5970 16568 8037 1106 2642 935 84 91 20 0 00 0 00 16 1036 5602 16568 7782 1037 1814 1 659 96 93 20 0 00 0 00 gt 7 975 6453 16568 7951 Jan ss Selected value In 18 921 4987 16568 8268 922 0542 2 553 60 95 90 0 00 0 00 the M isotopi 19 873 0518 16568 8382 873 5794 3 234 57 9540 0 00 0 00 e IVIONOISOLOpIC 20 829 4496 16568 8551 829 9507 3 078 31 96 90 0 00 0 00 Mass for This 21 790 0000 16568 8553 790 4772 321091 96 70 0 00 9 70 U Charge column The
326. um Protein Deconvolution Barwa Respects fotonica reoh ati sate IgG source cid raw Run Queue Parameters Chromatogram ff ECELAS Sample Comparison Reporting Process Save ResultAs Reset Method Save as Reference gt Main Parameters ReSpect Results Y Saved Xtract Results Saved ReSpect Results DefaultMethodReSpect_1089_1094 DefaultMethodReSpect_1129 131 DefaultMethodReSpect_36_57_10 DefaultMethodReSpect_68_356_10 DefaultMethodReSpect_844_874 1 DefaultMethodReSpect_893_900_1 DefaultMethodReSpect_900_942_1 DefaultMethodReSpect_942_1020 ReSpect3537 Load Result Source Spectrum Chromatogram 2761 2696 RT 3 41 100 SE 171 2618 5381 2977 7551 100 w 2373 1261 3167 1426 504RT 0 36 erst A 362 50 a 1383 1409 1659 4854 ull _3535 4380 172 417 a7 at 290 S S 1093 nas 5 01 6 00 0 TT eeannananas Be T T T T 1000 1500 2000 2500 3000 3500 4000 1 3 3 4 5 6 miz RT min Deconvolved Spectrum NL i 151814 672 in 20 NL 2 3 8068 60 s 40 amp 51560 344 149595583 20 37290 301 73219 359 100997 500 30895 424 141 asaza S saho 60244 000 pA 0995 T T T T 7 t T T T T T T 1 20000 40000 60000 0000 100000 120000 140000 160000 Mass Results Number of q Relative Fractional nella No Average Mass Intensity pees Mass Std Dev PPM Std Dev Delta Mass memme aes RT Range Al CEE 151814672 368 946 048 00 46 188 1240 0 00 100 0000 17 5152 3 304 3 630 340 e
327. urce spectrum into a mass spectrum and displays it in a new pane labeled with mass units rather than with the mass to charge ratio on the x axis For information on the Xtract algorithm see Xtract Algorithm on page 2 Thermo Scientific Protein Deconvolution User Guide 13 2 Manually Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Setting Up a Manual Xtract Protein Deconvolution Setting Up a Manual Xtract Protein Deconvolution First select the Xtract deconvolution algorithm a raw data file and a method To set up a manual protein deconvolution with the Xtract algorithm 1 Start the Protein Deconvolution application according to the instructions in Starting the Protein Deconvolution Application on page 8 2 Click the Method Selection tab if it is not already selected The Method Selection page contains three panes e Experiment Types pane Displays the available workflows and a command for loading the saved results of previous deconvolutions Load Raw Data File pane Displays the list of the available raw data files for the selected algorithm e Methods pane Displays the available methods 3 In the Experiment Types pane click Manual Xtract Isotopically Resolved 4 In the Load Raw Data File pane select the raw data file that contains the spectral data for your sample a In the Raw Data Directory box type the path of the raw data file or click the Browse button to browse t
328. us of the deconvolution e Queued The data file is awaiting analysis e Processing The Protein Deconvolution application is in the process of analyzing the data file e Completed The Protein Deconvolution application has finished analyzing the data file and has generated the report e Completed Report Not Concatenated The Protein Deconvolution application has finished analyzing the data file but has not generated the report because of size constraints Comparing Samples Generated with the Automatic Xtract Algorithm Thermo Scientific To compare a reference spectrum generated by the automatic Xtract algorithm to a source spectrum use the procedure in Comparing Samples on page 70 with one exception You must save the reference spectrum to the method that was used to generate the reference spectrum For best results use the same method to generate the source spectrum Without deconvolving a spectrum on the Process and Review page you can select a reference spectrum on the Sample Comparison page to evaluate it Protein Deconvolution User Guide 111 3 Automatically Deconvolving Isotopically Resolved Mass Spectra with the Xtract Algorithm Displaying the Results To compare samples generated with the automatic Xtract algorithm 1 Create a new method in the Manual Xtract workflow see Creating an Xtract Method on page 18 or change an existing method in the Auto Xtract workflow see Setting Up a Manual Xtrac
329. ve abundance is 1 percent and no peaks below an absolute abundance of 10 will appear in the deconvolved spectrum The minimum value is 0 which displays all results and the maximum value is 100 which displays only the most abundant component The default is 0 Protein Deconvolution User Guide 125 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Creating a ReSpect Method 126 Protein Deconvolution User Guide Calculate XIC Determines whether the Protein Deconvolution application calculates an extracted ion chromatogram from a range of deconvolved spectra Default Selected Calculates an extracted ion chromatogram for each detected component Cleared Does not calculate an extracted ion chromatogram for each detected component The application saves this setting to the method Using the Calculate XIC parameter can result in a much longer analysis time so you might avoid using it with complex data or with data where the displayed extracted ion chromatograms are unnecessary Quality Score Threshold Specifies a minimum protein quality score that components must have to be displayed in the Results table The application discards components that have a score beneath this threshold For more information on the protein quality score see Calculating a Protein Quality Score on page 173 You can enter any floating point number The default is 0 m z Range Specifies the portio
330. view page as a profile in mass and intensity along with a set of peak labels It also displays the component list in the Results pane as a table of masses intensities charge state information and mass shifts See Figure 77 on page 166 The values in the columns of the Results table represent the outputs of the deconvolution You can expand each entry in this table to display detailed information about the individual charge states that the entry contains Note When you do any of the following the layout dimensions of the Process and Review page remain fixed e Navigate away from the Process and Review page to another page of the Protein Deconvolution application e Open results from the Run Queue e Load previous results e Switch algorithms from Xtract to ReSpect or from ReSpect to Xtract However the layout dimensions do not remain fixed when you close the application and reopen it They revert to the default dimensions Thermo Scientific Protein Deconvolution User Guide 165 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Displaying the Results See the following sections e Displaying the Results on the Process and Review Page e Selecting a Reference Mass to Calculate Mass Differences Calculating a Protein Quality Score Specifying the Output Mass Range Adjusting the Deconvolution Results Displaying the Results on the Process and Review Page The Protein Deconvolution a
331. volution on page 181 Process and Review Page Parameters for the ReSpect Algorithm The Process and Review page displays parameters that you can set for the protein deconvolution the source spectrum the deconvolved spectrum and the Results table Table 32 describes the types of information available on the Process and Review page for ReSpect deconvolutions Table 32 Process and Review page information for ReSpect deconvolution Sheet 1 of 4 Parameter Main Parameters ReSpect pane Description The parameters in the Main Parameters ReSpect pane are the same as those in the Main Parameters ReSpect pane of the Parameters page For information on the parameters on this page see Table 27 on page 135 Apply Implements the parameter settings that you selected Saved Xtract Results pane Displays each set of Xtract results that you saved from a given raw data file Saved ReSpect Results pane Displays each set of ReSpect results that you saved from a given raw data file Load Result Loads the selected previous results into the Source Spectrum Deconvolved Spectrum and Results panes Source Spectrum pane Displays the selected spectrum before deconvolution Relative Intensity y axis Displays the ratio of the intensity of a specific peak to the intensity of the peak with the highest intensity m z x axis Displays the mass to charge ratio of ions formed from molecules
332. want to continue D d Click Yes Although you can load a raw data file with a maximum size of 34 GB processing and reporting issues might result when you load files this large Thermo Fisher Scientific recommends that you work with files up to 2 GB Several more tabs appear along the top of the Protein Deconvolution window and the available methods including a default method appear in the Methods pane as shown in Figure 63 Note The Run Queue tab is not available unless you select Auto Xtract or Auto ReSpect in the Experiment Types pane Thermo Scientific Protein Deconvolution User Guide 119 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Setting Up a Manual ReSpect Protein Deconvolution Figure 63 Method Selection page for manual ReSpect deconvolution after the raw data file is loaded Protein Deconvolution Manual Xtract Isotopically Resolved Auto Xtract Isotopically Resolved Auto ReSpect Isotopically Unresolved Load Results SCIENTIFIC eco ae Run Queue g Parameters E Chromatogram B Process and Review f Sample Comparison Reporting gt Select an experiment type a data file and a method Experiment Types Load Raw Data File Methods Manual ReSpect Isotopically Unresolved gt Raw Data Directory C Program Files Protein Deconvolutior Description DefaultMethodReSpect Default method for ReSpect Read Only Method for Native
333. wing information e The name of the raw data file for example IgG_source_cid NL The intensity of the most abundant peak in the entire LC MS run for example 3 10E9 F The scan filter used during the LC MS run The scan filter indicates the type of mass analyzer used to acquire the data in the raw data file and the ionization technique used If this field is blank no scan filter was used 162 Protein Deconvolution User Guide Thermo Scientific 4 Manually Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Deconvolving the Spectrum Source Spectrum Pane Shortcut Menu When you right click in the Source Spectrum pane a shortcut menu appears that contains all of the commands in Table 31 on page 162 except Mode but they apply to the Source Spectrum pane rather than the Chromatogram pane For information on these commands see Table 31 Source Spectrum Pane Header The header in the Source Spectrum pane displays the following information e Name of the raw data file for example IgG_source_cid e Scan number or range of scan numbers for example 1138 1290 e RT Retention time which is the time in the mass chromatogram when any particular precursor ion is observed for example 3 37 3 52 e NL single scans The intensity of the most abundant peak in the entire LC MS run for example 3 83E5 e AV multiple scans The number spectra that were averaged to create the source spectrum for example 39
334. within an individual spectrum The higher the score the better the component quality is Better components in one spectrum have higher scores than worse ones in another spectrum For example a score of 100 in one spectrum represents a more reliable fit than a score of 50 in another spectrum As another example a score of 83 in spectrum A is better than a score of 54 in spectrum B even if 83 is the lowest score in spectrum A and 54 is the highest score in spectrum B In general components with higher molecular weights higher intensities or both that might tend to have more charge states receive higher scores You might not be able to specify ahead of time what the best threshold for a particular spectrum might be For high quality spectra you might want to ignore components with scores below 40 For lower quality spectra this threshold might be 10 or lower In general scores indicate the following e In almost all cases scores below 0 signify bad components In particular scores significantly below 100 are associated with harmonics and other unreliable fits that you should ignore e Scores between 0 and 20 indicate components that might be questionable The precise value of this threshold depends on the quality of the spectra themselves For clean spectra with well defined and widely separated components all results with a score greater than 0 might be good As the noise increases and the spacing between components dec
335. wn in Figure 108 The data file or files that you selected appear on the Run Queue page with a status of Queued Figure 108 Run Queue page Auto ReSpect isotopically Unresolved l ly Help X Thermo Protein Deconvolution SCIENTIFIC v Method Selection v Run Queue am Parameters hromatogra m roce j Review B Sample Comparison eporting o Process methods defined in the work queue Queue Manipulation Set Priority Open Result Open Report Number of Chromatographic Components Completion Time Peaks Experiment Submit Time Method Name Raw Data File Type 1 Normal 10 14 2013 4 14 49PM_ DefaultMethodReSpect C Program Files Protein Deconvolution source files RSP_AUTO 9 Optional When you want to add spectra to the run queue return to the Method Selection page and repeat the previous steps You can process up to 1000 samples Thermo Scientific Protein Deconvolution User Guide 219 5 Automatically Deconvolving Isotopically Unresolved Mass Spectra with the ReSpect Algorithm Running Jobs in the Queue 10 If you want to run the job or jobs start the jobs by following the instructions in the next section Running Jobs in the Queue Running Jobs in the Queue By default the Protein Deconvolution application processes multiple jobs in the queue in the order that you submitted them If you want to change the processing order you can assign each deconvolution job a priority level For instructions see
336. y the automatic ReSpect algorithm to a source spectrum use the procedure in Comparing Samples on page 185 with one exception You must save the reference spectrum to the method that was used to generate the reference spectrum For best results use the same method to generate the source spectrum Without deconvolving a spectrum on the Process and Review page you can select a reference spectrum on the Sample Comparison page to evaluate it To compare samples generated with the automatic ReSpect algorithm 1 Create a new method in the Manual ReSpect workflow see Creating an Xtract Method on page 18 or change an existing method in the Auto ReSpect workflow see Setting Up a Manual Xtract Protein Deconvolution on page 14 2 Click the Sample Comparison tab 3 Select a reference spectrum from the Reference Spectrum Library to add to the method 4 Click Add to Method You do not need to deconvolve the data first 5 In to the Method Selection pane select the method in the Methods pane but it is not necessary to click Edit Method 6 In the Load Raw Data File pane select the raw data file and click Add to Queue Displaying the Results After the Protein Deconvolution application completes the analysis of a raw data file you can open the results of that job If you want to adjust the chromatogram after you view the results you must do it manually and rerun the job To display the results of a job 1 On

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