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The ProSightPC Application

1. Maximum hits exceeded fragment list table suppressed To view fragment information use sequence gazer or change threshold in options IMPORTANT Absolute mass and biomarker searches return one result list for each precursor ion 162 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in the Tab Controller Each result list displays the number of protein isoforms found Click the side arrow that precedes the results list to display the results contained in the result table Each result table contains complete information about each matching protein isoform Information in the result table is organized into the three regions shown in Figure 64 Table 32 describes these regions Figure 64 Search result table elements Description of protein isoform gt TOM1_YEAST Q03280 RecName Full E3 ubiquitin protein ligase TOM1 EC 6 3 2 AltName Full Suppressor of snRNA protein 2 AltName Full Temperature dependent organization in mitotic nucleus protein 1 Type basic Signal Peptide false Propep false Y L D E K Y E D Y A F D V M K V L N D Q L E N L N D F L N S P N D R S F F L E R D G E N S V R S C H S K L C R L A A I LEN I V T N V Y I D L TETEL S C K R I M Q I Y S Y F D K R 6 Fragment map E e o e A E E es Ss Sa E T F q ID Gene Length Mass Mass Diff PPM Diff C Ions Zions Total Ions PDE Score E Value C 2731369 2841 95 11226 6 03 2
2. Saving Puf files to the same directory as each raw file Running search tree logic Search Tree Options Repository yeast_topdown_etd Search Tree yeast_topdown_etd Number of Searches 1 Level 1 yeast absolute mass 14 Click Process The searching begins and can take a several minutes depending on the length of the RAW file the complexity of the database and the parameters of the search When the ProSightPC application finishes the search it opens the repository report see Figure 70 on page 171 For information on manipulating the data in this report see Viewing the Results in a Repository Report on page 168 Creating a Two Level Search Tree You might want to create a two level search tree on the Success side For example you might have run a preliminary general search but want to search the modifications on the experiments that found matches Thermo Scientific ProSightPC User Guide 43 2 Getting Started Processing LC MS MS Data Files You might also want to perform a two level search on the Failure side Suppose that you ran a first level search and some of the experiments found no matches that is the experiments failed the conditions To obtain good results you might then want to run a different search such as a broader search or a search of a different database a search with different parameters or a search in a different mode But if some of the experiments found matches you
3. intensity Specifies the abundance of the precursor ion id Specifies a unique in each MS MS experiment ProSightPC assigned numerical identification of the precursor ion Table 46 lists the columns in the Fragment Mass List area Table 46 Fragment Mass List area parameters Sheet 1 of 2 Parameter mz_monoisotopic Description Specifies the monoisotopic mass to charge ratio m z value of the fragment ion mz_average Specifies the average mass to charge ration m z value of the fragment ion mass_monoisotopic Specifies the monoisotopic mass of the fragment ion Thermo Scientific Thermo Scientific 7 Displaying Data in the Data Manager Editing Mass Values Table 46 Fragment Mass List area parameters Sheet 2 of 2 Parameter Description mass_average Specifies the average mass of the fragment ion intensity Specifies the abundance of the fragment ion id Specifies a unique in each MS MS experiment ProSightPC assigned numerical identification of the fragment ion Select any value from either of the two mass lists Click Delete or use the backspace key to remove the old value Type a new value in the blank space Click Ld to save the changes In the Save Masses Confirmation box click Yes The ProSightPC application returns you to the Data Manager To add a row to a mass list Click the last row of the Precursor Mass List and Fragment Mass List which is ma
4. 2013 Thermo Fisher Scientific Inc All rights reserved ProSightPC ProSight PTM and ProSightHT are trademarks of Proteinaceous Inc in the United States Q Exactive is a trademark and LTQ FT Orbitrap and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc in the United States Sequence Gazer and PTM Warehouse are registered trademarks of the Board of Trustees of the University of Illinois in the United States ITRAQ is a registered trademark of AB Sciex Pte Ltd in the United States TMT is a registered trademark of Proteome Sciences plc RESID is a service mark of John S Garavelli The following are registered trademarks in the United States and other countries MySQL is a registered trademark of MySQL AB Microsoft Excel Visual C and Windows are registered trademarks of Microsoft Corporation The THRASH procedure is based on routines in Numerical Recipes The Art of Scientific Computing published by Cambridge University Press and is used by permission All other trademarks are the property of Thermo Fisher Scientific Inc and its 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
5. Table 7 lists the parameters on the Running Highthroughput Logic page of the High Throughput Wizard shown in Figure 20 on page 36 Table 7 Running Highthroughput Logic page parameters Sheet 1 of 2 Parameter Repository Description Specifies the name of the repository where the search results will be saved New Repository Opens the New Repository dialog box shown in Figure 33 on page 67 so that you can specify the name of the repository where the search results will be saved Search Tree Name Specifies the name of the new or existing search tree Save Opens the Save Search Tree dialog box shown in Figure 25 on page 42 so that you can save a search tree under a new name Experiment Filter Filters out experiments that will not yield matches Min fragments Max fragments Specifies the minimum number of fragments to search for Default 10 Specifies the maximum number of fragments to search for Default 500 Min Intact Mass Specifies the minimum intact mass Default 750 Da Mass type list Specifies the mass type e Monoisotopic Specifies that the mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the mass is average which is the mass of the most abundant isotope of the protein peptide or fragment ion Add search Opens the Edit Add Searches for HT dialog box
6. If you want to load a targeted RAW file see Importing Targeted RAW Files on page 73 Setting Processing Options To set the processing options in the High Throughput Wizard follow this procedure To set the processing options in the High Throughput Wizard 1 Choose ProSightHT gt HighThroughput Wizard or click the HT Wizard icon Hy i The Process a Dataset page of the High Throughput Wizard appears as shown in Figure 18 28 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Figure 18 Initial Process a Dataset page of the High Throughput Wizard wrought Cae Process a dataset Select the files you want to process RAW or PUF Process Raw files Choose a Process Choose a Process Option Algorithm 5 Middle Down wd Ue 5 Top Down MS3 Se Top Down MS2 Custom E Save a copy of the puf files for future processing Same directory as raw file Browse Thrash and Xtract are both algorithms that interpret resolved isotopic distributions and output neutral mass E Skip search tree logic values 5 Process Puf files 2 Select the Process Raw Files or Process Puf Files option depending on the type of data that you want to import e Process Raw Files Converts LC MS MS RAW files to PUF files using an extension of the THRASH or Xtract algorithm designed to analyze high resolution profile LC MS MS data collected on Thermo
7. L 3 4 dihydroxyphenylalanine Sort the columns of Included PTMs in ascending or descending order by clicking the header Thermo Scientific 9 Using ProSightPC Tools Locating and Selecting PTMs with the PTM Tier Editor PTM Tier Editor Dialog Box Parameters Table 54 lists the parameters in the PTM Tier Editor dialog box shown in Figure 101 on page 236 Table 54 PTM Tier Editor dialog box parameters Parameter Description Included PTMs Lists the included PTMs Name Specifies the RESID name of the included PTM Tier Specifies the current tier assignment of the included PTMs Resid ID Specifies the RESID identifier of the included PTMs Excluded Lists all the presently excluded PTMs Update Applies the changes that you made in the dialog box Including PTMs All presently excluded PTMs are listed in the Excluded PTMs area Use the PTM Tier Editor to reclassify an excluded PTM as included To include a PTM in the database 1 In the Excluded PTMs section of the PTM Tier Editor dialog box select the check box to the left of the each PTM that you want to include e To select more than one adjacent row hold down the SHIFT key and click the first and last rows e To select more than one row where the rows are not adjacent hold down the CONTROL key and click the appropriate rows 2 Click Update to make the changes The PTMs appea
8. Q D M I N E V D Saree y lon clon ape b clon y r lon Beneath the ProSightPC Font Equivalent box are six buttons that correspond to the N and C terminal fragment marks used for b y and c z fragment ions 3 To add fragmentation tick marks position the cursor between the two amino acid letters and click the appropriate box 4 To display a complementary pair click the appropriate N terminal fragment and then click the appropriate C terminal fragment 5 To transfer the converted font to another application paste text from the ProSightPC Font Equivalent to the other application You can resize the ProSightPC font after a paste operation Depending on your system configuration the font information might not transfer during a paste operation and might be displayed in another font Correct this by selecting the incorrectly displayed output and manually changing the font to the ProSightPC application Thermo Scientific ProSightPC User Guide 243 9 Using ProSightPC Tools Converting Text to ProSightPC Font with the Font Converter Font Converter Dialog Box Parameters The Font Converter dialog box contains the parameters shown in Table 56 Table 56 Font Converter dialog box parameters Parameter Description Sequence Specifies the amino acid sequence to be converted ProSightPC Font Displays the ProSightPC application font equivalent of the Equivalent sequence displayed in the Sequence box
9. Select the files you want to process RAW or PUF Process Raw files C Program Files ProSightPC Test Data ETDfraction03 rew Choose a Process Choose a Process Option Algorithm Middle Down ai We Top Down MS3 D Ama Top Down MS2 Custom Remove Add Advanced Settings V Save a copy of the puf files for future processing Same directory as raw file Thrash and Xtract are both algorithms that interpret resolved isotopic distributions and output neutral mass E Skip search tree logic values Process Puf files fil 3 huma t n tral mas al f J 2 Database searcl te rch ift I 8 Click Next Process a Dataset Page Parameters Table 6 lists the parameters in the Process a Dataset page of the HighThroughput Wizard Table 6 Process a Dataset page parameters Sheet 1 of 2 Parameter Description Process Raw files Converts LC MS MS RAW files to PUF files using an extension of the THRASH or Xtract algorithm designed to analyze high resolution profile LC MS MS data collected on Thermo Scientific Fourier Transform instruments such as the LTQ FT Remove Removes the selected RAW file displayed in the box Add Opens a dialog box so that you can browse for a RAW file to process 34 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 6 Process a Dataset page parameters Sheet 2 of 2 Parameter Description C
10. You can select experiments in the following ways e Double click each experiment e Select the box to the extreme left of each experiment e Use the SHIFT key to select consecutive experiments e Use the CTRL key to select separate experiments Thermo Scientific ProSightPC User Guide 175 5 Viewing Search Results Viewing the Results in a Repository Report e Right click an experiment and choose either Select All or Check Selected Rows from the shortcut menu You can also choose Unselect All or Uncheck Selected Rows to clear rows 2 Click Import in the Actions area or right click the selected experiments in the page and choose Import from the shortcut menu If the data grid already contains experiments you are prompted to replace the current experiments in the data grid 3 Click Yes No Yes to All or No to All You can also import experiments from a repository by choosing File gt Import Data from Repository Exporting Experiments to a Repository Once you have imported the experiments shown on the repository report page into the PUF file and performed operations on them you can export them back to the same repository You can export experiments from a repository by using the following procedure or by using the procedure outlined in Exporting Experiments to a Repository on page 71 To export experiments to a repository 1 In the repository report page select the experiments that you want to export to the rep
11. e You can enter the output of a sequence tag search in a series into a gene restricted search to perform a hybrid search which frequently identifies and characterizes a protein e A sequence tag search is frequently the first step in MS experiments e Manually enter unresolved amino acid pairs such as isoleucine and leucine as a pipe separated list in square brackets with no spaces for example I L Setting Sequence Tag Search Preferences 136 ProSightPC User Guide When you add new sequence tag searches set the default values on the Sequence Tag Preferences page of the Options dialog box For information on sequence tag searches see Searching for Sequence Tags on page 136 Thermo Scientific 4 Searching Databases Searching for Sequence Tags To set sequence tag search preferences 1 Click Sequence Tag in the Options dialog box The Sequence Tag Preferences page of the Options dialog box opens as shown in Figure 57 Figure 57 Sequence Tag Preferences page in the Options dialog box Options 5 General E Grid Columns Sequence Tag Preferences Thrash re este Se ST E eg eee Search Parameters jg 57 Absolute Mass 27 Biomarker Sequence Tag Database Demo database for ProSight PC Single Protein Default Sequence Tag Search Parameters Fragment Mass X Lowe Defau Uppe Minimum Tag 0 2 20 Compiler Tolerance in 1 10 100 Minimum Tag Size 2 4 100
12. 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 3 of 8 Parameter Precursor Selection Options Description Minimum S N Specifies the lowest signal to noise ratio that the THRASH algorithm considers when trying to assign neutral mass to a charged mass to charge ratio 7 z species while analyzing the precursor ions Range 1 no maximum Default 3 0 Minimum RL THRASH only Specifies the minimum confidence level Range 0 1 0 Default 0 90 Minimum Charge State THRASH only Specifies the smallest charge state to be considered for the precursor Range 0 no maximum Default 1 Maximum Charge Specifies the maximum charge used by the algorithm while analyzing the precursor ions Range 0 no maximum Default 40 Maximum Mass kDa THRASH only Specifies the highest mass to consider for the precursor Range 0 no maximum Default 60 Minimum Fit Xtract only 60 ProSightPC User Guide Specifies the minimum fit parameter used by the algorithm while analyzing the precursor ions Range 0 100 Default 40 Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 4 of 8 Parameter Description Remainder Threshold Specifies the remainder of the fit that is left in the scan Xtract only during analysis of the precursor ions The Remainder T
13. 8306 1 2626 1 2232 1 3902 2 4438 2 0758 6 0575 5 1108 4 7382 5 0491 5 0764 5 4919 ProSightPC User Guide Max Frags i Min Frags 167 5 Viewing Search Results Viewing the Results in a Repository Report The report presents all relevant data for a search in a printable form similar to that of the Data Manager but only contains information from the selected search To generate a best hit report e Choose Tools gt Reports gt Best Hit Report The report shown in Figure 68 appears in a Web browser window Figure 68 Best hit report Data for Experiment 43 Custom created Experiment To edit this comment click Experiment Tools Edit CommentPuf Filter This file passed the following filters Max Frags 1 Min Frags 10 Min Intact Mass 750 Search 1 Biomarker Search Sample BioMarker Search Results for Intact Ion 1 Protein forms found 1 1 gt 39 RS28_HUMAN P62857 40S ribosomal protein 28 Type basic Signal Peptide false Propep false ID Length Mass Mass Diff PPM Diff C Ions Z Ions Total Ions 10 39 4337 2 0005 A071 24 16 Search 3 GRBM Search Sampie Gene Restricted BioMarker GRB amp M Search GREM and GRAM Searches are created using results from other searches Results for Intact Ion 1 Protein forms found 2 gt 39 RS28_HUMAN P62857 40S ribosomal protein 28 Type basic Signal Peptide faise Propep false Length Mass Mass Diff PPM Diff C Ion
14. B Using the ProSightPC Interface The ProSightPC Interface 268 ProSightPC User Guide Figure 115 Grid Columns page of the Options dialog box Options bo E General p E Grid Columns Grid Columns er Thrash E A ENS en Search Parameters V Pending Search F Successful Search V Marked 7 Matching Forms Exp Comment 7 Best Expectation E Search Comment E Best P Score V Search Type Best PDE E First Precursor Mono E Highest Total lons E First Precursor Avg b c lons E Largest Precursor Mono F y z lons E Largest Precursor Avg E Fragments E First m z Mono E Precursors First m z Avg First Abundance E Largest m z Mono E Largest Abundance Largest m z Avg Best Seq Score experiment grid Select the check boxes next to the names of the columns to be displayed by default in the For information on these columns see Using Filters in the Show Columns Area on page 267 Click OK To temporarily change the columns displayed in the data grid Click the Grid Display Preferences tab In the Show Columns area select the check boxes next to the names of any columns that you want to display in the data grid Click Refresh to display the columns that you selected in the data grid The columns that you selected appear in the data grid To remove a column from the data grid Click the Grid Display Preferences tab Clear th
15. Importing or Creating a Proteome Database on page 23 2 Use the High Throughput Wizard to do the following a Import a RAW or PUF file into the ProSightPC application b Ifyou import a RAW file select the Xtract or THRASH algorithm to interpret resolved isotopic distributions and output neutral mass values in a PUF file c Define an iterative search tree d Create a repository in which to store the search results e Perform the search For information on using the High Throughput Wizard see Processing LC MS MS Data Files on page 27 3 Search for neutral mass data against the proteome warehouse The ProSightPC application identifies and characterizes the observed proteins For information on conducting searches see Searching Databases on page 99 4 View the results in the user interface view the reports or generate a repository report For information on viewing the results of the search see Viewing Search Results on page 161 Figure 5 illustrates this flow Thermo Scientific ProSightPC User Guide 7 1 Introduction to the ProSightPC Application LC MS MS Workflow Figure 5 ProSightPC wizard workflow Perform LC MS MS experiments on Load proteome database with intact intact proteins protein sequences and PTMs Spectral data in RAW file FASTA or UniProtKB flat file ProSightPC application Import a RAW file Apply PTM information through shotgun annotation Convert
16. In the Absolute Minimum Intensity box enter the minimum intensity to be accepted for fragmentation peaks The ProSightPC application excludes any deisotoped peaks below this threshold so it removes low intensity fragment ions that might be spurious The minimum value is 1 and there is no maximum value The default is 100 In the Get Top X box specify the number of the most intense peaks per window size that the ProSightPC application considers This parameter works with the Window Size parameter to filter the deisotoped and decharged data The default settings mean that the ProSightPC application considers only the most intense 5 peaks in a 100 Da window Therefore this setting removes low intensity fragment ions that might be spurious The minimum value is 1 and there is no maximum value The default is 5 In the Window Size box specify the size of the window containing the number of the most intense peaks that the ProSightPC application considers This parameter works with the Get Top X parameter to filter the deisotoped and decharged data ProSightPC User Guide 57 2 Getting Started Processing LC MS MS Data Files 58 ProSightPC User Guide The minimum value is 1 and there is no maximum value The default is 100 o Click OK Advanced Settings Dialog Box Parameters Table 10 lists the parameters in the Advanced Settings dialog box which is shown in Figure 31 on page 53 for the THRASH algorithm and in Fi
17. Parameter Description ID Displays a unique in each MS MS experiment ProSightPC assigned numerical identification of the fragment m z type Displays the mass to charge ratio m z value corresponding to the fragment ion The type is monoisotopic or average depending on which you selected during the last rescoring Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Demonstrating the Sequence Gazer Table 43 Non matching fragments table parameters Sheet 2 of 2 Parameter Description Mass type Displays the observed mass of the fragment ion measured in Da The type is monoisotopic or average depending on which you selected during the last rescoring Intensity Displays the abundance of the fragment ion Demonstrating the Sequence Gazer Thermo Scientific Following is a demonstration that shows how to use the Sequence Gazer to refine a good result returned by an absolute mass search with a large mass error into an excellent result with no mass error as evidenced by decreasing the score by several orders of magnitude Click the button below to view the demonstration To enlarge the demonstration once you start it right click and choose Full Screen Multimedia ProSightPC User Guide 203 6 Using the Sequence Gazer to Search for Single Proteins Demonstrating the Sequence Gazer 204 ProSightPC User Guide Thermo Scientific ne Displaying Data in the Data Manager This chapter describes the
18. Table 75 Custom Filters section parameters Filter Description Hide Show Determines whether a search is displayed in the data grid if it meets the specified condition and is hidden if it does not meet the specified condition Color Applies colors to specific columns on the basis of the conditions that you set Left list Specifies the filter Middle list Specifies the operator Right list Specifies a value Add Adds a newly defined filter Cancel Resets the Custom Filters section to the configuration shown in Figure 114 on page 267 Use Selects the filter to apply to a search If Specifies the filter Is Specifies the operator Value Specifies a value Then Specifies what happens when a search meets the specified condition Otherwise Specifies what happens when a search does not meet the specified condition Refresh Displays the columns selected in the Show Columns area in the data grid Restore Defaults Reinstates the default settings in the Show Columns area Apply Executes the filters that you set in the Quick Filters and Custom Filters sections Table 76 describes the commands on the menu that appears when you right click in the Customer Filters section Thermo Scientific B Using the ProSightPC Interface Setting Default Options Table 76 Custom Filters shortcut menu Filter Description New Changes the configuration of the Custom Filters section to that
19. 644 3520 286 6895 1 757669E 06 8 837 2324 672 4502 5 759769E 05 770 7935 309 3587 1 029393E 06 8 836 9712 343 8559 5 000406E 05 i 669 7905 343 9159 2 680947E 05 127 917 2463 664 9562 3 936574E 06 3 753 0334 760 1306 3 602783E 06 132 954 0024 811 9804 9 018943E 06 637 7026 820 1718 9 765919E 06 783 0251 910 0891 8 039561E 05 14 980 5316 918 0974 4 276783E 06 5 784 6289 918 1084 0 0003204599 7g 814 0439 065 1829 7 371869E 06 144 1048 7888 191 1261 1 140738E 05 12 908 0963 4535 4453 8 282956E 06 Matching Non matching fragments table Search Parameter Display Thermo Scientific V L GTERLT G SLQLGI I ILREN VIKLG P v Methionine Information Position N 1 C 69 Amino Acid M RESID 49 Start PTM Acetylation PTM Choices None wi Custom Tier 1 I Formylation WE scetylation I methylation mono Fixed Modifications Cysteine None Acrylamide Cysteine Vinylpyridine Cysteine Ethanol Cysteine BME Cysteine Todoacetamide Cysteine Methionine Sulfone Methionine Sulfoxide Methionine Navigating the Sequence Gazer Fragments Explained box Amino acid information box Fixed Modifications box The search parameter display shows the data options and tolerances that you selected during the last round of scoring User defined selections appear in red You can change these by clicking on a new selection The new selection appears in red
20. B Using the ProSightPC Interface The ProSightPC Interface To import data into the data grid e See Working with Experiments on page 91 To perform and modify a search e See Searching Databases on page 99 To open the relevant Data Manager from the data grid e Double click a search in the data grid For more information about the Data Manager see Displaying Data in the Data Manager on page 205 Data Grid Shortcut Menu Commands Table 69 describes the commands available in the main data grid shortcut menu Table 69 Data grid main shortcut menu Sheet 1 of 2 Parameter Description Refresh Grid Redisplays the contents of the data grid Mark Marks an experiment by placing the ProSightPC symbol to the left of the experiment and an asterisk in the Marked column This mark can differentiate a particular experiment Append Predefined Searches Opens the Append Predefined Searches to Experiment X dialog box shown in Figure 47 on page 104 so that you can add more than one predefined search to the experiment For information on how to select options in this dialog box see Adding Predefined Searches to an Experiment on page 104 Append Predefined Search Opens a submenu with all of the predefined searches Clicking one of them adds it to the selected experiment Edit Search x Opens the Edit Search in Experiment X dialog box for that type of search this dialog box is the same as the New Search i
21. Calculates the intact mass if only the mass to charge ratio and the charge are known It opens the Intact Mass Calculator dialog box shown in Figure 38 on page 75 Specifies the mass type of the precursor ion if you select Manual in the Type list The mass type can be one of the following e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Precursor Ion Data Text File Specifies the path and name of an ASCII text file if you select Upload in the Type list Enter the path and name of the ASCII text file or files containing the fragment ion data or click Browse to browse for them These files must be properly formatted Experiment Comments Displays any comments to help you remember or understand details about the experiment that you just added Create Creates a new experiment from all the values entered for intact mass and fragment masses and adds it to the data grid Thermo Scientific Thermo Scientific 2 Getting Started Entering Data Manually Table 19 Experiment Adder dialog box parameters Sheet 2 of 2 Parameter Fragment Ion Data Type Description Specifies the method of inputting the fragment ion data You can select Manual or Upload from the Type list e
22. cee eee eee 217 Exporting a Proteome Database or Repository 0 000 e eee eee 218 Removing a Proteome Database or Repository 0 00 eee eee 219 Changing Views hve mee be wees hese k E wea ek see A 220 Creating a Proteome Database aniaogtaP i wta ahser ntalathea bacon ea ReneS 220 Create New Database Wizard Parameters 0 000 c cee eee eee 229 Linking to the UniProt Database sir Gea intes exten ah biteaioo da naaes 232 Using ProSightPC Tools ananena naaa 235 Locating and Selecting PTMs with the PTM Tier Editor 235 PIMS naea e ET E ae EE E T E n a A a te Uh 235 Accessing the PTM Tier Editor aciseseo dicate tac orem eens 236 Incudine PTM cetti ertai aet they a ty ated aeiaai atid 237 Excluding PIMS saa msue tronar Sh ieee Pie EA Ea EA 237 Moving PTMs Between Tiers vis acceso bases cet a arsenals besten wide ae 238 Viewing Fragments Ions with the Fragment Predictor 238 Fragment Predictor Window Parameters n u 04 ones oe eke ene ee ee 241 Converting Text to ProSightPC Font with the Font Converter 241 Font Converter Dialog Box Parameters ici ia ssk ee eee Cet 244 ProSightPC Reference cece cece eee eee eee eee eeeeneee 245 File CO eye iar OSs fearless 8 cep E e Gee Garasnte Mon wreak ured AE E oes 245 UAV eie glides oe las Pe eae a ail a tu Rk al Dah Di 247 View Menta nit yonan eaaa Asus ates Ait cea E ecco ast Gear 247 Expe
23. B3 B4 BS B6 B7 BS B9 B10 Bil B12 Y4 YS Y6 Y YS Y9 Y10 ahal Y 12 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 431 1628 544 2467 631 2786 760 3211 861 3683 932 4048 1031 4734 1118 5060 1217 5714 1354 6330 469 2649 568 3332 639 3703 740 4177 869 4599 956 4913 1069 5750 1232 6360 1363 6792 431 1630 544 2470 631 2790 760 3210 861 3690 932 4060 1031 4700 1118 5100 1217 5700 1354 6300 469 2670 568 3350 639 3720 740 4200 869 4630 956 4950 1069 5800 1232 6400 1363 6800 gt Show Non Matching Fragments Total 59 fragments 200 Error Da Error ppm 4m 0 0001 0 0001 0 0002 0 0003 0 0008 0 0014 0 0012 0 0006 0 0036 0 0009 0 0019 0 0020 0 0020 0 0023 0 0027 0 0033 0 0037 0 0060 0 0033 0 2319 0 1837 0 3168 0 3946 0 9288 1 5015 1 1634 0 5364 2 9567 0 6644 4 0489 3 5191 3 1281 3 1063 3 1054 3 4501 3 4593 4 8676 2 4199 The interactive fragment map and matching fragments table are linked for convenient data browsing Click a fragment name in the table to select the terminal amino acid in the fragment map You can also select the terminal amino acid of a fragment in the map to highlight the corresponding fragment name in the matching fragments table Table 42 describes the columns in the matching fragments table Table
24. D M Zubarev R A and McLafferty F W Automated Reduction and Interpretation of High Resolution Electrospray Mass Spectra of Large Molecules J Am Soc Mass Spectrum 2000 11 320 332 Thermo Scientific 2 Getting Started Importing Targeted RAW Files To use this option see Importing a Targeted RAW File with the Post Xtract Option on page 74 e Profile Uses the THRASH algorithm to process the input file This algorithm takes raw mass to charge m z data and finds the neutral mass values At its most basic level the THRASH algorithm infers monoisotopic or average masses from both precursor and data dependent MS MS scans and combines these mass lists into experiment sets precursor mass and its corresponding fragments masses These lists are then converted into a set of experiments in a ProSightPC upload format PUF file for searching with the ProSightPC suite of applications The THRASH algorithm is a little faster than the Post Xtract algorithm To use this option see Importing a Targeted RAW File with the Profile Option on page 79 e Manual entry method To use this option see Entering Data Manually on page 84 Table 14 summarizes the differences between the THRASH and Post Xtract methods of importing mass values Table 14 Comparison of Post Xtract and THRASH methods S N AIM equivalency Data type Speed Peaks A approximate Post Xtract Profile centroid Faster Slightly more 3 1 THRASH Profile
25. Predefined Searches on page 100 b To add a search click Ke in the dialog box The New Predefined Search dialog box opens Follow the instructions in Creating a Predefined Search on page 101 to create a new predefined search c To edit a predefined search select the name of the search and click a The Edit Predefined Search dialog box opens Follow the instructions in Editing a Predefined Search on page 105 to edit a predefined search d To remove a predefined search select the name of the search and click o e Click Save in the Edit Add Searches for HT dialog box 4 To set the conditions for the first search click Conditions in the purple circle in the Level 1 area of the Running Highthroughput Logic page The Condition dialog box appears as shown in Figure 23 You can use operators and values to create conditions for the search Thermo Scientific ProSightPC User Guide 39 2 Getting Started Processing LC MS MS Data Files Figure 23 Condition dialog box Condition elas E Value MIR 7 D AND OR End Condition Save Cancel e a From the left list select Number of Hits or E Value e Number of Hits Specifies the number of matches for an intact ion in the search e E Value Specifies the expectation value e value for the results of the search If at least one search result received an e value of less than 1e 4 the search is loaded to the good category For info
26. Tools gt Reports Tools gt Options 252 ProSightPC User Guide Generates the following types of reports e Status Report Gives a summary of every search in the open PUF file including search type and best score e Printable Report Contains all of the information related to one search formatted for easy printing e Best Hit Report Displays the match with the best score for each search that was run for each experiment in the data grid e Repository Report Lists all the experiments that a repository contains Opens the Options dialog box which you can use to set default values for most of the interface elements in the ProSightPC application See Setting Default Options on page 21 for instructions on setting default values Thermo Scientific Help Menu A ProSightPC Reference Help Menu Table 64 lists the commands on the Help menu Table 64 Help menu commands Command Help gt Help Description Opens the Help for the ProSightPC application Help gt Manage License Opens the License Information dialog box so that you can request a a new license activation code Help gt About Opens a dialog box that displays the release version the release date and the trademark information Help gt Report a Bug Opens an e mail form addressed to developers of the ProSightPC software so that you can report an issue Manuals Data Grid Shortcut Menu Opens the PDF file of
27. 1027 48219 11098 5193 1227 56189 11374 6303 1502 72526 1631 76785 1702 80496 1789 83699 1902 92105 2049 98946 12165 0164 2293 11136 E E Q I A E F K E A S L F D K D R S L G Q N P T E A E L Q D I G D n a a a a M 2 D Y Ions C Ions Z Ions y1 133 0375 a c1 88 06346 21 117 01878 a y2 232 10591 c2 203 0904 7 22 216 08719 y3 361 1485 3 331 14898 23 345 12978 y4 475 19143 c4 444 23304 24 459 17271 y5 588 27549 E c5 545 28072 25 572 25677 y6 719 31598 c6 674 32331 26 703 29726 y7 834 34292 c7 803 3659 27 818 3242 y8 962 4015 8 931 42448 28 946 38278 y9 1075 48556 c9 1044 50854 29 1059 46684 y10 1204 52815 10 1115 54565 210 1188 50943 y11 1275 56526 c11 1244 58824 211 1259 54654 y12 1404 60785 12 1391 65665 212 1388 58913 y13 1505 65553 13 1519 75161 213 1489 63681 y14 1602 70829 c14 1648 7942 214 1586 68957 y15 1716 75122 15 1719 83131 215 1700 7325 y16 1844 8098 c16 1806 86334 216 1828 79108 y17 1901 83126 17 1919 9474 217 1885 81254 y18 2014 91532 c18 2067 01581 218 1998 8966 y19 2101 94735 19 2182 04275 219 2085 92863 y20 2258 04846 c20 2310 13771 X 220 2242 02974 7 Optional Click Start Over to return to step 2 Fragment Predictor Window Parameters The Fragment Predictor window contains the parameters shown in able 55 Table 55 Fragment Predictor window parameters Parameter Description Please Enter Your Displays the protein sequence where yo
28. 1114 04 from retention time min 28 64 207 28 84 209 with FT de 74 1 Absolute Mass no 6 6e 83 3 ETD fragmentation for precursor at m z 1112 34 from retention time min 28 64 207 28 84 209 with FT de 75 1 Absolute Mass no 6 43e 10 3 ETD fragmentation for precursor at m z 927 12 from retention time min 28 92 210 29 14 212 with FT de 76 1 Absolute Mass no 2 52e 48 3 ETD fragmentation for precursor at m z 1117 73 from retention time min 28 92 210 29 14 212 with FT de 7 1 Absolute Mass no 2 49e 69 5 ETD fragmentation for precursor at m z 1083 53 from retention time min 29 23 213 29 75 218 with mafa 78 1 Absolute Mass no 31 5 1 ETD fragmentation for precursor at m z 1194 92 from retention time min 29 23 213 29 44 215 with FT de 79 1 Absolute Mass no 5 95e 60 5 ETD fragmentation for precursor at m z 1085 72 from retention time min 29 54 216 29 75 218 with FT d 80 1 Absolute Mass no 2 28e 69 5 ETD fragmentation for precursor at m z 1085 82 from retention time min 29 84 219 30 05 221 with FT d 81 1 Absolute Mass no 3 24e 56 5 ETD fragmentation for precursor at m z 1087 22 from retention time min 29 84 219 30 05 221 with FT di 82 1 Absolute Mass no 46e 43 3 ETD fragmentation for precursor at m z 1117 94 from retention time min 30 14 222 30 37 224 with FT de 83 1 Absolute Mass no na 0 ETD fragmentation for precursor at m z 1081 82 from retention time min 30 14 222 30 37 224
29. 152 ProSightPC User Guide Thermo Scientific Thermo Scientific 4 Searching Databases Performing Gene Restricted Searches Because the query is limited to the gene identifications listed a gene restricted biomarker mass search runs much quicker than a simple biomarker search To search for a gene restricted biomarker Perform any search Double click an experiment in the Data Manager to view it Click the arrow next to Search x Click Results for Precursor Ion 1 to view its results From the results list view click the Add Gene Restricted Search icon f circled in Figure 60 on page 147 The New Search in Experiment X dialog box opens as shown in Figure 62 ProSightPC User Guide 153 4 Searching Databases Performing Gene Restricted Searches Figure 62 New Search in Experiment X dialog box for biomarkers Search Type Gene Restricted Biomarker Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic X Precursor Tolerance 10 Da Fragment Mass Type Monoisotopic hg Fragment Tolerance 15 ppm v Am Mod Include Dea Modified Forms Hit Filtering v Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score 0 Max Proteins to Return all Fixed Modifications Cysteine a Methionine p 4 Lysine E F koleuci Leuci
30. 231 N terminal fragment ions 7 163 241 251 N terminal methionines 231 0 observed intact ion mass 125 Open Data Manager icon 260 Open Existing PUF File icon 93 Open icon 93 259 Options dialog box Absolute Mass Preferences page 115 117 accessing 21 252 Biomarker Preferences page 127 129 General Preferences page 21 purpose 277 Sequence Tag Preferences page 136 138 Single Protein Preferences page 143 145 Thrash Preferences page 79 80 Orbitrap based mass spectrometers 1 overlapping peaks 56 62 P p score calculating 193 gene restricted biomarker searches 155 in absolute mass searches 114 in General Preferences dialog box 22 23 in gene restricted absolute mass searches 149 in Quick Filters section 273 in reference article 193 in results list 164 in Show Columns section 270 partial characterization 16 pasting text 247 PDE McLuckey score calculation of 196 in General Preferences dialog box 22 23 in Quick Filters section 272 273 in reference article 193 in search results list 163 in Show Columns section 270 pending searches 212 Pending status 265 peptides containing disulfide bonds 126 converting to neutral masses 73 79 deconvolution 56 62 eliminating from search 38 Thermo Scientific fragmenting 10 middle down bottom up databases 3 222 229 multiply protonated 10 represented in FASTA files 221 top down proteomics 12 Poisson distribution 194 polymorphisms discarding 226 including in biomarker se
31. 5 Viewing Search Results Viewing the Results in the Tab Controller Table 33 Result statistics table elements Sheet 2 of 2 Parameter Description E Value Displays the expectation score e value For information on this value see Expectation Value e value on page 194 P Score Displays the p score For information on this value see p Score on page 193 Each result has the three context sensitive buttons as described in Table 34 Table 34 Result buttons Button Description Take to Sequence Creates a new single protein search based on the result See Gazer Chapter 6 Using the Sequence Gazer to Search for Single Proteins for details RESID Displays a RESID annotated sequence SEQ Displays the sequence Click the text in the header column to sort the results list in ascending or descending order Click again to reverse the order The ProSightPC application automatically generates a color coded legend An amino acid bearing a PTM is color coded according to this legend Cysteines are always colored yellow The matching fragment table contains a summary of all fragment ions matching the protein For information on the interactive fragment map see Interactive Fragment Map on page 198 Absolute mass biomarker single protein gene restricted absolute mass and gene restricted biomarker mass searches all return similar results You can perform a gene restricted search for any results list in the
32. Adder Contents e Starting the ProSightPC Application e Closing the ProSightPC Application e Setting Default Options e Importing or Creating a Proteome Database e Editing Modifications e Processing LC MS MS Data Files e Using Repositories e Importing Targeted RAW Files e Entering Data Manually e Importing Experiments e Searching the Proteome Warehouse for Matches For detailed information on the ProSightPC features noted in this chapter see Using the ProSightPC Interface on page 257 Thermo Scientific ProSightPC User Guide 19 2 Getting Started Starting the ProSightPC Application Starting the ProSightPC Application Open the ProSightPC application by choosing a Start menu command or clicking a desktop icon To start the ProSightPC application From the Start menu choose Programs gt Thermo ProSightPC or click the ProSightPC icon E on your desktop The ProSightPC main window opens as shown in Figure 13 Figure 13 ProSightPC main window ProSightPC Untitled puf File Edit View Experiment Tools Databases ProSightHT Tools Help Del Adnr Bse peg XY Ut Exp D Search ID Marked Search Type Pending Search Best Expectation Matching Forms Name Status Notes ProSightPC 3 0 Software for Precision Proteomics SG Bano For information on the features of this window and how to customize them see Using the ProSightPC Interface on page 257
33. Annotation The ProSightPC application relies on an analysis process called shotgun annotation to take PTM events on a single protein and precalculate all possible combinations regardless of whether the particular combination has ever been observed Shotgun annotation includes two components constructing databases and searching databases Constructing Databases Thermo Scientific Creating a shotgun annotated database is based on the following principle A given protein has x sites of modification that is sites where particular residues are observed or predicted to be modified in some way A residue that can be modified is called a site In a particular instance of a given protein the modified sites are active and the unmodified sites are inactive This instance is called a protein form Because you do not necessarily know which sites are simultaneously active in a living organism within practical limitations you want to precompute the masses and identities of all possible forms of a given protein resulting in 2 forms where 7 is the number of sites of modifications on the protein For proteins with a limited number of sites this growth rate is feasible for proteins with a large number of sites it is impractical to store all possible forms for highly modified proteins To address this issue the ProSightPC application first determines if the protein is going to need more than 1000 records to fully describe it If it does the ProSi
34. Data Type list select the mass type of the y yp P precursor ion in the Mass Type box and enter the precursor mass in the box beneath it e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion If you select Upload in the Precursor Ion Data Type list enter the path and name of the ASCII text file or files containing the precursor ion data in the Text File box or click Browse to browse for them These files must be properly formatted Optional Click m z to have the ProSightPC application calculate the intact mass if only the mass to charge ratio and the charge are known The Intact Mass Calculator dialog box opens as shown in Figure 38 i In the Precursor m z box type the mass to charge ratio m z value of the precursor ion ii In the Charge State box type the charge state z to assign to the mass to charge m z data that was found in the data files iii Click OK 4 Optional In the Experiment Comments box enter any comments to help you remember or understand details about the experiment that you just added 5 In the Fragment Ion Data area select the method of inputting the fragment ion data a b In the Type list select Manual or Upload If you select Manual in the Fragm
35. Description Filters the search by search type e Absolute Mass Biomarker e GRAM gene restricted absolute mass e GRBM gene restricted biomarker e Sequence Tag e Single Protein Pending Search Filters the search by whether a search is pending or not e Yes A search is pending e No A search is not pending Marked Filters the search by whether a search is marked or not e Yes A search is marked e No A search is not marked Best Expectation Filters the search by expectation value e value For information about the expectation value see Expectation Value e value on page 194 Matching Forms Filters the search by the number of matching forms Best PDE Filters the search by PDE McLuckey score For information about this scoring method see PDE McLuckey Score on page 196 Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface Table 74 Quick filters area parameters Sheet 2 of 2 Filter Only Experiments Where number Search Has option operator value Description Filters the search by experiments that meet the conditions set Number can be e At Least One Displays at least one search meeting the criteria e All Displays all searches meeting the criteria e No Does not display any of the searches meeting the criteria Option can be e Best Expectation value e value For information about the expectation value see Expectati
36. Editor so that you can add terminal modifications to the ends of proteins Tools gt Fixed Modification Editor Opens the Fixed Modification Editor so that you can add fixed modifications which apply the same specific mass to all occurrences of the named amino acid Tools gt Font Converter Tools gt Fragment Predictor Thermo Scientific Converts text into the ProSightPC fragment map font used to display N terminal and C terminal fragments You can use the Font Converter to generate fragment maps for inclusion in publications and presentations This command opens the Font Converter dialog box shown in Figure 107 on page 242 Adds post translational modifications PTMs or arbitrary custom masses to any amino acid in a known protein sequence and displays the predicted b y c and z fragment ion masses It opens the Fragment Predictor window shown in Figure 102 on page 239 ProSightPC User Guide 251 A ProSightPC Reference Tools Menu Table 63 Tools menu commands Sheet 2 of 2 Command Tools gt Experiment Manager Description Opens the Experiment Manager shown in Figure 43 on page 92 so you can manipulate experiments as objects copy individual experiments between PUF files or save them in their own PUF file Tools gt Batch Run Processes several predefined searches automatically You can queue and run a large number of searches over any number of experiments in a single action
37. Existing PUF File e Adding Experiments to PUF Files e Copying Experiments from One PUF File to Another e Removing Experiments from PUF Files e Saving a Changed PUF File e Changing the Experiment Display Deleting PUE Files Experiments in PUF Files The ProSightPC application operates on a single PUF file that contains experiments When you open the PUF file the application loads it into memory and makes the data visible in the data grid You can use the data grid to manage the experiments in a single PUF file To manage experiments in multiple files use the Experiment Manager An experiment is defined as one or more precursor masses one or more fragment masses and all related searches Thermo Scientific ProSightPC User Guide 91 3 Working with Experiments Creating a New PUF File Creating a New PUF File You can create a PUF file by creating an empty file and importing data into it or by using the Experiment Manager To create a PUF file by importing data into an empty file 1 Choose File gt New or click the New icon f The experiments in any previously opened PUF file disappear from the data grid and you are prompted to add an experiment or import data 2 Add experiments or import data to the PUF file See Adding Experiments to PUF Files on page 94 To create a PUF file by using the Experiment Manager 1 Choose Tools gt Experiment Manager The Experiment Manager opens as shown in Figure 43 Usually
38. F D KtD GtD GtT I T T K B b G 9 V8 R S L G6 O1N P 7 E A E LTN 1 8 E V D A 0 gt 6 N G T I D F P E F L T M M A R K M K D T D S E E E I R E A FTER V FED KtD G N G Y I S A A E L R H V M TEN L G E K L T D E S L S 8 PEL 1 5S S F C P R t F P C t R P 3D A F 1 5S ID Length Mass Mass Diff PPM Diff C Ions Z Ions Total Ions PDE Score Expectation 674572 146 16406 900000 372 647000 22208 400000 11 1 12 89 300000 7 83E 11 Take to Sequence Gazer The New Search in Experiment X dialog box opens as shown in Figure 53 on page 120 6 In the Search Type list select Gene Restricted Absolute Mass The appearance of the dialog box changes as shown in Figure 61 Thermo Scientific ProSightPC User Guide 147 4 Searching Databases Performing Gene Restricted Searches Figure 61 New Search in Experiment X dialog box for gene restricted absolute mass Search Type X Gene Restricted Absolute Mass Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic Fragment Mass Type Monoisotopic Fragment Tolerance 15 ppm Am Mode Disulfide Hit Filtering v Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score Max Proteins to Return all X Fixed Modifications Cysteine H Lysine H Isoleucine PTM Handling E A PTMs __ High priority PTMs Tier 1 Terminal Mods N Term
39. Fragment Tolerance box specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Indicate whether it is expressed as absolute measured in daltons or relative measured in parts per million An observed fragment ion matches a theoretical fragment ion if the observed fragment ion mass is within plus or minus the fragment tolerance of the theoretical fragment ion mass Select the Am Mode check box if you want to conduct the search in delta m Am mode For details see in Performing Searches in Delta m Mode on page 110 Select the Disulfide check box if you know that the protein s cysteines are oxidized The ProSightPC application looks for only one disulfide bond Select the Include Modified Forms check box if you want to include PTMs and polymorphisms when you perform a biomarker search To detect biomarkers with modifications on them select this option however analysis time increases as a result In the Hit Filtering section set at least one of the following filters otherwise the ProSightPC application returns all protein forms that are searched even proteins that have no matching fragments a Select the Min of Matching Fragments check box if you want the search algorithm to find only protein forms containing a minimum number of matching ion fragments these protein forms are called hits Then specify the minimum number of
40. Large Icons Lists the experiments from left to right in the pane using larger icons than the Small Icons command does This icon on the left displays the way experiments in the source PUF file are displayed and this icon on the right displays the way experiments in the destination PUF file are displayed Sends a copy of the selected experiments from the source PUF file to the destination PUF file O Sends a copy of the selected experiments from the destination PUF file to the source PUF file 98 ProSightPC User Guide Thermo Scientific M Searching Databases This chapter describes the ProSightPC search modes and how to use them Contents e Search Types e Performing Searches e Searching for Absolute Mass e Searching for Biomarkers e Searching for Sequence Tags e Searching for Single Proteins e Performing Gene Restricted Searches e Performing MS Hybrid Searches e Analyzing MS MS Experiments Search Types The ProSightPC application has four basic types of searches Each search mode represents a specific mechanism used to compare imported data to a proteome database in the proteome warehouse The four search modes are the following e Absolute mass search e Biomarker search e Sequence tag search e Single protein search In addition you can use the ProSightPC application to perform the following types of advanced searches e Gene restricted absolute mass search e Gene restricted biomarker se
41. Manual Inputs the precursor ion data e Upload Loads the precursor ion data from an ASCII text file or files Fragment Ion Data Mass Type Specifies the mass type of the fragment ion if you select Manual in the Type list The mass type can be one of the following e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion e Intensities Specifies the intensity of the fragment mass Fragment Ion Data Text File Specifies the path and name of an ASCII text file if you select Upload in the Type list Enter the path and name of the ASCII text file or files containing the fragment ion data or click Browse to browse for them These files must be properly formatted Intensities Specifies the abundance of the fragment ions Please Check Any Selects any predefined searches to add to an experiment a ae e Demo Search Searches the demonstration database included Included with Your in the installation of the ProSightPC the software Experiment R Opens the New Predefined Search dialog box so that you can create a new predefined search Ei Opens the Edit Predefined Search dialog box so that you can edit the parameters for the search Removes the selected predefined search from the lis
42. Ms Every form containing an unselected PTM is excluded from the interrogation In the Terminal Mods area select the fixed terminal modification for each terminus A fixed terminal modification is a chemical modification that is present on the terminus of the observed protein e N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus Click Save The new search appears in the data grid with yes appearing in the Pending Search column To execute the search from the data grid right click the pending search and then choose Run Search number Thermo Scientific 4 Searching Databases Performing Gene Restricted Searches New Search in Experiment_X Dialog Box Parameters for Biomarkers Table 31 lists the parameters in the New Search in Experiment X dialog box for biomarkers shown in Figure 62 on page 154 Table 31 New Search in Experiment X dialog box parameters for biomarkers Sheet 1 of 2 Parameter Search Type Database Description Description Specifies the type of search to perform Absolute mass See Searching for Absolute Mass on page 113 BioMarker See Searching for Biomarkers on page 125 Sequence Tag See Searching for Sequence Tags on page 136 Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins Gene Restricted Absolute Mass See S
43. N terminal of the sequence C applies the modification to the C terminal of the sequence d In the Monoisotopic Mass box type the monoisotopic mass of the terminal modification e In the Average Mass box type the average mass of the terminal modification f Click Save The window closes The modification appears when you create searches To edit fixed modifications 1 Choose Tools gt Fixed Modification Editor to open the Fixed Modification Editor shown in Figure 17 Thermo Scientific ProSightPC User Guide 25 2 Getting Started Editing Modifications 26 ProSightPC User Guide Figure 17 Fixed Modification Editor i i Mass 71 03711 105 05781 57 02146372 75 99829 15 99492 31 98984 44 0262 75 998285 125 047679 58 005479 352 247441 O Acrylamide Cystei RILALRAIANLNIANSA S e alraya 2 In the dialog box do the following a Scroll down to the last row which is marked by an asterisk b In the Name box type the name of the modification c In the Amino Acid box type the symbol of the amino acid being modified d In the Monoisotopic Mass box type the monoisotopic mass of the chemical formula of the modification e In the Average Mass box type the average mass of the chemical formula of the modification f In the Description box type a brief description of the modification 3 Click Save The window closes The
44. PDE McLuckey score of any match in the search results For more information on the calculation of this score see PDE McLuckey Score on page 196 Highest Total Ions Displays a column showing the highest total number of ions that matched the ions in the database b c Ions Displays a column showing the number of b and c fragment ions that matched in the database ylz Ions Displays a column showing the number of y and z fragment ions that matched in the database Fragments Displays a column with the total number of theoretical fragments present Precursors Displays a column with the total number of theoretical precursors present First Abundance Displays a column with the abundance of the first precursor Largest Abundance Displays a column with the abundance of the precursor with the largest abundance Best Seq Score Displays a column with the best sequence tag score Refresh Displays the columns selected in the Show Columns area in the data grid Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface Table 73 Show Columns area parameters Sheet 3 of 3 Parameter Description Restore Defaults Reinstates the default settings in the Show Columns area Apply Executes the filters that you set in the Quick Filters and Custom Filters areas Using the Filters in the Quick Filters Area You can use the Quick Filters area to quickly define conditions with which to filter the experiments and sear
45. ProSightPC User Guide 201 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Figure 84 Non matching fragments table MUM eVUIUSLELTIUALVUESt vind K gt Show Matching Fragments Total 19 fragments YShow Non Matching Fragments Total 59 fragments m z Monoisotopic Mass Monoisotopic Intensity 202 ID on one 10 12 13 16 17 18 etl 22 23 24 25 26 27 28 29 32 33 34 35 36 37 38 39 40 41 43 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 0 0000 413 1521 415 1480 516 2519 550 3231 576 3017 594 3126 612 3230 613 2681 621 3600 631 3233 722 4074 732 3266 768 4018 810 3869 814 3857 818 3804 825 3465 828 3977 833 3992 843 3582 851 4497 876 4053 894 4150 896 3853 904 4115 905 4589 913 4535 914 3948 918 3890 923 4701 931 4641 938 4808 8127 41 431 09 1161 64 321 61 252 34 102 72 418 44 796 79 277 86 349 58 1520 2 200 95 430 13 1352 94 1667 18 185 72 264 93 1073 84 202 9 1656 58 1112 78 295 28 270 54 484 73 186 5 213 78 277 77 2378 14 239 61 1282 07 1000 97 3119 16 The non matching fragments table displays the columns shown in Table 43 Table 43 Non matching fragments table parameters Sheet 1 of 2 ProSightPC User Guide
46. Rese S ee Er eet 1 The ProSightPC Application 2005 s ute eee eee peste ee sors oboe 3 Proteome Watchouse ss cheeks tiessdias eee phe eNews een diese 3 Search LY Pele nade herbs hinieadelorencestonteat owed bseat e 4 Iterative SEAL CUIIE ou cees AwReeedee Rien pe KEEL LKS ee E 4 Database Manager s 1345 85 cee eripe eee Ces eae Rha ee BOS 5 Data Manager sheets tee Ps pases pee eee Hees Lee eee ees 6 Segucnce Gazer pals erain a a E ET EE E A EE dad S 6 Experiment Manager ascii Kaew eae ee iai rre raria wares aeh 6 PTM lier Editor ps so katie asia tae a a ETE an E OERA 6 Fragment Predictor sp cccnibeteneki eter bbascis kbevids keawee betes 6 Font COnVEltEr sili ene nda ee i a sl tee tod eee dha wed eee he knee es 7 LC MS MS Workflow iscc4 capone Ripe ce pone eee ee eee Medea 7 Trips and OUR PUES oc tao nenat ranae a a Rae meeRae sd 9 JApUtS eer icc ces nieren eee dia tha aed HRS RSG FRG ERE Ha eed S 9 OUtpUtS sic ede aha ie de Meds Maw ae hae als 9 Fragmentation Methods 56 vein eas cekd nd ep eee Bee ee hd epee 9 lon TYPES civ cir doipere iriri eke ee Sheena 10 Introduction to Proteomics o cite Lie Keene eho ee hee ewes heme ws 10 Middle Down Bottom Up Proteomics 0 c eee cee eee 11 Top Down Proteomics lt i cocinncsseeane dune eedeawhs aimee ade 12 Shotgun Annotation 2 2322 teheeded ehaedeceken dee bee Sade seee 13 Search Modes and the Top Down Funnel 0 0 0 0 c eee eee 15 Chapter2 Getting Started 0 cece
47. Scans Specifies a value to filter out low quality spectra Range 1 no maximum Default 1 In some cases you might want to consider only precursors that have been fragmented twice or more Minimum Fragmentation Base Peak Intensity 64 ProSightPC User Guide Specifies a value that will filter out noise and poor quality data during analysis of the fragment ions Range 1 no maximum Default 100 THRASH 1000 Xtract Thermo Scientific 2 Getting Started Using Repositories Table 10 Advanced Settings dialog box parameters Sheet 8 of 8 Parameter Absolute Minimum Intensity Description Specifies the minimum intensity that the ProSightPC application accepts for fragmentation peaks The application excludes deisotoped peaks below this value so it removes low intensity fragment ions that might be spurious Range 1 no maximum Default 100 Get Top X Specifies the number of the most intense peaks per window size that the ProSightPC application considers This parameter works with the Window Size parameter to filter the deisotoped or decharged data The default settings mean that the application considers only the most intense 5 peaks in a 100 Da window Therefore this setting therefore removes low intensity fragment ions that might be spurious Range 1 no maximum Default 5 Window Size Using Repositories Specifies the size of the window containing the number of the most intense peak
48. Searches icon 101 260 manually importing MS MS experiment data 84 mass diagrams 198 mass values adding a row to list 211 copying to external application 211 editing 208 248 removing a row from list 211 matching fragments table 200 matrix assisted laser desorption ionization MALDI 11 McLuckey score See PDE McLuckey score menu bar 258 Merge Hits dialog box 180 Middle Down processing option 30 35 middle down bottom up databases 3 23 222 226 middle down bottom up experiments 2 10 11 minimum tag score 137 MS experiments 12 MS MS experiments 12 84 159 MS 3 experiments 159 MS experiments 136 MS hybrid searches 159 multiplexed scoring 56 multiplexing fragmentation data 56 62 MySQL relational databases 3 N terminal applying modifications to 25 27 cleavage at a proline 196 fragment marks in Font Converter 243 in Amino Acid Information box 199 in delta m searches 110 neutral masses 12 New icon 92 259 New Predefined Search dialog box 88 101 103 105 184 New Repository dialog box 36 49 67 69 New Search in Experiment X dialog box for absolute mass searches 120 123 for biomarker searches 131 134 for gene restricted absolute mass searches 147 151 for gene restricted biomarker searches 153 157 for sequence tag searches 140 142 for single protein searches 186 189 284 ProSightPC User Guide N formylmethionine 224 non matching fragments table 201 N terminal acetylation 224 231 N terminal formylation 224
49. Slower Slightly less 10 1 Importing a Targeted RAW File with the Post Xtract Option The Post Xtract algorithm averages the data from all fragmentation scans and only analyzes the averaged fragmentation data once This option reduces analysis and search time and should give better results This option is the default To import a targeted RAW file with the Post Xtract option follow this procedure To view a demonstration of this procedure see Demonstrating Targeted Raw File Importation with Post Xtract on page 78 To import a targeted RAW file with the Post Xtract option 1 Choose File gt Import raw gt Post Xtract or click the Import Xtract icon EA The Build Experiment from Post Xtract RAW Data dialog box appears as shown in Figute 37 Thermo Scientific ProSightPC User Guide 73 2 Getting Started Importing Targeted RAW Files 74 ProSightPC User Guide Figure 37 Build Experiment from Post Xtract RAW Data Build Experiment from Post Xtract RAW Data a 2 In to 3 In Post Xtract RAW File C Program Files ProSightPC Test Data Top Down Class CiDfraction01 raw Precursor Mass Predefined Search Please check any predefined searches gt E o moar A 5 Average Mass Til Monoisotopic Mass Fragmentation lon Data Fragmentation CID v Method Average Mass Monoisotopic Mass Check Al Uncheck Al the Post Xtract RAW File box type the pat
50. Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Mass Type Specifies the mass type of the fragment ions to use e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Tolerance Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Thermo Scientific ProSightPC User Guide 189 Table 37 New Search in Experiment X dialog box parameters for a single protein Sheet 2 of 2 Parameter Description Am Mode Determines whether the ProSightPC application conducts the search in delta m Am mode Performing Searches in Delta m Mode on page 110 explains this mode Fixed Modifications Specifies the chemical modifications present on all instances of a given type of amino acid in the observed protein Sequence Specifies the sequence You can either type the sequence or use a sequence from another source Save Saves the search information Navigating the Sequence Gazer Fragment ion information in the Sequence Gazer interfac
51. The experiment page and all pages related to it such as the Sequence Gazer disappear from the screen To close all Data Manager windows e Choose View gt Close All Data Tabs To close all Data Manager windows except for the currently selected window e Choose View gt Close All Data Tabs But Selected Thermo Scientific ProSightPC User Guide 207 7 Displaying Data in the Data Manager Adding or Editing an Experiment Comment Adding or Editing an Experiment Comment You can use an experiment comment to record information relating to all the searches information about which liquid chromatography fraction the data came from and information regarding the mass spectra used to create the mass list To add or edit an experiment comment 1 Click Edit Comment in the Data Manager choose Experiment Tools gt Edit Comment or click the Edit Comment icon abc 2 Type the comment in the box that opens in the Data Manager 3 Click Save to save the comment Editing Mass Values The Data Manager includes a facility for reviewing and editing mass values in an experiment If you would like to review the mass values you can export them to an external application such as an Excel spreadsheet You can also edit mass values by adding more precursor or fragment masses deleting existing precursor or fragment masses or changing values for precursor or fragment parameters In addition you can change the fragmentation method
52. User Guide Thermo Scientific 8 Using Proteome Databases Creating a Proteome Database Figure 96 Input File page of the Create New Database Wizard Create New Database Ea Input File Specify the location and type ofthe input data file A database may be created either from a simple Fasta format file which contains only descriptions and sequences or from a Swissprot format file which contains additional information about sequence features File Location 6 In the File Location box type the name and path of the file containing the sequence information or browse to it by clicking the Browse Folder icon In the Open dialog box activated by the Browse Folder icon you can select a FASTA file or a UniProtKB flat file for the input file For descriptions of these files see Creating a Proteome Database on page 220 The ProSightPC application generates the database from this data file Before you load the file open it in a text editor to ensure that it is free from errors Most errors in loading result from bad input files A good source for input files is the Web site of the UniProt consortium If you are going to create your own input file make sure that the encoding is correct Notepad can sometimes mishandle the encoding of newline characters in the file If you receive errors try a proper text editor The name of the data file must be unique 7 Click Next The Initial Methionines page of the Cre
53. a H E Lysine P Isoleucine H E Leucine gt PTM Handling JAI PTMs GE High priority PTMs Tier 1 Terminal Mods N Terminal Mod None z C Terminal Mod None 3 In the Database Description list select a description of the database that you want to search 4 In the Precursor Mass Type list select the type of precursor ion mass to use e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Thermo Scientific ProSightPC User Guide 131 4 Searching Databases Searching for Biomarkers 132 ProSightPC User Guide 10 11 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Precursor Tolerance box enter the range value for tolerance when testing all protein forms for biomarker peptides Indicate whether it is expressed as absolute measured in Da or relative measured in ppm In the Fragment Mass Type list select the mass type of the fragment ions to use e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the
54. all running searches you can click the Abort All Jobs icon oe Job Queue Parameters The job queue contains the areas shown in Table 71 Table 71 Job queue areas Area Name Description Lists the MS MS search identifiers associated with the job Status Displays the status of the current search e Pending indicates that the search has yet to be run e Running indicates that the search is currently running e Completed indicates that the search has been successfully run e Failed indicates that the search ended abnormally Notes Displays additional information about searches For example the notes explain why a search has failed Right click the job queue pane to display the commands described in Table 72 Thermo Scientific ProSightPC User Guide 265 B Using the ProSightPC Interface The ProSightPC Interface Table 72 Job queue shortcut menu Command Description Run Performs a search Abort Stops a search Clear Finished Jobs Removes all jobs that have finished For information on performing searches see Searching Databases on page 99 Tab Controller Many of the more complex interface elements of the ProSightPC application appear in the tab controller Double click an experiment in the data grid to display the experiment in the Data Manager in the tab controller area e Right click a page to hide the page e Right click a page and choose Close to close the pa
55. at 1154 1126 Da observed fragment ions of 1154 1090 Da 0 0034 Da from theoretical and 1154 1167 0 0041 Da from theoretical fall within the tolerance but 1154 2312 0 1222 does not because the mass difference is greater than the tolerance that you set 6 Click OK Single Protein Preferences Page Parameters Thermo Scientific Table 29 lists the parameters on the Single Protein Preferences page of the Options dialog box shown in Figure 59 on page 144 Table 29 Single Protein Preferences page parameters Sheet 1 of 2 Parameter Description Precursor Mass Specifies the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Mass Specifies the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion ProSightPC User Guide 145 4 Searching Databases Performing Gene Restricted Searches Table 29 Single Protein Preferences page parameters Sheet 2 of 2 Parameter Description Delta m Mode Determines
56. box See Expectation Value e value on page 194 for more information on the e value e lt Indicates that the first value is less than or equal to the second value This setting is the default e gt Indicates that the first value is greater than or equal to the second value d From the Max Proteins to Return list select the maximum number of proteins to return in the search With this option you can truncate the results of a search because the data from all of the similar matching proteins do not need to be returned You can load the results faster In the Fixed Modifications box select no more than one fixed modification per amino acid type A fixed modification is a chemical modification that is present on all instances of a given type of amino acid in the observed protein In the PTM Handling box select the PTMs that you want to search for The PTM Handling box displays PTMs arranged in one or more tiers based on the selected proteome database The ProSightPC application only queries theoretical protein forms containing exclusively selected PT Ms Every form containing an unselected PTM is excluded from the interrogation In the Terminal Mods area select the fixed terminal modification for each terminus A fixed terminal modification is a chemical modification that is present on the terminus of the observed protein N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Termin
57. data to neutral mass domain with THRASH or Xtract Save in a proteome database Create a PUF file optional Create a repository Define an iterative search tree Search mass data against the proteome warehouse 8 ProSightPC User Guide Thermo Scientific 1 Introduction to the ProSightPC Application Inputs and Outputs Inputs and Outputs The ProSightPC application works with the following formats Inputs The ProSightPC application works with three unique input file types e ProSightPC upload PUF files in XML format are used to store and transport ProSightPC results Each PUF file can contain many MS MS experiments and each MS MS experiment can contain searches A single MS experiment can contain mass lists for both precursor and fragment ions extracted from the MS and MS MS spectra Only one PUF file can be open at a time The active PUF file appears at the top of the Data Manager window Each experiment is identified by a number that is unique in the PUF file e Proteome warehouse PWF files are used to move databases and repositories from one computer to another You can download prebuilt databases by choosing Databases gt Download ProSightPC Databases in the ProSightPC application or by going to ftp prosightftp gsX 1gON prosightpc northwestern edu These databases include trypsin Lys C and top down databases for all the major model organisms These databases are constructed and maintained quarterly in t
58. do not need to run another search To create a two level search tree 1 Follow the instructions in Creating or Editing a One Level Search Tree on page 38 and select Run Search in the Success or Failure list for the first level search A second level search tree opens as shown in Figure 28 Figure 28 Second level search tree Running Highthroughput Logic Select a repository to load results to and select create a search tree with Repository _ yeast_topdown_etd New Repository Search Tree Name yeast_topdown_etd x Save Experiment Filter Min fragments 6 lt yeast absolute mass Max fragments 500 sS Min Intact Mass 750 Monoisotopic Y 2 Starting with Add Search perform the same steps as for the first level search however the Success and Failure lists are not available in second level searches so you must skip this step If you want to create a search tree of more than two levels see the next section Creating a Search Tree with Three or More Levels on page 45 44 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Figure 29 shows a completed second level search Figure 29 Completed second level search tree Running Highthroughput Logic Select a repository to load results to and select create a search tree with Repository
59. enters the search tree at the top as shown in Figure 20 The ProSightPC application conducts the first search according to the definitions in that box The results of that search come back and are graded by the conditions set for that search node Generally the application uses the condition that the best expectation score returned by the search is less than 0 0001 If the expectation value is lower than 0 0001 the ProSightPC application loads the results into the good category but if the results are greater than 0 0001 the application tries another search with looser search parameters that is a larger precursor search window biomarker mode or delta m Am mode This usually means that the search will take longer You now move down to the next node of the search tree as shown in Figure 21 The ProSightPC application checks the results of that second search against the conditions that you set again for example where the expectation value is less than 0 0001 and if the results meet the conditions the application loads the results to the good category If they do not meet the conditions the application loads them to the bad category and you can manually try to run them again Good and bad are arbitrary category names for searches that pass or fail the conditions set in the search tree respectively You can add results from searches such as biomarker or delta m Am mode searches to the results repositor
60. expands Figure 73 Expanded Custom Filters section Custom Filters V Show Custom Filters Use F Is Value Then Search sequen Show In Grid Theoreti gt 0002 Show In Grid 4 m r Merge Hits Add Custom Fitter Apply Fitters 178 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report In the Use column select the filter that you want to apply Click Apply Filters To add a new custom filter In the Custom Filters section of the Actions area see Figure 70 on page 171 select the Show Custom Filters check box The Custom Filter section expands as shown in Figure 73 on page 178 Click Add Custom Filter The Custom Filters section resembles Figure 74 Figure 74 Expanded Custom Filters section Custom Filters V Show Custom Filters 4 m P Add Custom Fitter Apply Filters From the list on the left select the parameter The parameters in this list are the same as the column names described in Display Columns in the Repository Report on page 173 4 From the middle list select an operator e Equal to e lt Less than e gt Greater than e lt Less than or equal to e gt Greater than or equal to e Not Not equal to 5 In the box on the right specify an appropriate value 6 Click Add Thermo Scientific The Custom Filters sec
61. experiments as needed The search parameters in predefined searches are persistent until you modify or delete them e Creating a Predefined Search e Adding Predefined Searches to an Experiment e Editing a Predefined Search e Running a Predefined Search e Cancelling a Predefined Search e Removing a Predefined Search e Removing Search Results from a Search e Removing an Experiment from the Data Grid Thermo Scientific 4 Searching Databases Performing Searches Creating a Predefined Search To create a predefined search 1 Choose Tools gt Manage Predefined Searches or click the Manage Predefined Searches Figure 45 Predefined Search Manager dialog box P W Predefined Search Manager oo ENC Search Name Type Database Demo Search Absolute Mass Demo database All existing predefined searches appear in the Predefined Search Manager dialog box In the example in Figure 45 a search of the demonstration database included in the installation of the ProSightPC software is available 2 Click the Create New Search icon ga in the Predefined Search Manager dialog box or right click the view area and choose New from the shortcut menu The New Predefined Search dialog box opens as shown in Figure 46 Thermo Scientific ProSightPC User Guide 101 4 Searching Databases Performing Searches Figure 46 New Predefined Search dialog box boba Search Name Search Type X Absolu
62. false ID Length 123 11 Mass 1247 62 Fragment Details Ion Fragment ID Observed Mass Da Mass Diff 0003 PPM Diff Theoretical Mass Da 2445 B Ions Y Ions 6 Mass Error Da 6 Total Ions 12 PDE Score 257 Expectation 1 47E 18 Mass Error PPM Delta M B3 B4 BS B6 B7 B8 Y4 YS Y6 Y7 Ys Y9 1 6 10 24 33 37 3 7 18 27 35 40 Fragmentation Method CID Intact Mass List 361 1634 475 206 572 2587 719 3268 818 3942 915 4471 429 2218 528 2901 675 358 772 4101 886 4524 1001 4783 Ion Type BY 361 164 475 207 572 259 719 328 818 396 915 449 429 224 528 293 675 361 772 414 886 457 1001 48 ID MZ Monoisotopic MZ Average Mass Monoisotopic Mass Average i Fragment Mass List ID MZ Monoisotopic MZ Average woronpwone BR RO Thermo Scientific 1247 6183 Mass Monoisotopic Mass Average 361 1634 411 2114 429 2218 447 2113 458 1797 475 206 528 2901 555 232 559 2785 572 2587 587 2735 589 3254 626 3058 656 295 659 3507 674 3052 675 3088 675 358 676 3616 684 2893 0003 0006 0007 001 002 0019 0026 0027 0032 0039 0045 0055 Intensity 962700 06 Intensity 768 2 1490 77 32657 78 606 08 1182 41 3657 39 2738 81 2209 89 542 87 1123 25 358 68 653 22 259 8 684 98 764 86 6875 22 956 34 3691 4 594 9 458 39
63. fragment ion mass is within plus or minus the fragment tolerance of the theoretical fragment ion mass Select the Am Mode check box if you want to conduct the search in delta m Am mode For more information on delta m Am mode see Performing Searches in Delta m Mode on page 110 Select the Disulfide check box if you know that the protein s cysteines are oxidized The ProSightPC application looks for only one disulfide bond In the Hit Filtering section set at least one of the following filters otherwise the ProSightPC application returns all protein forms that are searched even proteins that have no matching fragments a Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum number of matching ion fragments these protein forms are called hits Then specify the minimum number of matching ion fragments in the box to the right b Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum percentage of matching ion fragments Specify the percentage of matching ion fragments in the box to the right c Select the Min Score check box to determine whether the search algorithm finds only proteins with a p score that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right box See p Score on page 193 for more
64. in Figure 25 Thermo Scientific ProSightPC User Guide 4 2 Getting Started Processing LC MS MS Data Files Figure 25 Save Search Tree dialog box 11 If you created a new search tree type the name of the search tree and click OK If you made changes to an existing search tree a prompt box appears to confirm that you want to replace the existing search tree Click Yes 12 Click OK in the message box that appears The completed Running Highthroughput Logic page now resembles Figure 26 Figure 26 Completed Running Highthroughput Logic page of the High Throughput Wizard Running Highthroughput Logic Select a repository to load results to and select create a search tree with Repository yeast_topdown_etd y New Repository Search Tree Name yeast_topdown_etd X Save Experiment Filter Min fragments 6 Max fragments 500 W Min Intact Mass 750 Monoisotopic X 13 Click Next on the Running Highthroughput Logic page 42 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files A summary of the parameters that you have set appears as shown in Figure 27 Figure 27 Summary page of the High Throughput Wizard This is the summary of this job Input Files Processing raw files C Program Files ProSightPC Test Data ETDfraction03 raw Raw File Options Thrash Custom
65. mass 1 In absolute mass searches the queries all protein forms in a user defined window of the observed intact mass ProSightPC User Guide 113 4 Searching Databases Searching for Absolute Mass 114 ProSightPC User Guide MS MS O values O O The ProSightPC application finds all proteins in the database with intact mass within the tolerance of the search window For each protein it calculates all theoretical fragment ions It compares theoretical fragment ions with observed fragment ions It calculates its scores An observed ion matches a theoretical ion if the two masses are within a user defined tolerance The probability of the observed number of fragment ions matching by chance is then determined and reported as a p score For information on the calculation of the p score see p Score on page 193 Figure 51 shows this process graphically Figure 51 Absolute mass search process Database of known protein forms Observed data 0 O C O Oo P Score results C 9O C 8 O O O O IH i Use the following strategies when running absolute mass searches e Use a 1000 Da precursor search window search as the first search for an unknown protein If there are few modifications on the unknown protein not in the proteome warehouse a 1000 Da intact search will frequently identify but not characterize the protein A large number of ions matching one terminal in a protein is evidence of
66. methodology Thermo Scientific 1 Introduction to the ProSightPC Application Features Figure 4 Iterative searching in the ProSightPC application 20305 8 3 exp Iterative Searching Intact 10 Protein 20305 9 73 the Y63 14 At Yoo MS MS experiment y25 I 10 4586 36 2 9 human db all SNPs amp PTMs 5 ppm on fragment ions Predefined search Success Failure If passed load to the If failed run next Good category in search repository Condition rule E Value lt E 4 Repository of Results If the rule failed for all searches load to the Bad category in the repository for manual interpretation For a detailed explanation of the ProSightPC iterative search tree see Creating a Search Tree on page 37 The ProSightPC interface supports two levels of searching but advanced users can define a search tree with unlimited levels by editing the xml file that contains the search tree Database Manager The Database Manager provides a point and click environment for managing the proteome warehouse and repositories It imports and exports ProSightPC proteome warehouse PWF files enabling you to create your own proteome databases The PWF files are in a custom format that holds databases patches and repositories For details on the functionality of the Database Manager see Chapter 8 Using Proteome Databases Thermo Scientific ProSightPC User Guide
67. modification appears when you create searches Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Terminal Modification Editor Parameters Table 4 lists the parameters in the Terminal Modification Editor Table 4 Terminal Modification Editor dialog box parameters Parameter Description Modification Specifies the name of the modification for example Carboxymethyl cysteine Name Specifies the descriptive name of the terminal modification for example carboxymethyl cysteine Terminus Specifies the terminus to apply the modification to e N applies the modification to the N terminal of the sequence e C applies the modification to the C terminal of the sequence Monoisotopic Mass Specifies the monoisotopic mass of the chemical formula of the modification Average Mass Specifies the average mass of the chemical formula of the modification Fixed Modification Editor Parameters Table 5 lists the parameters in the Fixed Modification Editor Table 5 Fixed Modification Editor dialog box parameters Parameter Description Name Specifies the name of the modification Amino Acid Specifies the symbol of the amino acid being modified Monoisotopic Mass Specifies the monoisotopic mass of the chemical formula of the modification Average Mass Specifies the average mass of the chemical formula of the modification Description Briefly describes the modification Processi
68. most abundant isotope of the protein peptide or fragment ion ProSightPC User Guide 117 4 Searching Databases Searching for Absolute Mass Table 23 Absolute Mass Preferences page parameters Sheet 2 of 3 Parameter Description Fragment Mass Specifies the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Delta m Mode Determines whether the ProSightPC application conducts the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 Precursor Search Specifies the dimensions of the precursor search window of the observed intact ion mass in the selected units For intact ion masses the dimensions are always in daltons but for fragments they can be in daltons or parts per million Set the following parameters e Lower Sets the minimum value for a precursor search window that will not trigger an out of range warning e Default Sets the default value for a precursor search window e Upper Sets the maximum value for a precursor search window that will not trigger an out of range warning 118 ProSightPC User Guide Thermo Scientific 4 Searching Databases Searching for Absolute Mass
69. of the largest precursor ion First mz Mono First mz Avg Displays a column showing the monoisotopic mass to charge ratio m z value of the first precursor ion for each experiment Displays a column showing the average mass to charge ratio m z value of the first precursor ion for each experiment Largest mz Mono Displays a column showing the largest monoisotopic mass to charge ratio m z value of all precursor entries for each experiment ProSightPC User Guide 269 B Using the ProSightPC Interface The ProSightPC Interface 270 ProSightPC User Guide Table 73 Show Columns area parameters Sheet 2 of 3 Parameter Description Largest mz Avg Displays a column showing the largest average mass to charge ratio m z value of all precursor entries for each experiment Pending Search Displays a column indicating whether a search has been performed Successful Search Displays a column indicating whether a match in the database was found Matching Forms Displays a column showing the number of matching forms Best Expectation Displays a column with the best lowest expectation score of any match in the search results Best P Score Displays a column with the best lowest p score of any match in the search results For more information on the calculation of this score see p Score on page 193 Best PDE Displays a column showing the best highest
70. of the protein peptide or fragment ion In the Precursor Search Window box specify the tolerance that determines whether comparing an observed precursor mass to a theoretical precursor mass is considered a match and indicate whether it is expressed as absolute measured in daltons or relative measured in parts per million The window is one number For example if you type 10 and select Da the ProSightPC application queries 10 Da and 10 Da around the observed precursor for a total range of 20 Da The ProSightPC application queries all protein forms with a theoretical mass within this range In the Fragment Mass Type list select the type of fragment ion to search for e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Tolerance box specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicate whether it is expressed as absolute measured in daltons or relative measured in parts per million An observed fragment ion matches a theoretical fragment ion if the observed fragment ion mass is within plus or minus the fragment tolerance of the theoretical fragment ion mass Select th
71. on fragment map 163 moving between tiers 238 removing 183 RESID designation 199 searching for 122 133 150 156 sorting in PTM Tier Editor 236 specifying for all proteins 224 tiers 199 PUF files adding experiments to 94 building new experiment manually 260 with Profile algorithm 260 with Xtract algorithm 260 changing display in Experiment Manager 96 98 closing 245 copying experiments from one file to another 94 97 98 creating 92 97 245 259 deleting 97 displaying search information in data grid 261 exporting experiments to repositories 176 importing experiments from repository 175 importing into ProSightPC 7 27 35 91 importing mass spectral data from 2 input to High Throughput Wizard 29 input to ProSightPC 3 9 opening from Experiment Manager 93 97 from File menu 93 245 259 Grid Display Preferences page 266 last experiment 260 most recently opened 246 output by ProSightPC 9 purpose and contents 9 removing experiments from Experiment Manager 95 97 shortcut menu 95 removing from High Throughput Wizard 35 reverting to last saved version 96 97 saving 33 35 96 97 245 260 saving under other name 245 PWF databases 23 PWF files exported by Database Manager 5 218 imported into Database Manager 5 217 218 220 input to ProSightPC 9 output by ProSightPC 9 purpose and contents 9 Thermo Scientific Q Q Exactive mass spectrometers 1 Qual Browser 73 246 quick filters 271 Quick Filters s
72. on the complexity of the protein mixture For samples containing only a few proteins you can separate the proteins by gel electrophoresis or chromatography Enzymatic digestion breaks them down into smaller peptides with the aid of proteolytic agents such as trypsin or Lys C For complex samples containing many different proteins the proteins can be digested into peptides and then separated by several orthogonal methods before electrospray mass spectrometry ESI MS The left side of Figure 7 illustrates these two methods In either case these peptides are then introduced to the mass analyzer Figure 7 Comparing top down and bottom up proteomics Bottom up Top down Extract cellular proteins amp 9 ip FIRA al Shotgun digest NA Nar ya a G in i a Separate 242 gt lt 2 _ 2 B x _ SS 5 90 X y X a N ee C In top down proteomics electrospray ionization ESI or matrix assisted laser desorption ionization MALDI ionize intact proteins The proteins are then introduced into a mass analyzer where they are subjected to gas phase fragmentation The right side of Figure 7 illustrates this methodology Thermo Scientific ProSightPC User Guide 11 1 Introduction to the ProSightPC Application Introduction to Proteomics Top Down Proteomics Top down proteomics is a technique for protein identification and characterization Combining top down proteomics searches with the sh
73. one or more mass measurements of intact protein ions and the masses of one or more fragment ions that result from the disruption of those intact ions Although many ProSightPC search modes accept multiple intact masses associated with a fragment ion mass list performance improves when an MS MS experiment consists of a single intact ion mass and a corresponding list of fragmentation masses You must add complete MS MS experiments in the ProSightPC application You can add them to an existing PUF file or create a new PUF file for them You can also edit existing MS MS experiments The application queries each experiment against the ProSightPC proteome warehouse to identify and characterize the proteins A search is a predefined query against the ProSightPC proteome warehouse All experiments are associated with at least one search By defining searches in the search logic wizard or during importations you can use the ProSightPC application in a batch mode that facilitates high throughput proteomic research The ProSightPC tool suite consists of the ProSightPC application and a small number of secondary applications to aid in managing the proteome database and experimental results Thermo Scientific 1 Introduction to the ProSightPC Application Features The ProSightPC Application The ProSightPC application can process a large number of searches to assist you in protein and peptide identification and characterization for high resolution data I
74. or Tools gt Reports gt Repository Report or click the Repository Report icon H The Repository Report dialog box opens as shown in Figure 69 Figure 69 Repository Report dialog box Repository Report foes Repository PheATE_demo_repository V Category good Z Files C Program Files ProSightPC Test Data Carbonic anhydrase ca puf C Program Files ProSightPC Test Data ETDfraction03 ET Dfraction03 puf Export directly to file Report only best hit per search 2 From the Repository list select the name of the repository to generate a report for 3 Select the Category option and then select the name of the category from the list to the right of the option You assign a search category on the Running Highthroughput Logic page of the High Throughput Wizard For more information on selecting a search category see Creating a Search Tree on page 37 4 Optional To generate a report on the experiments in a specific file or files select the File option and then select the name of the file or files from the list to the right of the option or type the name of the file in the box When you use the High Throughput Wizard or the Export to Repository command or button the ProSightPC application automatically adds the names of the PUF files used to the Repository Report dialog box Because each file represents the data that you obtained from an instrument in a specific run
75. same search without delta m Am mode 110 ProSightPC User Guide Thermo Scientific 4 Searching Databases Performing Searches By carefully observing the pattern of fragments with and without delta m Am mode you can frequently locate the delta m For example if a particular result returns with the two smallest N terminal fragments matching without the delta but all other matching N terminal fragments contain the delta the unknown PTM must be on an amino acid between the second and third N terminal fragments As shown in Figure 49 the ProSightPC application first checks the observed precursor mass against the theoretical precursor masses of every protein in the specified precursor mass window and calculates and stores the mass difference delta m Next it doubles the theoretical fragment ion list for each protein for each b y or c z ion The ProSightPC application checks both the original fragment mass and the modified fragment mass plus the delta against the observed fragment ion mass list As a result the ProSightPC application returns any observed fragment ions having the same mass shift as the precursor protein as positive matches Figure 49 Schematic of Am mode Am 2 Da b btAm gt gt 9998Da y yeAm Am 1 Da 9999 D b btAm y ytAm 10000 Da AM 0 Da wi 10000Da gt P bem 2500 Da y y Am 7500 Da Observe
76. search_name the header reads Edit Predefined Search x 106 ProSightPC User Guide Thermo Scientific 4 Searching Databases Performing Searches Figure 48 Edit Predefined Search dialog box oe C man Search Name Demo Search Search Type Absolute Mass Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic Precursor Search Window 22 Da Fragment Mass Type Monoisotopic X Fragment Tolerance 15 ppm Am Mod F Disulfide e Hit Filtering v Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score J 0 Max Proteins to Return all ad Fixed Modifications Cysteine Methioni Lysine Isoleucine Leucine anm 4 D 0A00A Handling All PTMs E F High priority PTMs Tier 1 z 7 q Terminal Mods N Terminal Mod None C Terminal Mod None X 2 Edit the parameters in the dialog box See the following sections for more information e Absolute mass search See Searching for Absolute Mass on page 113 e Biomarker search See Searching for Biomarkers on page 125 e Sequence tag search See Searching for Sequence Tags on page 136 Thermo Scientific ProSightPC User Guide 107 4 Searching Databases Performing Searches e Single protein search See Chapter 6 Using the Sequence Gazer to Search for S
77. sequences in the database If Nis the number of protein forms considered during a search the e value currently reported by the ProSightPC application is e Nx p n Consider ubiquitin carboxyl terminal hydrolase 12 EC 3 1 2 15 This protein has 355 amino acids and a theoretical intact mass of 41201 daltons Consider a hypothetical MS MS experiment that results in 32 fragment ions of which n number matches this protein with a mass accuracy of plus or minus 2 5 daltons To calculate the p score of this assignment apply the equation shown in p Score on page 193 to find the sum of the Poisson distribution for i 0 to m 1 with lambda f x or 32 2 5 4 111 1 which is subtracted from 1 Figure 81 shows p m for all values of n between 0 and 32 As the number of matching fragments increases it becomes highly unlikely that the fragment ion matching is due to chance Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Figure 81 Poisson value versus Poisson value or greater Poisson Model Value vs Model Value or Greater Nh 2 5 f 32 Probability gt gt had gt gt gt gt e amp pos an a J oo o D 012 3 4 6 6 7 3 9 1011 12 13 1415 1617 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Number of Matching Fragnerts Poisson Value Poisson value or greater To find the p score for 12 matching fragments sum the first 11 values as follows 0 0
78. shown in Figure 22 on page 39 so that you can add a predefined search to your search Conditions 48 ProSightPC User Guide Opens the Condition dialog box shown in Figure 23 on page 40 so you can sets the conditions for the search Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 7 Running Highthroughput Logic page parameters Sheet 2 of 2 Parameter Description Success Specifies the action to take on the results that passed the condition e Load Loads the results to the selected category if the experiment passed the condition e Run Search Opens a second level search tree if the experiment passed the condition as shown in Figure 28 on page 44 The experiment will be re searched with the second level search and will later be loaded to the categories that depend on the conditions See Creating a Two Level Search Tree on page 43 for instructions on creating a two level search Category Specifies that the searches that pass the conditions set in the search tree be stored in the repository in that specific category Failure Specifies the action to take on the results that failed the condition e Load Loads the results to the selected category if the experiment failed the condition e Run Search Opens a second level search tree if the experiment failed the condition as shown in Figure 28 on page 44 The experiment will be re searched with the second level search and will later be
79. single user defined amino acid sequence Setting Single Protein Search Preferences When you add new single protein searches you can set the default values on the Single Protein Preferences page of the Options dialog box For more information on single protein searches see Using the Sequence Gazer to Search for Single Proteins on page 183 To set single protein search preferences 1 Click Single Protein in the Options dialog box The Single Protein Preferences page of the Options dialog box opens as shown in Figure 59 Thermo Scientific ProSightPC User Guide 143 4 Searching Databases Searching for Single Proteins 144 ProSightPC User Guide Figure 59 Single Protein Preferences page of the Options dialog box Options General i 7 Grid Columns Single Protein Preferences Thrash Te E E ee E ech ela eed ede 27 Search Parameters E o Default Single Protein Mode Search Parameters 2 Sequence Tag Precursor Mass X E Fragment Mass Monoisotopic X Delta m Mode o Lowe Defau Uppe Fragment Tolerance 10 100 ppm v In the Precursor Mass box specify the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Frag
80. specified and with an intact mass tolerance within the number of ppm specified 180 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report Demonstrating Repository Report Generation The following demonstration shows you how to generate a repository report filter its data and save the data to an Excel file Click the button below to view the demonstration To enlarge the demonstration once you start it right click and choose Full Screen Multimedia Thermo Scientific ProSightPC User Guide 181 ee i Using the Sequence Gazer to Search for Single Proteins This chapter describes how to use the Sequence Gazer to perform single protein searches You can fit any MS MS experiment data to a single protein and hypothesize various permissible PTMs As you test these different hypotheses you can save the results The ProSightPC application stores each saved result as a single protein mode search result Contents e Sequence Gazer e Accessing the Sequence Gazer e Navigating the Sequence Gazer e Demonstrating the Sequence Gazer Sequence Gazer The Sequence Gazer is an interactive environment for comparing MS MS data to a known protein sequence The Sequence Gazer characterizes previously identified proteins by selectively adding or removing PTMs or custom masses to amino acids in a protein sequence Once you have made all your modifications to the amino acids you can reevaluate t
81. the precursor scan a From the Fragmentation MSz Analysis Level list select the level of analysis that includes your fragmentation data e ms2 For data dependent LC MS MS experiments e ms3 For ion trap marching experiments b To specify the start of the chromatographic time range in which to analyze the data select the Specify Start Time check box and select the start time in the box underneath it This value is the start of the first scan The default is 10 minutes c To specify the end of the chromatographic time range in which to analyze the data select the Specify End Time box and select the end time in the box underneath it This value is the end of the first scan The default is 80 minutes If you do not specify a time range every scan in the RAW file is analyzed d Inthe Mass Tolerance check box specify an m z tolerance that determines which scan filters are summed together If the mass and retention time is within the tolerance the ProSightPC application combines the scan filters ProSightPC User Guide 53 2 Getting Started Processing LC MS MS Data Files The minimum value is 0 01 and the maximum value is 1 0 m z The default is 0 05 m z e In the Retention Time Tolerance box specify a retention time tolerance in minutes that determines which scan filters are summed together You can specify a value of at least 0 1 minutes and there is no maximum value The default is 2 0 minutes f Ifyou
82. the C terminus Click Save The new search appears in the data grid with yes appearing in the Pending Search column To execute the search from the data grid right click the pending search and then choose Run Search number Thermo Scientific 4 Searching Databases Performing Gene Restricted Searches New Search in Experiment X Dialog Box Parameters for Absolute Mass Table 30 lists the parameters in the New Search in Experiment X dialog box for gene restricted absolute mass shown in Figure 61 on page 148 Table 30 New Search in Experiment X dialog box parameters for absolute mass Sheet 1 of 2 Parameter Description Search Type Specifies the type of search to perform e Absolute Mass See Searching for Absolute Mass on page 113 e BioMarker See Searching for Biomarkers on page 125 e Sequence Tag See Searching for Sequence Tags on page 136 e Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins e Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 e Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Database Description Describes the database that you want to search Precursor Mass Type Specifies the type of precursor ion mass to use e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment i
83. the data grid For more information on removing experiments see Removing an Experiment from the Data Grid on page 110 Table 70 describes the command on the secondary data grid shortcut menu which appears when you right click the area to the right of the columns in the data grid as shown in Figure 112 on page 262 Table 70 Data grid secondary shortcut menu Command Description Columns Determines the columns that appear in the data grid The job queue shown in Figure 113 displays the status of any previously run or currently running searches in the ProSightPC session While a search is running a status bar displays the progress of that search Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface Figure 113 Job queue Name Status Notes Retriever 1 4 Aborted Aborted Retriever 1 4 Aborted Aborted Retriever 1 4 Running Retriever 1 5 Waiting Processing es To display the job queue pane e Choose View gt Job Queue To rerun a search in the job queue e Ifa job in the job queue has finished but you want to rerun it right click the search in the job queue and choose Run from the shortcut menu To cancel a search in the job queue e Ifa search is running right click the search in the job queue and choose Abort or click the Abort Running Job icon gt in the toolbar The search ends and the status changes to Failed e Ifyou want to cancel
84. the selected ProSightPC user manual Table 65 lists the commands on the data grid shortcut menu which appears when you right click an experiment in the data grid Table 65 Data grid shortcut menu commands Sheet 1 of 3 Command Description Refresh Grid Redisplays the contents of the data grid Mark Marks an experiment by placing the ProSightPC symbol to the left of the experiment and an asterisk in the Marked column This mark can differentiate a particular experiment Append Predefined Searches Thermo Scientific Opens the Append Predefined Searches to Experiment X dialog box shown in Figure 47 on page 104 so you can add more than one predefined search to an experiment For information on how to select options in this dialog box see Adding Predefined Searches to an Experiment on page 104 ProSightPC User Guide 253 A ProSightPC Reference Data Grid Shortcut Menu Table 65 Data grid shortcut menu commands Sheet 2 of 3 Command Append Predefined Search Description Opens a submenu with all of the predefined searches Clicking one of them adds it to the selected experiment Edit Search x Edit Mass List Opens the Edit Search in Experiment X dialog box for that type of search this dialog box is the same as the New Search in Experiment X dialog box for that search type For information on how to edit a search see Editing a Predefined Search on page 105 This command is o
85. want the ProSightPC application to process only fragmentation data from Thermo Scientific Fourier Transform instruments select the Analyze Only FTMS Fragmentation option or If you want the ProSightPC application to process fragmentation data from Thermo Scientific Fourier Transform and ion trap instruments select the Analyze Ion Trap and FTMS Fragmentation option You can select either the Analyze Only FI MS Fragmentation parameter or the Analyze Ion Trap and FTMS Fragmentation parameter When you select one parameter the other parameter is automatically cleared 3 In the Precursor Selection Options area specify the parameters for analyzing precursor ions a In the Minimum S N box enter the lowest signal to noise ratio that the algorithm considers when trying to assign neutral mass to a charged mass to charge ratio m z species while analyzing precursor ions The minimum value is 1 and there is no maximum value The default is 7 b In the Maximum Charge box enter the maximum charge to be used by the algorithm The minimum value is 1 and there is no maximum value The default is 40 c Inthe Minimum Fit box enter the minimum fit parameter used by the Xtract algorithm Xtract only The minimum value is 0 and the maximum value is 100 The default is 40 d Inthe Minimum RL box enter the minimum confidence level THRASH only The minimum value is 0 and the maximum value is 1 0 The default is 0 90 e In th
86. whether the ProSightPC application conducts the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 Fragment Tolerance Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a fragment tolerance that does not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that does not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm Performing Gene Restricted Searches Gene restricted searches look at all protein forms of explicitly listed genes They can only be made from the results or match list of a previously completed absolute mass biomarker or sequence tag search Gene restricted searches are most often performed with a sequence tag search to form a hybrid search The ProSightPC application automatically generates a gene ID list from the results of a previous search Gene restricted searches consist of two functionally different but closely related types of searches e Gene restricted absolute mass e Gene restricted biomarker mass Searching for Gene Restricted Absolute Masses 146 ProSightPC User Guide U
87. z value of the precursor ion b In the Charge State box type the charge state z to assign to the mass to charge m z data found in the data files c Click OK 12 In the Fragmentation Method list select one of the following fragmentation methods e CID e ECD e ETD e HCD e IRMPD For information on these methods see Fragmentation Methods on page 9 13 Optional In the Predefined Search box add a predefined search to the new MS MS experiment by selecting the check box next to the search name 14 Click OK You can change the THRASH default preferences by using the Options dialog box Build Experiment from Profile RAW Data Dialog Box Parameters Table 18 lists the parameters in the Build Experiment from Profile RAW Data dialog box shown in Figure 40 on page 81 Thermo Scientific ProSightPC User Guide 81 2 Getting Started Importing Targeted RAW Files ProSightPC User Guide Table 18 Build Experiment from Profile RAW Data dialog box parameters Sheet 1 of 2 Parameter Description RAW File to Be Specifies the name of the RAW file to import THRASHed Minimum Specifies the lowest signal to noise ratio that the THRASH Signal to Noise Ratio algorithm will consider when trying to assign neutral mass to data in your RAW files Maximum Mass Specifies the cutoff point for the THRASH algorithm when searching for masses First m z Specifies the lowest mass to charge ratio m z value consider
88. 0 Add Gene Restricted Search icon 147 153 Advanced Settings dialog box 35 parameters on 59 THRASH 53 Xtract 53 Advanced Settings processing option 35 Amino Acid Information box 199 amino acids adding PTMs to 198 238 251 adding virtual PTMs to 199 fixed modifications in absolute mass searches 122 in biomarker searches 133 in gene restricted absolute mass searches 150 in gene restricted biomarker searches 156 in sequence tag searches 141 in single protein searches 187 isobaric 141 number in protein form 163 pairs 136 reevaluating ion data 6 183 removing PTMs from 199 sequences 242 244 analysis to infer mass AIM 12 73 246 Append Predefined Searches to Experiment X dialog box 104 105 184 248 253 263 Thermo Scientific aspartic acid 196 averagine pattern 56 57 62 63 b fragment ions displayed in Show Columns section 270 in CID HCD and IRMP analysis 207 in Font Converter 243 244 in interactive fragment map 198 returned by Fragment Predictor 6 238 241 batch mode 252 batch mode searches 111 Batch Run icon 112 bc ions in Font Converter 244 best hit reports 165 168 252 Biomarker Preferences page 127 129 biomarker searches methodology 125 parameters for 134 performing 130 precursor search tolerance 126 results list 162 setting default values for 127 steps performed in 125 strategies used in 126 bottom up databases See middle down bottom up databases bottom up experiments 1 Build Experiment from Post Xtrac
89. 0ccs4 iopesastiavad ee das ove 165 Viewing the Results in a Repository Report 2 0 0 c ee eee eee eee 168 Display Columns in the Repository Report 0 6 2 0 00 eee eee eee 173 Repository Report Dialog Box Parameters 00000 eee eee eee 174 Using the Repository Report To Import Experiments from a Repository into the PUP Pilea ca teenie eae nae 175 Exporting Experiments to a Repository 06 0 cece eee eens 176 Exporting Experiments to an Excel Spreadsheet 0 2 00004 177 Applying Filters to Repository Report Data 0 00 cece eee eee t77 Demonstrating Repository Report Generation 00000000 181 Chapter6 Using the Sequence Gazer to Search for Single Proteins 183 Sequence Gazette 4 0 41Gieal sna alee espe Oa aa Sade eRe 183 Accessing the Sequence Gazer cas 6 canes Saeteanes Aol oie eae Sd Be esas Pees 184 Searching for Single Proteins and Accessing the Sequence Gazer 184 Identifying a Protein and Accessing the Sequence Gazer 187 New Search in Experiment X Dialog Box Parameters for a Single Proteine diare adere wag lao Hila ale Sle SiR Mw tne Sods whe evade ar dls 189 Navigating the S quence Gazer cick Sah area es ee ae EN 190 Search Parameter Display reti sprecare yprenti Seach enVeeak edie ks 191 COLES DOM ats thts usin b E EAEE a Ale aca atin Oa cen wien we tea ele whee ge 193 Fragments Explained Doxa nad tater titer nates teh As 197 Mass Di
90. 12428 623 13156 2199 4 13 06 1244 51595 623 26525 1272 6 13 06 1243 61129 622 81292 974 8 13 06 1241 72878 621 87167 2232 2 13 06 1249 43234 625 72345 1964 1 2502 23830 626 56685 186900 7 625 23826 626 24554 1053 0 629 25930 630 26657 3176 8 629 23987 630 24715 2670 7 1256 60220 629 30838 2373 4 1255 56226 628 78840 1889 8 628 24892 629 25620 1092 3 1259 61607 630 81531 1334 8 630 16128 631 16856 857 7 1264 58001 633 29728 1427 8 2525 28362 632 32818 2406 5 658 25446 659 26174 2155 5 1315 62380 658 81918 1429 0 1977 67508 660 23230 4424 6 2644 29794 662 08176 34522 5 664 30459 665 31187 3359 1 1327 76041 664 88748 6162 6 665 24714 666 25442 8875 8 666 32679 667 33407 11126 8 665 36896 666 37623 4714 2 3688 72984 923 18974 16092 7 825 27227 826 27954 4065 9 1651 88663 826 95059 44181 8 827 18883 828 19610 6921 9 2480 05961 827 69382 17459 4 826 26679 827 27407 2930 7 3317 44753 830 36916 14474 3 2491 34380 831 45521 8397 8 832 27181 833 27908 10545 1 835 36520 836 37248 4099 1 625 00154 626 00882 21759 4 625 33595 626 34323 25604 7 1250 52981 626 27218 3397 2 627 12798 628 13526 5060 6 1252 54509 627 27982 1843 4 To change the columns in the repository report Select or clear the check boxes in the Columns to Display area in the lower right corner of the tab controller To change the order of the columns in the repository report e Select the column hea
91. 2 In the Database list select the name of the database to search 3 In the Fragment Mass list select the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion 4 In the Minimum Tag boxes specify the minimum tag score for protein forms matched in a sequence tag search The ProSightPC application scores any sequence found containing one or more of the sequence tags and reports any sequence scoring above this defined minimum tag score Set the following parameters e Lower Sets the minimum value for a minimum tag score that does not trigger an out of range warning Thermo Scientific ProSightPC User Guide 137 4 Searching Databases Searching for Sequence Tags e Default Sets the default value for a minimum tag score e Upper Sets the maximum value for a minimum tag score that does not trigger an out of range warning 5 In the Compiler Tolerance in ppm boxes enter the permissible error measured in ppm between two fragment ion masses that are still considered as matching an amino acid Set the following parameters e Lower Sets the minimum value for a compiler tolerance that does not trigger an out of range warning e Default Sets the default value for
92. 20 ProSightPC User Guide Thermo Scientific 2 Getting Started Closing the ProSightPC Application Closing the ProSightPC Application Save your changes before you exit the ProSightPC application because it does not prompt you To exit the ProSightPC application e Choose File gt Exit Setting Default Options You can use the General Preferences page of the Options dialog box to set default values for options that are available throughout much of the ProSightPC interface To set general preferences 1 Choose Tools gt Options to open the Options dialog box shown in Figure 14 2 From the left pane of the Options dialog box click the General folder to open the General Preferences page shown in Figure 14 Figure 14 General Preferences page of the Options dialog box amp Options E E Search Parameters Decimal Precision to Display l Maximum Hits to Display 3 Maximum Hits to Calculate 2000 ar ae Foes PDE Score P Score 3 In the Decimal Precision to Display box specify the number of decimal places to display most numbers in Thermo Scientific ProSightPC User Guide 21 2 Getting Started Setting Default Options 4 In the Maximum Hits to Display box specify the number of matching fragment tables to be displayed in the Data Manager Only the best matches up to this number are displayed 5 In the Maximum Hits to Calculate box specify the maximum number of protei
93. 30000 11 oO 11 85 700000 3 73E 09 gt gt CALM_HUMAN P62158 Calmodulin CaM Type conflict Signal Peptide false Propep false A D Q L T E E Q I A E F KtE A F S L F D KtD GtD GtT I T T K 188 ProSightPC User Guide Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer New Search in Experiment X Dialog Box Parameters for a Single Protein Table 37 lists the parameters in the New Search in Experiment X dialog box for a single protein shown in Figure 78 on page 186 Table 37 New Search in Experiment X dialog box parameters for a single protein Sheet 1 of 2 Parameter Description Search Type Specifies the type of search to perform e Absolute Mass See Searching for Absolute Mass on page 113 e BioMarker See Searching for Biomarkers on page 125 e Sequence Tag See Searching for Sequence Tags on page 136 e Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins e Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 e Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Precursor Mass Type Specifies the type of precursor ion mass to use e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average
94. 42 Matching fragments table parameters Sheet 1 of 2 Parameter Description ID Displays a unique in each MS MS experiment ProSightPC assigned numerical identification of the matching fragment Name Displays a name consisting of the ion type followed by the ProSightPC User Guide terminal amino acid number Thermo Scientific Non Matching Fragments Table Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Table 42 Matching fragments table parameters Sheet 2 of 2 Parameter Description m z type Displays the mass to charge ratio m z value corresponding to the fragment ion The type is monoisotopic or average depending on which you selected during the last rescoring Mass type Displays the observed mass of the fragment ion measured in Da The type is monoisotopic or average depending on which you selected during the last rescoring Theoretical Mass Displays the mass of the corresponding theoretical fragment ion Error Da Displays the difference between the fragment mass and the theoretical fragment mass measured in Da Error ppm Displays the difference between the fragment mass and the theoretical fragment mass measured in ppm Am Displays the word True if the corresponding fragment is a match considering the delta Am mode The non matching fragments table shown in Figure 84 lists every fragment that does not match the sequence
95. 5 1 Introduction to the ProSightPC Application Features Data Manager Sequence Gazer The Data Manager is part of the ProSightPC graphical user interface and provides a visual representation of all the information related to a single MS MS experiment Use it to view all information for a single experiment You can use its context sensitive controls to determine what information is displayed For more information on the Data Manager see Chapter 7 Displaying Data in the Data Manager The Sequence Gazer in the ProSightPC graphical user interface is an interactive environment for comparing MS MS data to a known protein sequence The Sequence Gazer characterizes previously identified proteins by selectively adding or removing PTMs or custom masses to amino acids in a protein sequence Once you have made all your modifications to the amino acids you can reevaluate the ion data You use the Sequence Gazer to test hypotheses regarding which PTMs are present You can also use it to fully characterize a protein Chapter 6 Using the Sequence Gazer to Search for Single Proteins explains how to perform single protein searches by using the Sequence Gazer Experiment Manager PTM Tier Editor The Experiment Manager provides a simple interface for managing multiple MS MS experiments in PUF files For more information about the Experiment Manager see Chapter 3 Working with Experiments The ProSightPC application groups all PTM
96. 56118597 0 161637722 0 232781598 0 223492684 0 160930825 0 092705426 0 044503055 0 018311659 0 006592857 0 002109925 0 000607719 0 000159128 0 999951 Then subtract 0 99951 from 1 000000 1 000000 0 99951 4 9E 5 Thermo Scientific ProSightPC User Guide 195 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Therefore the probability of 12 or more fragments matching by chance out of a fragment ion list with 32 masses and a tolerance of plus or minus 2 5 daltons is 4 9E 5 PDE McLuckey Score 196 ProSightPC User Guide The ProSightPC application also reports a score calculated according to McLuckey The McLuckey or PDE score is a way of scoring how well a set of observed fragment ions matches a protein It takes into consideration which amino acids would have to have been cleaved in order to match the observed fragment ion data with the theoretical ion masses from the database To find this score use the following equation McLuckey_score SnPX Ip T SnDX Ip 4nK Iy 2nEY Ig F nX Iy This equation includes the parameters nP nD nK nE nX In Specifies the number of product ions predicted with cleavage at an N terminal to a proline Specifies the number of product ions predicted with cleavage at a C terminal to an aspartic acid Specifies the number of product ions predicted with cleavage at a C terminal to a lysine Spe
97. 6061 0 2786 26 0 0 1114 344874 0 2150 27 0 0 1114 389972 0 2307 28 0 0 1114 603336 0 1406 29 0 0 1115 935301 0 2618 30 ProSightPC User Guide 209 7 Displaying Data in the Data Manager Editing Mass Values 210 ProSightPC User Guide Table 44 lists the parameters and icons at the top of the Edit Masses experiment_number page Table 44 Edit Masses experiment_number page parameters and icons Parameter ld Description Saves the edits that you made to the mass values Does not save any of the edits that you made to the mass values closes the Edit Masses experiment_number page and returns you to the Data Manager Fragmentation Method Specifies the fragmentation method For more information on fragmentation methods see Fragmentation Methods on page 9 The mass values are displayed in a series of columns in two areas Precursor Mass List and Fragment Mass List on the Edit Masses experiment_number page Table 45 lists the columns in the Precursor Mass List area Table 45 Precursor Mass List area parameters Parameter mz_monoisotopic Description Specifies the monoisotopic mass to charge ratio m z value of the precursor ion mz_average Specifies a column showing the average mass to charge ratio m z value of the precursor ion mass_monoisotopic Specifies the monoisotopic mass of the precursor ion Miass_average Specifies the average mass of the precursor ion
98. 64 Sequence Gazer accessing 184 190 accessing from any identified protein 187 accessing from single protein searches 184 adding virtual PTMs to amino acid 199 Amino Acid Information box 199 demonstration of use 203 evalue 194 features of 190 Fixed Modifications box 199 fragment ion information 190 Fragments Explained box 197 interactive fragment map 198 mass diagram 198 matching fragments table 200 non matching fragments table 201 p score 193 PDE McLuckey score 196 PTMs available to 235 purpose 6 183 Scores box 193 search parameter display 191 sequence tag score 197 window 191 Sequence Tag Preferences page 136 138 sequence tag score 138 139 140 197 sequence tag searches methodology 136 parameters for 142 performing 139 setting default values for 136 steps performed in 136 strategies used in 136 sequence variants 2 213 shotgun annotation database construction 13 database searching 14 definition 2 effect on new database creation 221 288 ProSightPC User Guide example 226 example sequence 15 formats of databases 220 in proteome databases 214 place in workflow 7 PTMs available for 221 purpose 13 213 Show Columns section 267 side arrows 205 signal to noise ratio 61 Single Protein Preferences page 143 145 single protein searches accessing Sequence Gazer 187 purpose 143 setting default values for 143 using Sequence Gazer 6 164 183 184 See also Sequence Gazer small icons 96 SNPs 2 213 225 231 splice varia
99. 6754 1 3 4 1 99 143 Statistics table Take to Sequence Gazer RESID SEQ De am a a ey fey a ee ee ey ee SS eee ee R ey SS R Se ee es ee Table 32 Result table elements Parameter Description Description Briefly describes the protein or peptide isoform Fragment Map Graphically represents the protein isoform showing the location of PTMs and matching fragment ions Statistics Table Organizes information relating to the search The statistics table is subdivided into the display elements shown in Table 33 Table 33 Result statistics table elements Sheet 1 of 2 Parameter ID Gene Description Displays the internal identifier for the protein form and the gene identifier Length Mass Mass Difference Displays the number of amino acids in the protein form Displays the theoretical precursor mass of the protein form Displays the observed mass minus the theoretical mass PPM Difference Displays the mass difference in parts per million N terminal Ions Displays the total number of matching N terminal ions C terminal Ions Displays the total number of matching C terminal ions Other ions Displays the ions that match the candidate sequence Total Ions Displays the total number of matching ions PDE Score Thermo Scientific Displays the PDE McLuckey score For information on this score see PDE McLuckey Score on page 196 ProSightPC User Guide 163
100. 7 Adds the sign for a b ion Adds the sign for az ion Adds the sign for ac ion Adds the sign for a bc ion Adds the sign for a yz ion L J Adds the sign for a y ion 244 ProSightPC User Guide Thermo Scientific es i ProSightPC Reference This appendix describes the commands on the ProSightPC menus They are listed in the order in which they appear on the menus Contents e File Menu e Edit Menu e View Menu e Experiment Tools Menu e Databases Menu e ProSightHT Menu e Tools Menu e Help Menu e Data Grid Shortcut Menu File Menu Table 57 lists the commands on the File menu Table 57 File menu commands Sheet 1 of 2 Command Description File gt New Clears the data grid so that you can create a new PUF file File gt Open Opens an existing PUF file File gt Close Closes a PUF file File gt Save Saves a PUF file File gt Save As Saves a PUF file under another name Thermo Scientific ProSightPC User Guide 245 A ProSightPC Reference File Menu Table 57 File menu commands Sheet 2 of 2 Command File gt Import Data from Repository Description Imports experiments from a repository into the ProSightPC application so that you can perform operations on them such as adding or changing searches or using the Sequence Gazer It activates the Import Data from Repository dialog box shown in Figure 35 on page 70 File gt Export Data to Re
101. 97 on page 224 Table 50 Initial Methionines page parameters Sheet 1 of 2 Parameter Description Ensure Initial Determines whether each isoform generates two forms for each Methionine Cleavage N terminal sequence one where the N terminal methionine is present and one where it is cleaved off e Default Selected Each isoform generates two forms for each N terminal sequence e Cleared Each isoform generates only the form where the N terminal methionine is present 230 ProSightPC User Guide Thermo Scientific 8 Using Proteome Databases Creating a Proteome Database Table 50 Initial Methionines page parameters Sheet 2 of 2 Parameter Apply N Terminal Acetylation Description Determines whether the ProSightPC application adds acetylation to N terminal methionines whenever it is possible regardless of whether the input includes it e Default Selected Adds acetylation to N terminal methionines e Cleared Does not add acetylation to N terminal methionines Apply N Terminal Formylation Complexity Page Parameters Determines whether the ProSightPC application adds formylation to N terminal methionines e Selected Adds formylation to N terminal methionines e Default Cleared Does not add formylation to N terminal methionines Table 51 lists the parameters on the Complexity page of the Create New Database Wizard shown in Figure 98 on page 225 Table 51 Complexity page parameters Paramete
102. A biomarker search is a two step process that is repeated for each base protein sequence in the proteome database 1 Identify a candidate entry matching an observed precursor mass 2 Calculate all possible theoretical fragment ions for the candidate entry then compare the theoretical fragment ion masses to the observed fragment ion masses Figure 54 shows the process involved in a biomarker search ProSightPC User Guide 125 4 Searching Databases Searching for Biomarkers 126 ProSightPC User Guide Figure 54 Biomarker searches Intact precursor mass See ea Ea Protein sequence database Sliding mass window FO Top down fragments Peptides within tolerance Query proteome subset Proteome subset Identified characterized polypeptide In a biomarker search the precursor search tolerance is an estimate of measurement error on the observed precursor mass The value is usually small compared to an absolute mass precursor search window Thermo Fisher Scientific recommends the following when you conduct biomarker searches Use a biomarker search if an absolute mass analysis fails to identify a protein The default biomarker search searches only for the basic forms with no known modifications If you want to search for modified forms you must select the Include Modified Forms check box in the New Search in Experiment X dialog box for biomarkers see Figure 56 on page 131 However searching for modified forms in
103. Biomarkers on page 152 Describes the database that you want to search Compile Sequence Tags Determines the sequence tags and compiles them before searching them This option is the default Compiler Tolerance in ppm Specifies the permissible error measured in ppm between two fragment ion masses that are still considered matching an amino acid Minimum Tag Size Specifies the lowest acceptable sequence tag score reported as a match Fragment Mass Type Fixed Modifications 142 ProSightPC User Guide Specifies the mass type of the fragment ions to use Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Specifies the chemical modifications present on all instances of a given type of amino acid in the observed protein Thermo Scientific 4 Searching Databases Searching for Single Proteins Table 28 New Search in Experiment X dialog box parameters for sequence tags Sheet 2 of 2 Parameter Description Manually Enter Select to enter sequence tags that you have determined possibly from manually analyzing a spectrum and to search them Save Saves the search information Searching for Single Proteins Single protein searches match MS MS data against a
104. Data Manager For information on this procedure see Performing Gene Restricted Searches on page 146 To enter and save information specific to the search e Click Edit Comment see Figure 63 on page 162 A box opens so that you can type your comments as shown in Figure 65 164 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Search Report Figure 65 Edit comment box aA Fragmentation Method ETD Ion Type CZ gt Precursor Mass List gt Fragment Mass List gt Search 1 Absolute Mass Search w Search 2 BioMarker Search Edit Comment Viewing the Results in a Search Report The ProSightPC application provides several batch processing and reporting tools for managing large numbers of MS MS experiments They simplify working with several experiments in a single PUF file This section describes how to use these tools to manage multiple experiments The following types of reports help you summarize your work e A status report gives a summary of every search in the open PUF file including search type and best score e A printable search report contains all of the information related to one search formatted for easy printing e A best hit report displays the search result with the best score for each search that was run for each experiment in the data grid e A repository report lists all the experiments that a repository contains For information on this report s
105. Data Manager in the ProSightPC graphical user interface Contents e Data Manager e Opening a Data Manager Window e Closing a Data Manager Window e Adding or Editing an Experiment Comment e Editing Mass Values e Running a Pending Search Data Manager The Data Manager shown in Figure 85 provides a visual representation of all the information related to a single MS MS experiment It appears when you double click an experiment in the data grid You can use it to view all information for a single experiment The context sensitive controls help you determine what information is displayed Figure 85 Data Manager Data Management for Experiment 1 Source C Share ProSightPCData Carbonic anhydrase CA_ETD1117_8ms_XT_00001_M_ raw Scan 2 29006 7 Scan Number 2 Retention Time 1 min Fragmentation Method ETD gt Precursor Mass List gt Fragment Mass List gt Search 1 Single Protein Search gt Search 2 Absolute Mass Search Each side arrow indicates hidden information related to a search Clicking the side arrow reveals the information by turning the side arrow down as shown in Figure 86 Thermo Scientific ProSightPC User Guide 205 7 Displaying Data in the Data Manager Data Manager Figure 86 Information revealed by down arrow in the Data Manager Data Management for Experiment 23 Source C Program Files ProSightPC data Example Raw Files PheATE_126kDa_Trypsin_Hi_Hi_Top3 raw 1247 6188 Pre
106. Displays the number of matching search results with the best e value For example if an experiment had five matching search results the best search result is the one that received the best lowest score For more information on the e value see Expectation Value e value on page 194 P Score Displays the p score of the best search result in a search For more information on the p score see p Score on page 193 Seq Tag Score Displays the sequence tag score of the best search result in a search You see a score only if the search type is sequence tag Repository Report Dialog Box Parameters 174 ProSightPC User Guide Table 36 lists the parameters in the Repository Report dialog box shown in Figure 69 on page 169 Table 36 Repository Report dialog box parameters Sheet 1 of 2 Parameter Description Repository Specifies the name of the repository to generate a report for Category Specifies the category of experiments in the repository to generate a report for Files Specifies the files to include in the report Select All Selects all the listed files This button is not available unless you select Files Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report Table 36 Repository Report dialog box parameters Sheet 2 of 2 Parameter Description Unselect All Clears all the listed files This button is not available unless you select Files T
107. E 64 MQIFVKTLT absolute_mass 1 92E 67 51 STLLKS absolute_mass 5 9E 100 51 STLLKS absolute_mass 5 73E 83 51 STLLKS absolute_mass 821E 18 51 STLLKS absolute_mass 7 61E 80 MQIFVKTLT absolute_mass 1 58E 11 MQIFVKTLT absolute_mass 524E 19 VSQLFEEK absolute_mass 274E 12 VSQLFEEK absolute_mass 276 85 MKYLAAYL absolute_mass 276 85 MKYLAAYL absolute_mass 456 MKYLAAYL absolute_mass 1 59E 71 MKYLAAYL absolute_mass 1 59E 71 MKYLAAYL absolute_mass 7 6E 80 MKYLAAYL absolute_mass 7 6E 80 MKYLAAYL absolute_mass 716 absolute_mass 2 95E 10 absolute_mass 1 79E 46 O ROD oO PO fm s2eRgrr ot oe adag OOOO LELELLELLLLLLLLEL ELLE SEER EE EEEEE BESNIJO Thermo Scientific ProSightPC User Guide 171 5 Viewing Search Results Viewing the Results in a Repository Report Figure 71 Repository report exported directly to a text file End Scan Number Start RT min End RT min Monoisotopic Mass Da Monoisotopic m z 1 36 1 51 2133 82100 1067 91777 582 4 1 36 1 51 1072 17572 1073 18300 6078 2 1 36 2 91 2142 87281 1072 44368 4208 6 1 36 1 51 1073 46172 1074 46900 464 2 11 69 22 27 797 11899 798 12627 613 3 11 69 12 27 796 17017 797 17745 922 8 22er 1598 21869 800 11662 6070 5 12 27 1598 73176 800 37315 490 6 32 27 803 40969 804 41696 2798 8 12 27 1608 66523 805 33989 734 8 13 06 622
108. Figure 36 Figure 36 Export Data to Repository dialog box Please select a project file and any number of experiments to export to the repository 6 Click OK For information on exporting experiments to a repository by using the repository report see Exporting Experiments to a Repository on page 71 You can also right click an experiment in the data grid and choose Export Experiment to Repository from the shortcut menu to export it back to the same repository from which it was imported without specifying the repository name project name and file name Thermo Scientific ProSightPC User Guide n 2 Getting Started Importing Targeted RAW Files Export Data to Repository Dialog Box Parameters Table 13 lists the parameters in the Export Data to Repository dialog box shown in Figure 36 on page 72 Table 13 Export Data to Repository dialog box Parameter Description Experiments Lists all the experiments in the selected RAW or PUF file so that you can select the experiments that you want to export to a repository Select All Selects all the experiments in the selected file for importation Repository Specifies which repository to export the experiments to Category Specifies the category in the repository to export the experiments to The categories available in the list include the default good and bad categories as well as any that you defined Set N
109. ITTKELGTVMRSLGQNPTEAELQDM INEVD Enter your sequence using single letter abbreviations You can use two methods of entering a protein sequence in the Protein Sequence box e Manually enter a protein sequence e Copy and paste a protein sequence from another source Note You can access the sequence from any successful search by clicking RESID or SEQ in the Data Manager You can also acquire the protein sequence from external sources 3 Click Continue after you enter the sequence in the area provided The Fragment Predictor displays a new window showing the protein sequences in an interactive sequence map as shown in Figure 104 Thermo Scientific ProSightPC User Guide 239 9 Using ProSightPC Tools Viewing Fragments lons with the Fragment Predictor Figure 104 Interactive sequence map in the Fragment Predictor window Gia Display Preferences Experiment 1 Fragment Predictor Fragment Predictor Alanine Information JH D Q L T E E Q 1 A E F K E A S L F D K D G D G T N 1C I T T K E L G T V M R S L G Q N P T E A E L Q D M amino Acid A RESID none I N E V D Start PTM None PTM Choices Get Fragments None Custom p Tier 1 E Trimethylation a Acetylation HB Methylation mono 4 Click the sequence to select an amino acid A black box around an amino acid indicates that it is selected For each selected amino acid common PTMs appear in
110. K To export a repository Click the Export Repository icon 1 in the toolbar in the middle of the Database Manager window In the Export dialog box shown in Figure 93 on page 218 select the database to export To combine multiple databases into a single export file hold down the CTRL key and select additional databases Type a destination PWF or browse to it Select a compression level by typing an integer ranging from 9 slow export but small file size to 0 fast export but large file size This number controls the final PWF size Click Export to execute the procedure and create a new PWF When the Export Complete message box appears click OK Removing a Proteome Database or Repository You can remove unwanted proteome databases and repositories from the proteome warehouse by using the Database Manager R kod 1 2 Thermo Scientific To remove a proteome database Select the proteome database to remove in the Database Manager Click the Remove Database icon a in the toolbar at the top of the Database Manager window In the Confirm Delete dialog box click Yes to remove the proteome database from the proteome warehouse IMPORTANT Removing a proteome database from the proteome warehouse is a SaR P permanent change and cannot be undone except by reloading the data from the original source into the proteome warehouse To remove a repository Select the repository to remove
111. LR PRS EFE GE E ETEA L N I V 1 N V Y I4D L T TtL S C K R I M Q 1 S Y F D K R G FSE Result tables 1D Gene Length Mass Mass Diff C Ions Total Ions 2731369 2841 95 11226 6 03 K 1 4 143 Take to Sequence Gazer SEQ gt TOM1_YEAST Q03280 RecName Full E3 ubiquitin protein ligase TOM1 EC 6 3 2 AltName Full Suppressor of snRNA protein 2 AltName Full Temperature dependent organization in mitotic nucleus protein 1 Type predicted Signal Peptide false Propep false Y L D E K Y E D Y A F M D V M K V L N D Q L E N L N D F L N S P N D R S F F L E R D G E N S V R S C H S K L C R L A A I L N I V T N V Y I D L T TEL S C K R I M Q I Y S Y F D K R G F S L I K 1D Gene Length Mass Mass Diff C Ions Z Ions Total Ions E Value 2731372 2841 95 11226 6 03 x 1 3 4 a 143 Take to Sequence Gazer SEQ gt TOM1_YEAST Q03280 RecName Full E3 ubiquitin protein ligase TOM1 EC 6 3 2 AltName Full Suppressor of snRNA protein 2 AltName Full Temperature dependent organization in mitotic nucleus protein 1 Type basic Signal Peptide false Propep false Y L D E K Y E D Y A F M D V M K V L N D Q L E N L N D F L N S P N D R S F F L E R D G E N S V R S C H S K L C R L A A I L N I V T N V Y I D L T TEL S C K R I M Q I Y S Y F D K R G F S L I K 1D Gene Length Mass Z Ions Total Ions 2731371 2841 95 11226 6 a 3 4 Take to Sequence Gazer
112. PC application conducts the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 Thermo Scientific ProSightPC User Guide 129 4 Searching Databases Searching for Biomarkers Table 25 Biomarker Preferences page parameters Sheet 2 of 2 Parameter Precursor Tolerance Description Specifies the tolerance that determines whether comparing an observed precursor ion mass to a theoretical precursor ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a precursor search window that does not trigger an out of range warning e Default Sets the default value for a precursor search window e Upper Sets the maximum value for a precursor search window that does not trigger an out of range warning Fragment Tolerance Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a fragment tolerance that does not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that does not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm Minimum Matches Searching for Biom
113. PWF files trigger the Import Proteome Database or Repository function e FASTA and UniProtKB flat files trigger the Create Proteome Database function Creating a Proteome Database The ProSightPC application supports the creation of top down and middle down bottom up databases You can create your own shotgun annotated proteome databases These databases are restricted to one of the following two input file formats e UniProtKB flat files which store a large amount of known modification information UniProtKB a curated biological database of protein sequences is a part of the UniProt database 220 ProSightPC User Guide Thermo Scientific Thermo Scientific 8 Using Proteome Databases Creating a Proteome Database All PTMs listed in RESID are available for shotgun annotation However only the PTM information in the source UniProtKB flat file can actually be processed into the proteome database FASTA files which contain no PTM information so only predicted PTMs can be processed to their sequences FASTA format represents either nucleic acid sequences or peptide sequences where single letter codes represent base pairs or amino acids A sequence in FASTA format begins with a single line description followed by lines of sequence data In FASTA format sequences of nucleic acids or peptides begin with a lone line description followed by lines of sequence data Single letter codes represent base pairs or amino acids IMPOR
114. Repositories dialog box 37 68 250 Edit Add Searches for HT dialog box 39 49 50 editing comments 248 260 editing mass values 248 260 electron capture dissociation See ECD electron transfer dissociation See ETD electrospray ionization ESI 11 electrospray mass spectrometry ESI MS 11 Enter Custom Mass function 238 Thermo Scientific Index E ETD 10 207 ex vivo proteolysis 222 229 Excel spreadsheets 177 expectation value See e value Experiment Adder dialog box 84 87 251 opening 84 251 260 purpose 251 260 Experiment Manager changing display of experiments 96 copying experiments from one PUF file to another 97 creating PUF files 92 97 deleting experiments from PUF files 95 displaying experiments in 93 opening 92 252 opening PUF files 93 97 parameters on 97 purpose 6 removing experiments from PUF files 97 reverting to last saved PUF file 96 97 saving PUF files 96 97 Experiment Tools menu 248 259 experiments adding predefined searches to 248 253 254 analyzing 104 copying 252 definition of 2 91 differentiating in data grid 253 263 269 displaying in tab controller 266 exporting to Excel spreadsheets 177 exporting to repository 246 filtering out low yielding 38 49 importing from repository 246 ion trap marching 54 59 LC MS MS 3 7 27 28 54 manipulating 252 manually importing data 84 MS 12 MS MS 12 MS 159 MS 136 removing from data grid 255 264 removing results of search 264 removing searche
115. Scientific Fourier Transform instruments such as the LTQ FT This option is the default Example RAW files are available in the Example Raw Files folder for demonstration purposes e Process Puf Files Processes PUF files 3 To add a RAW or PUF file click Add then browse to the file in the dialog box that opens and select the file 4 If you choose a RAW file select one of the following methods in the Choose a Process Algorithm area for importing the data files Thermo Scientific ProSightPC User Guide 29 2 Getting Started Processing LC MS MS Data Files 30 ProSightPC User Guide e Thrash Uses the THRASH algorithm to process the input file e Xtract Uses the Xtract algorithm to process the input file This option reduces analysis and search time and should give better results This option is the default processing algorithms Xtract Precursor Minimum S N Precursor Maximum Charge Precursor Minimum Fit Precursor Remainder Threshold Precursor Selection Criterion Allow Multiple Precursors Relative Precursor Threshold Precursor Add Remainder Afterwards Fragmentation Minimum S N Fragmentation Maximum Charge Fragmentation Minimum Fit Fragmentation Remainder Threshold Minimum Fragmentation Base Peak Intensity Fragmentation Add Remainder Afterwards THRASH Precursor Minimum S N Precursor Minimum RL Precursor Maximum Charge Precursor Maximum Mass kDa Precursor Selection Criterion Allow Multiple Prec
116. Searching Databases on page 99 Each search mode overcomes different issues of protein identification and characterization The fundamental unit of analysis in top down proteomics is the MS MS experiment In this experiment intact precursor protein molecules are ionized and subjected to mass spectrometry A single peak which usually represents one charge state of the unknown protein but sometimes represents a small number of isobaric proteins is isolated and subjected to fragmentation The accurate mass measurement of the resulting MS MS fragment ions provides the second vital piece of information This MS and MS MS mass data is then compared to prior information about protein sequences and known or predicted PTMs in order to identify and characterize the unknown protein Note The ProSightPC application includes the RESIDS database http www ebi ac uk RESID The system identifies all post translational modifications as a truncated form of their RESID identification number The ProSightPC application removes leading zeros and the letters AA from the start of a RESID identifier This identifier is placed in parentheses before the amino acid containing the modification For example AA0049 N acetyl L methionine becomes 49 and an acetylation of a methionine residue in a protein sequence is expressed as 49 M 12 ProSightPC User Guide Thermo Scientific 1 Introduction to the ProSightPC Application Introduction to Proteomics Shotgun
117. Sets one of two conditions that the results of the search must meet e Number of Hits Specifies the number of matches for an intact ion in the search e Default E Value Specifies the expectation value e value for the results of the search If at least one search result received an e value of less than e 4 the search is loaded to the good category See Expectation Value e value on page 194 for information on the expectation value Middle list Specifies the operator that indicates the relationship between the values in the left and right boxes e Default lt Indicates that the first value is less than or equal to the second value e gt Indicates that the first value is greater than or equal to the second value Right list Specifies the desired value AND Expands the Condition dialog box so that you can add another condition The search results must meet the first condition and the second OR Expands the Condition dialog box so that you can add another condition The search results must meet either the first condition or the second End condition Indicates that the search has only one condition or that this is the last condition Save Saves the condition or conditions for the search Save Search Tree Dialog Box Parameters The Save Search tree dialog box shown in Figure 25 on page 42 has one parameter for specifying the name of the search tree that you created Using Custom Setti
118. Single Protein Search et i Tab control er dit Comment E Search Parameters Fragment Tolerance 10ppm Fragment Type Monoisotopic Precursor Mass Type Monoisotopic A m Mode On Sequence AVSKVYARSVYDSRGNPTVEVELTTEKGVFRSIVPSGASTGVHEALEMRDGDKSKWMGKGVLHAVKNVNDVIAPA FVKANIDVKDQKAVDDFLISLDGTANKSKLGANAILGVSLAASRAAAAEKNVPLYKHLADLSKSKTSPYVLPVPF LNVLNGGSHAGGALAL QEFMIAPTGAKTFAEALRIGSEVYHNLKSLTKKRYGASAGNVGDEGGVAPNIQTAEEAL DLIVDAIKAAGHDGKVKIGLDCASSEFFKDGKYDLDFKNPNSDKSKWLTGPQLADLYHSLMKRYPIVSIEDPFAE DDWEAWSHFFKTAGIQIVADDLTVTNPKRIATAIEKKAADALLLKVNOIGTLSESIKAAQDSFAAGWGVMVSHRS GETEDTFIADLVVGLRTGQIKTGAPARSERLAKLNQLLRIEEELGDNAVFAGENFHHGDKL Run Search Se The following sections explain the features of the ProSightPC main window in detail e Toolbar e Data Grid e Job Queue e Tab Controller e Data Manager e Grid Display Preferences Page Menu Bar The ProSightPC menu bar shown in Figure 109 appears at the top of the ProSightPC graphical user interface It contains the menus shown in Table 67 For detailed descriptions of each command on these menus see Chapter A ProSightPC Reference 258 ProSightPC User Guide Thermo Scientific Toolbar Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface Table 67 Menus in the ProSightPC menu bar Parameter Description File Use these commands to manipulate a PUF file such as Open Edit Use these commands to edit files such as Cop
119. Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Am Mode Determines whether the ProSightPC application conducts the search in delta m Am mode Performing Searches in Delta m Mode on page 110 explains this mode Disulfide Indicates whether a protein s cysteines are oxidized Include Modified Indicates whether to include PTMs and polymorphisms when you Forms perform a biomarker search Min of Matching Determines whether the search algorithm finds only proteins Fragments containing a minimum number of matching ion fragments The box to the right specifies the minimum number of matching ion fragments Min of Matching Determines whether the search algorithm finds only proteins Fragments containing a minimum percentage of matching ion fragments The box to the right specifies the percentage of matching ion fragments Min Score Determines whether the search algorithm finds only proteins with a p score that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right box e Default lt Indicates that the first value is less than or equal to the second value e gt Indicates that the first value is greater than or equal to the second val
120. TANT Creating a proteome database can take several hours or in some extreme cases days to complete depending on your hardware and the data being processed by the shotgun annotation method To create a proteome database Choose Databases gt Create a Custom Database or click the Create Database icon on the Database Manager The Welcome to the New Database Wizard page of the Create New Database Wizard opens as shown in Figure 94 Figure 94 Welcome to the New Database Wizard page of the Create New Database Wizard W Create New Database ka Welcome to the New Database Wizard This wizard creates a new proteomic database ProSight Warehouse to search experimental data against in ProSight Please be aware that depending on the complexity of the database creation can take anywhere from minutes to hours or even days In general in silico digestion sequence polymorphisms and high numbers of permitted forms per input sequence will increase database creation time substantially Click Next to continue 2 Click Next ProSightPC User Guide 221 8 Using Proteome Databases Creating a Proteome Database The Database Type page of the Create New Database Wizard appears as shown in Figure 95 Figure 95 Database Type page of the Create New Database Wizard Database Type Specify the overall parameters of the database you wantto create Database Top Down No Sam
121. Table 23 Absolute Mass Preferences page parameters Sheet 3 of 3 Parameter Description Fragment Tolerance Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a fragment tolerance that will not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that will not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm Minimum Matches Specifies the minimum number of matching ion fragments that you want the search algorithm to find in proteins Set the following parameters e Lower Sets the minimum value for minimum matches that will not trigger an out of range warning e Default Sets the default value for minimum matches e Upper Sets the maximum value for minimum matches that will not trigger an out of range warning Searching for Absolute Mass To search for absolute mass 1 Start a search by following the instructions in Creating a Predefined Search on page 101 2 In the Search Type list in the New Predefined Search dialog box shown in Figure 53 select Absolute Mass if it is not already selected Thermo Scientific ProSightPC User Guide 119 4 Searching D
122. To edit mass values 1 Do one of the following e Choose Experiment Tools gt Edit Masses or e Click the Edit Masses icon el in the Data Manager or the ProSightPC toolbar or e Right click an experiment and choose Edit Mass List from the shortcut menu Each of these methods opens a new page in the tab controller as shown in Figure 88 208 ProSightPC User Guide Thermo Scientific Thermo Scientific Figure 88 Editing mass values in the tab controller 7 Displaying Data in the Data Manager Editing Mass Values Grid Display Preferences Fragment Predictor Experiment 1 Edt Masses Ex 1 Fragmentation Method ETD mz_monoisot mz_average mass_monois mass_averag intensity 0 29006 7 1 mz_monoisot mz_average mass_monois mass_averag intensity id gt 0 0 529 3202682 0 811 1 0 0 587 3288376 0 1287 2 0 0 606 2641948 0 2019 3 0 0 663 2856827 0 3856 4 0 0 806 4266933 0 797 5 0 0 839 4144198 0 445 6 0 0 883 3696584 0 7419 7 0 0 906 6391225 0 587 8 0 0 925 4850272 0 456 9 0 0 970 5567998 0 822 10 0 0 1011 464035 0 4734 11 0 0 1017 110454 0 97 12 0 0 1038 412555 0 374 13 0 0 1047 955442 0 1227 14 0 0 1048 068323 0 1017 15 0 0 1054 511307 0 484 16 0 0 1072 258938 0 564 17 0 0 1086 742093 0 685 18 0 0 1087 544839 0 773 19 0 0 1092 532645 0 307 20 0 0 1098 456195 0 439 21 0 0 1101 453736 0 174 22 0 0 1101 537711 0 413 23 0 0 1109 921973 0 864 24 0 0 1111 542553 0 368 25 0 0 1114 05
123. Upper Sets the maximum value for a precursor search window that does not trigger an out of range warning 7 In the Fragment Tolerance boxes specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a fragment tolerance that does not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that does not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm A fragment tolerance is a mass value either absolute or relative within which your observed masses must match the theoretical fragment mass For instance if you set your tolerance to 0 005 Da an absolute tolerance and your theoretical fragment ion is at 1154 1126 Da observed fragment ions of 1154 1090 Da 0 0034 Da from theoretical and 1154 1167 40 0041 Da from theoretical fall within the tolerance but 1154 2312 0 1222 does not because the mass difference is greater than the tolerance that you set 8 In the Minimum Matches boxes specify the minimum number of matching ion fragments that you want the search algorithm to find in proteins Set the following parameters 128 ProSightPC User Guide Thermo Scientific 4 Searching Database
124. You must click Rescore to implement the changes made in the search parameter display Table 38 lists the parameters in the search parameter display ProSightPC User Guide 191 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Table 38 Search parameter display parameters Parameter Description Precursor Mass Type Specifies the type of precursor ion mass to use e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Tolerance Displays the fragment tolerance and unit at the time that the search was last scored The fragment tolerance is the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match An observed fragment ion matches a theoretical fragment ion if the observed fragment ion mass is within plus or minus the fragment tolerance of the theoretical fragment ion mass Am Mode Indicates whether delta m Am mode has been selected Mass Type Specifies the type of ion mass fragment type to use e Monoisotopic Specifies that the mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the mas
125. _ yeast_topdown_etd v New Repository Search Tree Name yeast_topdown_etd X Save Experiment Filter 7 Min fragments s E Max fragments 530 W Min Intact Mass 750 Monoisotopic X lt Prev Next gt Finish Creating a Search Tree with Three or More Levels You can use the ProSightPC application to create two levels of searches in its user interface However you can create additional search levels by editing the XML file that contains the search trees After you create these levels in the XML file and run the High Throughput Wizard these levels appear on the Summary page of the Wizard as shown in Figure 30 Thermo Scientific ProSightPC User Guide 45 2 Getting Started Processing LC MS MS Data Files Editing a Search Tree Figure 30 Summary page for a five level search Input Files Processing raw files C Program Files ProSightPC dataHistone Raw Files Histone_H4_actyl25 scans_11299 409Da raw Raw File Options XtractNadvg Custom Not saving Puf files Running search tree logic Search Tree Options Repository repository12 Search Tree biomarkersearch_rattd_1 Number of Searches 5 Level 1 biomarker_1da Level 2 biomarker_500da Level 3 absolute_mass_narrow Level 4 absolute_mass_wide Level 5 sequence_tag_search If you want to edit the XML file contact the Thermo Fisher Scientif
126. a which is a numerical list of searches arranged by search identification following the instrument data Figure 87 gives an example The search type and status are displayed If the search is highlighted in blue the search is pending and has yet to be run Figure 87 Search numbers gt Search 1 Single Protein Search gt Search 2 Absolute Mass Search gt Search 3 Absolute Mass Search gt Search 6 Sequence Tag Search gt Search 7 GRAM Search gt Search 8 GRAM Search 206 ProSightPC User Guide Thermo Scientific 7 Displaying Data in the Data Manager Opening a Data Manager Window When an MS MS experiment is generated the fragmentation method used to generate the MS MS data is declared From this input the ProSightPC application determines which of the following ion types to use during searches e Electron capture dissociation ECD and ETD fragmentation is analyzed using c z ions e Collision induced dissociation CID HCD and IRMPD fragmentation is analyzed as b y ions Opening a Data Manager Window To open a Data Manager window e Do one of the following Double click an experiment displayed in the data grid or Select the experiment and choose View gt Open Data Manager or Click the Open Data Manager icon ae Closing a Data Manager Window To close a Data Manager window e Choose View gt Close Data Tab or e Click the page containing the Data Manager and choose View gt Close Data Manager
127. a compiler tolerance e Upper Sets the maximum value for a compiler tolerance that does not trigger an out of range warning 6 In the Minimum Tag Size boxes enter the lowest acceptable sequence tag score reported as a match Set the following parameters e Lower Sets the minimum value for a minimum tag size that does not trigger an out of range warning e Default Sets the default value for a minimum tag size e Upper Sets the maximum value for a minimum tag size that does not trigger an out of range warning 7 Click OK Sequence Tag Preferences Page Parameters Table 27 lists the parameters on the Sequence Tag Preferences page of the Options dialog box shown in Figure 57 on page 137 Table 27 Sequence Tag Preferences page parameters Sheet 1 of 2 Parameter Description Database Specifies the name of the database to search Fragment Mass Specifies the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion 138 ProSightPC User Guide Thermo Scientific 4 Searching Databases Searching for Sequence Tags Table 27 Sequence Tag Preferences page parameters Sheet 2 of 2 Parameter Description Minimum Tag Specifies the minimum tag score for prote
128. a grid shortcut menu which appears when you right click the area to the right of the columns in the data grid as shown in Figure 112 on page 262 Table 66 Data grid pane commands Command Description Columns Determines which columns appear in the data grid Thermo Scientific ProSightPC User Guide 255 ee i Using the ProSightPC Interface The ProSightPC application has a unique approach to the organization of elements in the graphical user interface This appendix describes the features of this interface Contents e The ProSightPC Interface e Setting Default Options The ProSightPC Interface Figure 109 shows some of the features of the ProSightPC graphical user interface Thermo Scientific ProSightPC User Guide 257 B Using the ProSightPC Interface The ProSightPC Interface Figure 109 ProSightPC window Grid Display Menu bar Toolbar Data grid Preferences page na or ial esa File Edit iew Experiment Tools Datal ProSightHT Tools Hel a a Cat AAP eo PAS ke UF Exp D SearchID Marked Search Type PendingSearch Best Expectation Matching Forms Name Status Notes 1 1 Single Protein yes na nla 2 1 Single Protein yes nla na _ _ _ _ _ rrr CC Job queue Data Management for Experiment 2 e _ Pata Manager Source enolase_ETD_60K_4ms_avg_1000 raw 46642 214 Fragmentation Method ETD Ton Type CZ gt Precursor Mass List gt Fragment Mass List w Search 1
129. a protein s identity You can use the Sequence Gazer for further characterization Thermo Scientific 4 Searching Databases Searching for Absolute Mass e Use delta m Am mode explained in Performing Searches in Delta m Mode on page 110 to locate unknown modifications near either terminus If the 1000 Da absolute mass search fails to identify a protein consider running another absolute mass search with a 1000 Da precursor search window in delta m Am mode Activating delta m Am mode increases the likelihood that the search will identify proteins with unknown modifications However this mode takes approximately three times longer than the corresponding absolute mass search Setting Absolute Mass Search Preferences Thermo Scientific Use the Absolute Mass Preferences page of the Options dialog box to set the default values used when you add new absolute mass searches For information about absolute mass searches see Searching for Absolute Mass on page 113 To set absolute mass search preferences 1 Click Absolute Mass in the Options dialog box The Absolute Mass Preferences page of the dialog box opens as shown in Figure 52 Figure 52 Absolute Mass Preferences page of the Options dialog box Options econ face 5 General 2 Grid Columns Absolute Mass Preferences Thrash ERE N Ala pat eee eae Para eee E Search Parameters sale p E Default Absolute Mass Search Parameters 7 Sequence Tag Da
130. ages result in the following peptides AAAK AAAK AAA If you select up to one missed cleavage the peptides are the following AAAK AAAK AAA AAAKAAA AAAKAAA Thermo Scientific ProSightPC User Guide 227 8 Using Proteome Databases Creating a Proteome Database 228 ProSightPC User Guide 19 20 21 If you select two missed cleavages the peptides are the following AAAK AAAK AAA AAAKAAA AAAKAAA AAAKAAAKAAA In the Minimum Peptide Mass Da box type the minimum mass that a peptide must have in daltons before the application puts it into the database No peptide less than the minimum peptide mass is put into the database any theoretical peptide less than this mass is discarded and ignored This parameter is useful because particularly small peptides sometimes cannot be identified but have a very strong impact on database size In the Maximum Peptide Mass Da box type the maximum mass that a peptide can have in daltons before the application puts it into the database No peptide greater than this mass is put into the database Click Next The Database Description page of the Create New Database Wizard opens as shown in Figure 100 so that you can enter identifying information about the database that you want to create Figure 100 Database Description page of the Create New Database Wizard Database Descnption Specify information about this database that will be displayed in the Database Manager an
131. agram sni tics eked oN Gentes Dak ieee TAAL Hee a ete 198 Interactive Fragment Map 0 ec eee eee 198 Amino Acid Information Box ti 6 14 6 eeied kaha bane ene taal 199 Fixed Modifications Box x 25 4 059 edad be ew dale a wee eee Ae 199 Matching Fragments abe so su ne tot cet ene eG hin eles ae 200 Non Matching Fragments Table osc baow setae aerate pitas 201 Demonstrating the Sequence Gazernig i Caves tors eee awn aan es 203 Thermo Scientific ProSightPC User Guide v Contents vi Chapter 7 Chapter 8 Chapter 9 Chapter A ProSightPC User Guide Displaying Data in the Data Manager 200eee cece e eee 205 DatasMana ger teri detaria nepa aos taventog tte ase aybed aay a e cheeks oye i 205 Opening a Data Manager Window secu taiwan Wa tan Va ees 207 Closing a Data Manager Window 1 2 0 0 cee cece cece ee eens 207 Adding or Editing an Experiment Comments 450 phones swe oA 208 Editing Mass Vales ais acne tate Shae ed bho k ota San ae ahaa edhe 208 Running a Pending Search wey hats acy hate ao Matai ane atime 212 Using Proteome Databases 2 ccc eee eee eee eee 213 Proteome Warehouse jcc annsa Fae vases MRR POW eee eae 213 Importing Data into the Proteome Warehouse 00 eee ee eee 214 Accessing the Database Manager oi eet eit puta hada wieder ss 214 Database Manager Window Parameters 0 00 cece eee eee 215 Importing a Proteome Database or Repository 0 0
132. ainder Afterwards THRASH Precursor Minimum S N Precursor Minimum RL Precursor Maximum Charge Precursor Maximum Mass kDA Precursor Selection Criterion Allow Multiple Precursors Relative Precursor Threshold Fragmentation Minimum S N Fragmentation Minimum RL 7 0 30 40 20 Highest Intensity Selected 10 Cleared 3 0 30 10 100 Selected 7 0 0 90 40 35 Highest Intensity Selected 10 30 0 ProSightPC User Guide 31 2 Getting Started Processing LC MS MS Data Files 32 ProSightPC User Guide Fragmentation Maximum Charge Fragmentation Maximum Mass kDA Minimum Number of Fragmentation Scans Minimum Fragmentation Base Peak Intensity 25 25 1 100 Top Down M82 Specifies the following default settings for the Xtract and THRASH processing algorithms Xtract Precursor Minimum S N Precursor Maximum Charge Precursor Minimum Fit Precursor Remainder Threshold Precursor Selection Criterion Allow Multiple Precursors Relative Precursor Threshold Precursor Add Remainder Afterwards Fragmentation Minimum S N Fragmentation Maximum Charge Fragmentation Minimum Fit Minimum Fragmentation Base Peak Intensity Fragmentation Add Remainder Afterwards 7 0 30 40 20 Highest Intensity Selected 10 Cleared 3 0 30 10 100 Selected Thermo Scientific 2 Getting Started Processing LC MS MS Data Files THRASH Precursor Minimum S N 7 0 Precursor Minimum RL 0 90 Precursor Maximu
133. al Mod Specifies the fixed terminal modification for the C terminus Click Save The new search appears in the data grid with yes appearing in the Pending Search column To execute the search from the data grid right click the pending search and then choose Run Search number To view the results of the search refer to Viewing Search Results on page 161 Thermo Scientific 4 Searching Databases Searching for Absolute Mass New Search in Experiment X Dialog Box Parameters for Absolute Mass Table 24 lists the parameters in the New Search in Experiment X dialog box for absolute mass shown in Figure 53 on page 120 Table 24 New Search in Experiment X dialog box parameters for absolute mass Sheet 1 of 2 Parameter Search Type Database Description Description Specifies the type of search to perform e Absolute Mass See Searching for Absolute Mass on page 113 e BioMarker See Searching for Biomarkers on page 125 e Sequence Tag See Searching for Sequence Tags on page 136 e Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins e Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 e Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Describes the database that you want to search Precursor Mass Type Specifies the type of precursor ion mass
134. amino acid in the observed protein 13 In the PTM Handling box select the PTMs that you want to search for The PTM Handling box displays PTMs arranged in one or more tiers based on the selected proteome database The ProSightPC application only queries theoretical protein forms containing exclusively selected PTMs Every form containing an unselected PTM is excluded from the interrogation 14 In the Terminal Mods area select the fixed terminal modification for each terminus A fixed terminal modification is a chemical modification that is present on the terminus of the observed protein e N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus 15 Click Save The new search appears in the data grid with yes appearing in the Pending Search column 16 To execute the search from the data grid right click on the pending search and then choose Run Search number To view the results of the search refer to Viewing Search Results on page 161 Thermo Scientific ProSightPC User Guide 133 4 Searching Databases Searching for Biomarkers New Search in Experiment X Dialog Box Parameters for Biomarkers Table 26 lists the parameters in the New Search in Experiment X dialog box for biomarkers shown in Figure 56 on page 131 Table 26 New Search in Experiment X dialog box parameters for biomarkers Sheet 1 of 2 Para
135. arch Thermo Scientific ProSightPC User Guide 99 4 Searching Databases Performing Searches e MS hybrid searches You can compare MS MS data to entries in a proteome database by adding a search to the MS MS experiment containing the MS MS data Performing Searches When you perform searches with the ProSightPC application start with a more specific search rather than a more general search The database search takes considerably longer with increasing search complexity so identifying as many spectra as possible with a quick simple search saves a lot of search time overall There is also another reason for starting with a more specific search The e value for a result is inversely proportional to the size of the searched database For the exact same spectrum a search against a smaller database produces a better identification than a search against a larger one assuming that the correct protein identification resides in both databases e Performing Predefined Searches e Performing Searches in Delta m Mode e Performing Searches in Batch Mode Performing Predefined Searches 100 ProSightPC User Guide Predefined searches are a strategy to simplify the repetition of identical searches on different sets of MS MS data They enable you to assign a name to a set of parameters that you can then add to any experiment Use a predefined search to set up defaults for frequently run searches Create a predefined search and add it to your
136. arch 132 135 155 158 polypeptides 125 Post Xtract AIM 73 74 246 post translational modifications See PTMs precursor intensity 56 63 precursor ions average mass of first 269 average mass of largest 269 average mass to charge ratio of 269 dimensions of search window 116 118 editing mass values 208 254 260 in experiments 12 91 input method 85 87 largest average mass to charge ratio of 269 largest monoisotopic mass to charge ratio of 269 listing mass values 9 210 264 mass type absolute mass search preferences 116 117 absolute mass searches 120 123 adding experiments 75 85 87 advanced settings 56 biomarker search preferences 127 129 biomarker searches 131 134 gene restricted absolute mass searches 148 151 gene restricted biomarker searches 157 importing raw file with Post Xtract 75 importing raw file with Profile 81 83 Sequence Gazer 192 single protein search preferences 144 145 single protein searches 186 189 mass to charge ratio 75 77 82 85 87 monoisotopic mass of first 269 monoisotopic mass of largest 269 monoisotopic mass to charge ratio of 269 multiplexing multiple 56 multiply charged 12 observed mass 113 125 163 173 observed mass versus theoretical mass 110 111 130 173 198 theoretical mass 113 146 163 173 THRASH parameters for analyzing 55 61 64 tolerance for comparison of observed to theoretical 128 using MS data as 159 using MS MS data as 159 Precursor Mass List 210 254 264 Thermo Scie
137. ards When you select Add Remainder Afterwards the spike shows up in the deconvolved spectrum because unassigned remainders are added to the corresponding pattern e Selected Adds the remaining intensities to the output spectrum during analysis of the precursor ions e Default Unselected Does not add the remaining intensities to the output spectrum during analysis of the precursor ions Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 6 of 8 Parameter Fragmentation Analysis Options Description Minimum S N Specifies the lowest signal to noise ratio that the algorithm considers when trying to assign neutral mass to a charged mass to charge ratio 7 z species while analyzing the precursor ions Range 1 no maximum Default 3 0 Minimum RL THRASH only Specifies the minimum confidence level Range 0 1 0 Default 0 90 Minimum m z Considered Specifies the low end of the m z range that the ProSightPC application analyzes Range 1 no maximum Default 50 Maximum Charge Specifies the maximum charge used by the algorithm while analyzing the precursor ions Range 1 no maximum Default 40 Maximum Mass kDa THRASH only Specifies the highest mass to consider for the precursor Range 1 no maximum Default 60 Maximum m z Considered Specifies the high end of the m z range that the Pr
138. arkers Specifies the minimum number of matching ion fragments that you want the search algorithm to find in proteins Set the following parameters e Lower Sets the minimum value for minimum matches that does not trigger an out of range warning e Default Sets the default value for minimum matches e Upper Sets the maximum value for minimum matches that does not trigger an out of range warning To search for a biomarker 1 Start a search by following the instructions in Creating a Predefined Search on page 101 2 In the Search Type list in the New Predefined Search dialog box shown in Figure 53 on page 120 select BioMarker 130 ProSightPC User Guide Thermo Scientific 4 Searching Databases Searching for Biomarkers The appearance of the dialog box changes as shown in Figure 56 Figure 56 New Search in Experiment X dialog box for biomarkers ew Search in Experimer oes Search Type bd Biomarker Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic h Precursor Tolerance 10 ppm v Fragment Mass Type Monoisotopic v Fragment Tolerance 15 ppm v Am Mode Include Disulfide Modified Forms Hit Filtering 7 Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score Max Proteins to Return 25 Fixed Modifications 4 Cysteine a E E Methionine
139. ases containing PTM and sequence variant information e PWF databases that are included on the ProSightPC DVD for various organisms e Top down databases e Bottom up databases You have two options for importing or creating a proteome database download databases from the ProSightPC FTP Web site or manually create a custom database To download a database from the ProSightPC FTP Web site 1 Choose Databases gt Download ProSightPC Databases This step takes you to ftp prosightftp gsX 1 gON prosightpc northwestern edu This site contains top down and bottom up databases Thermo Scientific ProSightPC User Guide 23 2 Getting Started Editing Modifications 2 Select the desired database to download it to your local computer 3 In the File Download dialog box shown in Figure 15 click Save Figure 15 File Download dialog box File Download 4 Do you want to open or save this file Name bos_taurus_2012_07_top_down_simple pwf Type pwf_auto_file 1 25KB From prosightpc northwestern edu Co a Ge 7 Always ask before opening this type of file a 1 While files from the Intemet can be useful some files can potentially harm your computer If you do not trust the source do not open or save this file What s the risk The downloaded database appears in the Database Manager 4 Follow the instructions in Importing a Proteome Database or Repository on page 217 To manually create a cust
140. ass data is scan 1 in the Post Xtract file e Monoisotopic Mass Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion The uncharged monoisotopic mass data is scan 2 in the Post Xtract file Thermo Scientific ProSightPC User Guide 75 2 Getting Started Importing Targeted RAW Files Table 15 Build Experiment from Post Xtract Raw Data dialog box parameters Sheet 2 of 2 Parameter Description Fragmentation Ion e Fragmentation Method Specifies one of the following Data fragmentation methods e CID e ECD e ETD e HCD e IRMPD For information on these methods see Fragmentation Methods on page 9 e Average Mass Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Monoisotopic Mass Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Predefined Search Displays the predefined searches set for an experiment Check All Selects all predefined searches Uncheck All Does not select any predefined searches Intact Mass Calculator Dialog Box Parameters Table 16 lists the parameters in the Intact Mass Calculator dialog box shown in Figure 38 on page 75 Table 16 Intact Mass Calculator dialog box parameters Parameter Description Precursor m z Specifies the mass to charge ratio m z value of
141. at you can perform operations on them such as adding or changing searches or using the Sequence Gazer To import experiments from a repository Choose File gt Import Data from Repository to open the Import Data From Repository dialog box In the Repository list select the name of the repository from which to import experiments into the ProSightPC application In the Category list select the category of results to import The categories available in the list include the default good and bad categories as well as any that you defined Thermo Scientific 2 Getting Started Using Repositories 4 In the File list select the name of the PUF or RAW file whose data formed the basis of the experiments 5 In the Experiments box select the experiments to import Click Select All to choose all the experiments in the repository Verify that the Import Data from Repository dialog box resembles the example in Figure 35 Figure 35 Import Data from Repository dialog box Please select a project file and any number of experiments to import into ProSightPC Repository yeast_topdown_etd X Category good X File Dfraction03 ET Dfraction03 ET Dfrection03 put A4 Experiments 51 81 53 82 57 6 Click OK The experiments are now listed in the data grid For information on importing experiments into the ProSightPC application by using the repository report see Using th
142. atabases Searching for Absolute Mass Figure 53 New Search in Experiment X dialog box for absolute mass ew Search in Experimen oe Search Type X Absolute Mass Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic bd Precursor Search Window 1000 Da Fragment Mass Type Monoisotopic hg Fragment Tolerance 15 ppm v Am Mod Disulfide Hit Filtering Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score 0 Max Proteins to Return 25 Fixed Modifications Cysteine Methioni Lysine Isoleucine Leucine tthe 4 Handling All PTMs High priority PTMs Tier 1 T E 2 B A B 8 oy Terminal Mods N Terminal Mod none m C Terminal Mod None x 3 In the Database Description list select a description of the database that you want to search 4 From the Precursor Mass Type list select the type of precursor ion mass to search for e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 120 ProSightPC User Guide Thermo Scientific Thermo Scientific 5 10 4 Searching Databases Searching for Absolute Mass e Average Specifies that the average mass is average which is the mass of the most abundant isotope
143. ate New Database Wizard appears as shown in Figure 97 Thermo Scientific ProSightPC User Guide 223 8 Using Proteome Databases Creating a Proteome Database 224 ProSightPC User Guide Figure 97 Initial Methionines page of the Create New Database Wizard a Initial Methionines Specify how initial methionines in the input file should be handled v Ensure Initial Methionine Cleavage This setting ensures that each isoform will generate two forms for each N terminal sequence one where the N terminal methionine is present and one where itis cleaved off S Apply N Terminal Acetylation Apply N Terminal Formylation These settings specify what PTMs should be presumed to potentially exist on all proteins even if they are not present in the input 8 Select the method of handling initial methionines e Ensure Initial Methionine Cleavage recommended Determines whether each isoform generates two forms for each N terminal sequence one where the N terminal methionine is present and one where it is cleaved off Default Selected Each isoform generates two forms for each N terminal sequence Clear Each isoform generates only the form where the N terminal methionine is present not contain them 9 Specify the PTMs that should be presumed to exist on all proteins even if the input does e Apply N Terminal Acetylation Adds N terminal acetylation whenev
144. ation information are stored in proteome databases in the ProSightPC proteome warehouse The sequence and PTM information are combined using shotgun annotation as explained in Chapter 1 Introduction to the ProSightPC Application Figure 89 shows all the known modifications such as SNPs and sequence variants that can be applied to a basic sequence Thermo Scientific ProSightPC User Guide 213 8 Using Proteome Databases Importing Data into the Proteome Warehouse Figure 89 Known modifications applied to a basic sequence N terminal processing in vivo half life signal peptides Polymorphisms Known Intron exon Unexpected modifications boundaries modifications The proteome warehouse consists of a collection of proteome databases and a small amount of metadata Proteome databases contain the shotgun annotation of all possible combinations of known modifications on each basic sequence in the proteome A protein form refers to any given possible combination of modifications on a basic sequence Each proteome is stored as a proteome database and is uniquely identified by an internal name consisting of one or more letters A Z without spaces or punctuation For example you might designate E coli UniProt as ecoli_uniprot Importing Data into the Proteome Warehouse Use either of the following two methods to import data into the proteome warehouse e Load databases from PWF files See Importing a Proteome Database or Repos
145. ay of 216 220 creating 216 220 downloading from Web site 23 exporting from proteome warehouse 215 218 importing into proteome warehouse 2 215 217 manually creating 24 removing from proteome warehouse 215 219 shotgun annotated 220 strain information 216 unmodified protein forms in 216 Proteome Discoverer application 1 proteome warehouse contents of 3 213 databases in 3 definition of 3 exporting proteome databases from 215 218 exporting repositories from 216 218 importing proteome databases into 2 7 214 215 217 importing repositories into 216 217 managing in Database Manager 249 removing databases from 219 removing proteome databases from 215 removing repositories from 216 219 searching for matches 89 searching for neutral mass data against 7 See also PWF files PTM Tier Editor accessing 236 assigning PTMs to tiers 235 customizing PTM tier assignment 240 excluding PTMs 235 237 including PTMs 235 237 moving PTMs between tiers 238 purpose 6 235 sorting PTMs 236 PTM Tier Editor dialog box 236 237 251 PTMs adding to amino acids 183 238 251 adding virtual 199 adding virtual to amino acids 199 annotating onto a protein 225 assigning to tiers 235 237 251 available in Swiss Prot files 221 excluded 237 excluding from database 235 237 grouping 235 included 237 including in biomarker search 132 135 155 158 286 ProSightPC User Guide including in database 226 231 235 including in PTM Tier editor 237 location
146. ber of matching ion fragments Min of Matching Fragments Min Score Determines whether the search algorithm finds only proteins containing a minimum percentage of matching ion fragments The box to the right specifies the percentage of matching ion fragments Determines whether the search algorithm finds only proteins with a p score that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right box e Default lt Indicates that the first value is less than or equal to the second value e gt Indicates that the first value is greater than or equal to the second value Max Proteins to Return Specifies the maximum number of proteins to return in the search Fixed Modifications Specifies the chemical modifications present on all instances of a given type of amino acid in the observed protein PTM Handling Specifies the PTMs that you want queried Terminal Mods Specifies the fixed terminal modification for each terminus e N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus Save Saves the search information Searching for Gene Restricted Biomarkers Use a gene restricted biomarker GRBM search to perform a biomarker search on all protein forms of the genes listed in the gene identification list
147. c ProSightPC User Guide 15 1 Introduction to the ProSightPC Application Introduction to Proteomics Figure 10 Results of full characterization from initial data Identified protein Partially characterized protein Identified and fully characterized protein Note Partial characterization occurs in cases where it is possible to determine which PTM must be present on the protein but the fragmentation data is not sufficient to determine on exactly which amino acid one or more of the PTMs must be You can narrow the list of possibly modified amino acids to one or two residues in a short subsequence of the protein In some MS MS experiments you will have sufficient fragmentation data to fully characterize the proteins with the first search If you shotgun annotate the exact protein form observed into the proteome database and the MS spectra contains sufficient fragmentation information to uniquely identify this form you can discover the correct answer by conducting an absolute mass search This situation occurs frequently In some well annotated proteomes unknown proteins are completely characterized on the first search as shown in Figure 10 Figure 11 illustrates another common situation when the initial search only identifies and perhaps partially characterizes the unknown protein In this case conducting a second search fully characterizes the protein Often the second search is either a biomarker or a single protein mode
148. c envelopes 4 In the Maximum Mass box type the cutoff point for the THRASH algorithm when searching for masses 5 In the First m z box type the lowest mass to charge ratio m z value considered 6 In the Minimum RL value box type the minimum confidence level 7 In the Maximum Charge box type the maximum charge to be used by the THRASH algorithm 8 In the Last m z box type the highest mass to charge ratio m z value considered 9 In the Summing Options area type the first scan number scanned in the Start Scan Number box and the last scan number scanned in the End Scan Number box 10 In the Type list in the Precursor Mass area select the mass type e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 80 ProSightPC User Guide Thermo Scientific 2 Getting Started Importing Targeted RAW Files e Average mass Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion 11 Optional Click m z to have the ProSightPC application calculate the intact mass if only the mass to charge ratio and the charge are known The Intact Mass Calculator dialog box appears as shown in Figure 41 Figure 41 Intact Mass Calculator dialog box Intact Mass Calculator Precursor miz Charge State Co a In the Precursor m z box type the mass to charge ratio m
149. can access the Sequence Gazer through one of two strategies e By performing a single protein search e By clicking Take to Sequence Gazer from any protein identification of a completed search See Demonstrating the Sequence Gazer on page 203 for a demonstration showing how to use the Sequence Gazer to find modifications in fragment ions Searching for Single Proteins and Accessing the Sequence Gazer To perform a single protein search and access the Sequence Gazer 1 Open the desired experiment in the Data Manager by double clicking the experiment in the data grid 2 Choose Experiment Tools gt Append Predefined Search The Append Predefined Searches to Experiment X dialog box opens 3 Click the Create New Search icon zm The New Predefined Search dialog box opens as shown in Figure 77 184 ProSightPC User Guide Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer Figure 77 New Predefined Search dialog box Sax Search Name Search Type Absolute Mass X Absolute Mass Search Database Description Demo Database for ProSighPC Precursor Mass Type Monoisotopic hd Precursor Search Window 1000 Da Fragment Mass Type Monoisotopic v Fragment Tolerance 15 ppm Am Mod Disulfide Hit Filtering v Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score Max Proteins to Retum x 4 Fixed Modifications
150. cece eee eee eee eens 19 Starting the ProSightPC Applicatioti i650 chccuredesivseeredenndv ena 20 Closing the ProsightPC Application i44504 4 16 08 2oacda bela tieeeds 21 Thermo Scientific ProSightPC User Guide iii Contents iv Chapter 3 Chapter 4 ProSightPC User Guide Setting Default Options dca datante eiaa ae a a klipe kite wanes 21 General Preferences Page Parameters chen accede a cuties Sok ele iidee aes 22 Importing or Creating a Proteome Database pou 2s aa aes ees eae 23 Editing Modifications 3 63 80 0 qebig ONS aes bales ee ees aie We 24 Terminal Modification Editor Parameters aunan seied os eek a ees 27 Fixed Modification Editor Parameters noun aie sega vie siya cone ess 27 Processing LC MS MS Data Files vce eave hbwate hand atlas 27 Using the High Throughput Wizatd sis sisted oaks ewes walt 28 Using Custom Settings in the High Throughput Wizard 52 Using Repositofiess fete see siei ee hdd Wore tall elo a east nue a RA hee ada 66 Creating a Repositety c icea 4st cee Goose hone ee Sacer s haces pecans 67 Editing a REPOSIE Lyi och thay 0G a Eohaed ted bintch afemdchacdrothanbaterdacded aye 4 68 Deleting a Repository ser wca wens hs aoe ees Da ease kaw aie Ses 69 Importing Experiments from a Repository 0000s eee eee 69 Exporting Experiments to a Repository 0 06 0 eee cece ene 71 Importing Targeted RAW Files sh weicg sca rte dp tees hea er 73 Importing a Targeted RAW File
151. ch modes Thermo Scientific ProSightPC User Guide 235 9 Using ProSightPC Tools Locating and Selecting PTMs with the PTM Tier Editor Note 1 You can enter tier assignments greater than 2 2 The PTM Tier Editor does not append PTM information to databases The PTM information must reside in the proteome database before the ProSightPC application analyzes MS data Accessing the PTM Tier Editor 236 ProSightPC User Guide To access the PTM Tier Editor Choose Tools gt PTM Tier Editor The PTM Tier Editor dialog box opens as shown in Figure 101 Figure 101 PTM Tier Editor dialog box S Name N N dimethyl L proline N4 N4 dimethyl L asparagine NG NG N6G trimethyl L lysine ___ N6 N6 dimethyl L lysine omega N omega N dimethyl L arginine 1 methyl L histidine 1 phospho L histidine 2 3 carboxamido 3 trimethylammonio propyl L histidine 3 methyl L histidine 5 methyl L arginine __ Disulfide Bond Homoserine Lactone l annartia A nhannhnarin snherdiida gt la as m as NS NS d d ad ed ek ld led Excluded To include a PTM check t E2 Zdidehyd sine 2 3didehyd sine 3 3 5 triiodo L a 3 4 dihydroxy L arginine 4 5 dihydroxy Ltysine S hydroxy N6 N6 N64rimethyl L4ysine cis 14 hydroxy 10 13 dioxo 7 heptadecenoic acid L aspartate ester L 2 4 5Stopaquinone L 3 4 5 trihydroxyphenylalanine
152. 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 Thermo Fisher Scientific Inc makes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissions damage or loss that might result from any use of this document even if the information in the document is followed properly 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 Conditions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history Revision A January 2013 Software version Thermo ProSightPC 3 0 requires Thermo Xcalibur version 2 1 For Research Use Only Not for use in diagnostic procedures B Contents it ee ee ere er eer re ree ix Related Documentation i s00 220 sas LO peed be OW ede RS es CO Bods ix Special NGUCE 64 cht sacked wndobwa three na cewey dace tenance E x System Requirements 0 cee nu rnrn nren ranr x Installine PiroSightG soirassa a ea a e Bose xi Contacting US Sorarrain See E hae E e a E EE ties dd oes xiii Chapter1 Introduction to the ProSightPC Application aana 1 Features i irrorrek ernie E E E ada Meo PE
153. ches displayed in the data grid Check one or more of the criteria to filter hide certain data grid rows Click an operator to change its value To define quick filters for a search 1 Access the Grid Display Preferences page 2 Optional In the Quick Filters area of the Grid Display Preferences page select the Search Type Pending Search or Marked check box For information on these parameters see Table 74 3 Optional Select the Best Expectation Total Fragments or Best PDE check box click the corresponding operator to set it and enter the appropriate value in the box to the right of the option 4 Optional Select the Only Experiments Where number search has option operator value check box Click number option and operator to display the choices available For information on these parameters see Table 74 Type the value in the box to the right of the operator 5 When you have set all the filters that you want click Apply To remove quick filters 1 Access the Grid Display Preferences page 2 Clear the check box next to the name of the filter that you want to remove Quick Filters Table 74 describes the quick filters available in the Quick Filters area of the Grid Display Preference page Thermo Scientific ProSightPC User Guide 271 B Using the ProSightPC Interface The ProSightPC Interface 272 ProSightPC User Guide Table 74 Quick filters area parameters Sheet 1 of 2 Filter Search Type
154. cifies the number of product ions predicted with cleavage at a C terminal to a glutamic acid Specifies the number of product ions predicted with cleavage at any other non specific residue Specifies the sum of the intensities of the corresponding 7 values just given Thermo Scientific Sequence Tag Scores 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer The ProSightPC application uses a scoring system to rank the matches between a set of sequence tags and a sequence The score for a single tag in a query that matches a sequence is calculated as follows score In PD where e In is the length of the sequence e p is the probability of the ith amino acid occurring in a protein Since multiple tags can match the sequence each tag is weighted by the number of independent possibilities for the tag to match the sequence This is approximated in the ProSightPC application as follows ies iy 4 score I PIG where e Lis the overall length of the sequence e n is the length of the sequences in the tag The final score for a query is then the sum of all tag scores that matched Fragments Explained Box Thermo Scientific The Fragments Explained box displays a percentage representing the number of matching fragments divided by the total number of fragments Table 39 lists the three additional controls in the The Fragments Explained box Table 39 Fragments Explained box paramet
155. coring functions to determine the best match see Scores Box on page 193 for detailed information about scoring methods Figure 8 illustrates the database searching component of shotgun annotation Every ball is a protein form that matches within a mass window Figure 8 Shotgun annotation search strategy Q Q 6 Q Observed Protein Form Correct Gene Family s Correctly Predicted Protein Form a Correct Gene C Falsely Predicted Protein Form Protein Form Most potentially matching protein forms have negligible scores that you can ignore They are represented by the gray balls outside any circle The balls within the blue circle share many fragments among the proteins encoded by a gene family but each identification is at best partial The balls in the concentric red circle represent better identifications because they match fragments that are unique to proteins encoded by a particular gene 14 ProSightPC User Guide Thermo Scientific 1 Introduction to the ProSightPC Application Introduction to Proteomics The blue balls are forms resulting from combinations of modifications that might be abiological The shotgun annotation algorithm created them but they do not exist in living organisms Usually you do not inherently know which protein forms in your database exist or do not exist in real life The green balls are forms resulting from combinations of modifications that do in fact exist in livin
156. creases the search run time Increasing the precursor tolerance results in longer run times Biomarker searches are well suited for identifying biologically relevant proteolytic products You can identify proteins or peptides containing disulfide bonds by setting the precursor search tolerance to 2 5 Da and running the search in delta m Am mode or you can select the Disulfide check box in the New Search in Experiment X dialog box for biomarkers see Figure 56 Thermo Scientific 4 Searching Databases Searching for Biomarkers Setting Biomarker Search Preferences When adding new biomarker searches you can set the default values on the Biomarker Preferences page of the Options dialog box For information on biomarker searches see Searching for Biomarkers on page 125 To set biomarker search preferences 1 Click Biomarker in the Options dialog box The Biomarker Preferences page of the Options dialog box opens as shown in Figure 55 Figure 55 Biomarker Preferences page of the Options dialog box Options 5 General 7 Grid Columns 5 Thrash Search Parameters D a Default BioMarker Search Parameters 27 Sequence Tag Database Demo database for ProSight PC 27 Single Protein Precursor Mass Monoisotopic Fragment Mass Monoisotopic X Delta m Mode Lowe Defau Uppe Precursor Tolerance 1 10 100 ppm v Fragment Tolerance 1 15 100 ppm v Minimum Matches 1 4 100 2 In
157. cursor at m z 624 82 from retention time min 27 62 29 27 75 32 with FT detectionPuf Filter This file passed the following filters Max Frags i Min Frags 10 Min Intact Mass 750 Fragmentation Method CID Ion Type BY k p Precursor Mass List gt Fragment Mass List w Search 1 Absolute Mass Search Edit Comment Search Parameters Precursor Search Window 2 2Da Precursor Type Monoisotopic Fragment Tolerance 15ppm Fragment Type Monoisotopic Database Demo database for Am Mode Off Disulfide Off ProSight PC Matching Proteins to Return Minimum Matches 4 Minimum Matches Percent 0 PTM List Formylation Acetylation Results for Precursor Ion 1 Protein forms found 1 f Add Gene Restricted Search ID Gene Length Mass Mass Diff PPM Diff B Ions Y Ions Total Ions PDE Score E Value gt gt PheATE unknown Trypsin peptide from AA 396 406 in with O missed cleavage sites Type basic Signal Peptide faise Propep false F VvV iD N P LF Lv P G E kK ID Gene Length Mass Mass Diff PPM Diff B Ions Y Ions Total Ions PDE Score E Value 123 21 11 1247 62 0002 1563 i 6 13 265 5 84E 21 Take to Sequence Gazer Information in the Data Manager is displayed in two functional groups e Instrument data which includes the mass values fragmentation method and ion type of the MS MS experiment If you have defined an experiment level comment it is displayed at the top of the Data Manager e Search dat
158. d Ame ay 10001 Da PDAM feo fragment y yt Am ion masses Am 2 Da y 10002 Da b b Am y yt Am Theoretical mass Theoretical fragment for each ion values for each protein candidate protein candidate Performing Searches in Batch Mode Thermo Scientific The ProSightPC application offers two ways of performing multiple searches at the same time e With batch processing you can queue and run a large number of searches over any number of experiments in a single action The ProSightPC application runs any search in the grid with pending searches Use batch processing when you have many pending searches in a PUF file and you would like to run all of them e The Run Searches command runs any searches that are selected highlighted in the data grid Ifa search is not selected the ProSightPC application does not run it ProSightPC User Guide 111 4 Searching Databases Performing Searches To perform searches in batch mode 1 Select all the desired experiments in the data grid e To help you sort entries in the data grid you can click the title row of the column to sort entries from lowest to highest value or highest to lowest e To select contiguous experiment names click the name of the first experiment hold down the SHIFT key and click the last experiment name that you want to select e To select noncontiguous experiment names click the name of the first experiment hold down the CTRL key and click each separate experime
159. d import it from the repository into the ProSightPC application and re run the searches until you obtained good results Then you save the results back to a repository either overwriting the current experiment in the same repository or saving the results in a different repository To export experiments to a repository 1 Choose File gt Export Data to Repository to open the Export Data to Repository dialog box 2 In the Experiments area of the dialog box select the experiments that you want to export Click Select All to choose all the experiments in the repository 3 In the Repository list select the name of the repository where you would like to export the experiments 4 From Category list select the category in the repository where the experiments will be exported The categories available in the list include the default good and bad categories as well as any that you defined Thermo Scientific 2 Getting Started Using Repositories 5 Optional Select the Set New File option if you want to change the PUF or RAW file on whose data the experiments were based Otherwise the ProSightPC application exports the experiments from the currently open PUF file If the file is not a PUF file the ProSightPC application names it untitled puf In the File box that opens select the name of the file or type the name of the file Verify that the Export Data to Repository dialog box resembles the example in
160. d other programs in the ProSight suite Database Name Description Organism Strain Owner Last Update Monday July 23 2012 v Thermo Scientific 8 Using Proteome Databases Creating a Proteome Database a In the Database Name box type the name of the database that you want to create Use only letters numbers and underscores b In the Description box type a brief description of the database c Inthe Organism box type the name of the organism for the proteome database that you are creating d Optional In the Strain box type the strain designation for the proteome database that you are creating e In the Owner box type either your name or the name of the data source f In the Last Update box type the date when the database was last updated or click the down arrow to display a calendar and select a different date 22 Click Finish 23 On the Ready to Load page click Go to create the new database Create New Database Wizard Parameters The pages of the Create New Database Wizard contain the following parameters Database Type Page Parameters Thermo Scientific Table 48 lists the parameters on the Database Type page of the Create New Database Wizard shown in Figure 95 on page 222 Table 48 Database Type page parameters Parameter Description Database Specifies the type of database to create Top Down No Sample Builds a database around whole intac
161. der and move it to the desired location 172 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report Display Columns in the Repository Report Thermo Scientific Table 35 lists the parameters in the repository report Table 35 Repository report columns Sheet 1 of 2 Parameter Description Check box Selects or clears an experiment for export to an Excel spreadsheet Repository Name Displays the name of the repository where an experiment belongs Category Name Displays the name of the category where the experiment is saved Experiment Number Search Type Accession Number Displays the number of an experiment Displays the type of search performed in an experiment absolute mass biomarker sequence tag single protein gene restricted absolute mass GRAM or gene restricted biomarker GRBM Displays the accession number used by the major protein databases such as UniProt to index a protein in a database E Value Specifies the expectation value e value of the best search result in the search See Expectation Value e value on page 194 for more information on the e value Sequence Displays the protein sequence that forms the basis of an experiment Number of Matching Displays the number of matching ion fragments in the Fragments protein identified PTMs Displays the name of the PTM and the RESID number the number of the am
162. e or fragment ion where all carbons are carbon 12 Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Thermo Scientific Thermo Scientific 4 Searching Databases Searching for Biomarkers Table 26 New Search in Experiment X dialog box parameters for biomarkers Sheet 2 of 2 Parameter Fragment Tolerance Description Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Am Mode Determines whether the ProSightPC application conducts the search in delta m Am mode This mode is explained in Performing Searches in Delta m Mode on page 110 Disulfide Indicates whether a protein s cysteines are oxidized Include Modified Determines whether to include PTMs and polymorphisms when Forms you perform a biomarker search Min of Matching Determines whether the search algorithm finds only proteins Fragments containing a minimum number of matching ion fragments The box to the right specifies the minimum number of matching ion fragments Min of Matching Determines whether the search algorithm finds only proteins Fragments containing a minimum percentage of matching ion fragments The box to the right specifies the percentage of matching ion fra
163. e 68 ProSightPC toolbar Sheet 2 of 3 Icon Menu equivalent File gt Save Function Saves a PUF file File gt Import raw gt Post Xtract Builds a new experiment in the current PUF file by using Post XTRACT raw data lia File gt Import raw gt Profile Builds a new experiment in the current PUF file by using high resolution raw data obtained in profile mode Tools gt Experiment Adder Builds a new experiment in the current PUF file by using manually input MS and MS MS data Pe lel View gt Open Data Manager Opens the last experiment using the open PUF file if there are no Experiment tabs open in the Tab Controller Ti Experiment Tools gt Append Predefined Search Adds a predefined search to the selected experiment This icon is available only when an experiment is open and shown in the Tab Controller Pa Experiment Tools gt Edit Masses Experiment Tools gt Edit Comment Changes MS and MS MS data in the ProSightPC application This icon opens a new page showing the precursor and fragment masses of the current experiment This icon is available only when an experiment is open and shown in the Tab Controller Enables you to edit the comment at the top of the current experiment a View Database Information Opens the Database Manager so that you can view information about the proteome databases in the proteome warehouse Tools gt Ma
164. e Am Mode check box if you want to conduct the search in delta m Am mode For more information on delta m Am mode see Performing Searches in Delta m Mode on page 110 Select the Disulfide check box if you know that the protein s cysteines are oxidized Note The ProSightPC application looks for only one disulfide bond In the Hit Filtering section set at least one of the following filters otherwise the ProSightPC application returns all protein forms that are searched even proteins that have no matching fragments a Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum number of matching ion fragments these protein forms are called hits Then specify the minimum number of matching ion fragments in the box to the right b Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum percentage of matching ion fragments Specify the percentage of matching ion fragments in the box to the right ProSightPC User Guide 121 4 Searching Databases Searching for Absolute Mass 122 ProSightPC User Guide 11 12 13 14 15 c Select the Min Score check box to determine whether the search algorithm finds only proteins with an e value that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right
165. e Maximum Mass kDa box enter the highest mass to be considered for the precursor THRASH only The minimum value is 1 and there is no maximum value The default is 60 f In the Minimum Charge State box select the smallest charge state to be considered for the precursor THRASH only The minimum value is 1 and there is no maximum The default is 1 54 ProSightPC User Guide Thermo Scientific Thermo Scientific g 2 Getting Started Processing LC MS MS Data Files In the Remainder Threshold box enter the remainder of the fit that is left in the scan Xtract only The Remainder Threshold option as a percentage determines whether a packet is further processed after an averagine pattern is subtracted This option is important if overlapping peaks are analyzed If there is an overlapping pattern of two peptides and the first pattern has been identified the first averagine pattern is subtracted The remaining pattern is only processed if its peaks the remainder have an intensity that is greater than that specified by the Remainder Threshold option Setting the Remainder Threshold option to 100 percent disables deconvolution of overlapping patterns The ProSightPC application recognizes only the first most intense pattern and ignores overlapping less intense patterns Setting Remainder Threshold to 10 percent allows the deconvolution of a peptide even if it is overlapped by a peptide pattern with 10 fold intensity The minimu
166. e Repository Report To Import Experiments from a Repository into the PUF File on page 175 Thermo Scientific ProSightPC User Guide 69 2 Getting Started Using Repositories Import Data from Repository Dialog Box Parameters Table 12 lists the parameters in the Import Data from Repository dialog box shown in Figure 35 on page 70 Table 12 Import Data from Repository dialog box parameters Parameter Description Repository Specifies the repository from which to import the experiments Category Specifies the category of experiments in the repository to import This list is not available unless you select the repository first The categories available include the default good and bad categories as well as any that you defined File Specifies the RAW or PUF file containing the data on which the experiments were based This list is not available unless you select the category first Experiments Lists all the experiments in the selected file so that you can select the experiments to import Select All Selects all the experiments in the selected file for importation Exporting Experiments to a Repository 70 ProSightPC User Guide You can export the experiments in the ProSightPC data grid into a repository For example suppose that you processed a RAW file and then viewed the repository report that was generated You decide to further investigate a specific experiment that you find interesting You woul
167. e check box for any of the columns that you want to hide in the data grid Click Refresh to hide the columns Thermo Scientific Thermo Scientific To restore default columns B Using the ProSightPC Interface The ProSightPC Interface To reinstate the default settings click Restore Defaults Show Columns Area Parameters Table 73 describes the parameters available in the Show Columns area of the Grid Display Preferences page Table 73 Show Columns area parameters Sheet 1 of 3 Parameter Description Exp ID Displays a column showing the ProSightPC assigned experiment number Search ID Displays a column showing the ProSightPC assigned search number Marked Displays a column showing experiments marked by an asterisk These experiments are also marked by a ProSightPC symbol to the left of the experiment Exp Comment Displays a column showing a brief description of the experiment Search Comment Displays a column showing a brief description of the search Search Type Displays a column showing the type of search First Precursor Mono Displays a column showing the monoisotopic mass of the first precursor ion First Precursor Avg Displays a column showing the average mass of the first precursor ion Largest Precursor Mono Displays a column showing the monoisotopic mass of the largest precursor ion Largest Precursor Avg Displays a column showing the average mass
168. e in the mass spectrometer you can search both precursor ions against the same set of fragment ions e Default Selected Multiplexes fragmentation data e Unselected Creates a new experiment for each precursor detected Thermo Scientific ProSightPC User Guide 61 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 5 of 8 Parameter Relative Precursor Threshold Description Specifies the threshold for selecting the precursor intensities when there are multiple precursors within the window The ProSightPC application selects only precursors with intensities within the top x percent of the top precursor Range 1 100 Default 10 Add Remainder Afterwards Xtract only 62 ProSightPC User Guide Determines whether the ProSightPC application adds the remaining intensities to the output spectrum during analysis of the precursor ions If a pattern is identified during the processing of the input file with the Xtract algorithm the corresponding averagine pattern is subtracted from the input spectrum The remaining intensities or remainders are then processed again with the Xtract algorithm so that Xtract can find an overlapping low intensity pattern If there is no overlapping second pattern but a small spike in the first pattern the spike is not visible in the deconvolved spectrum but will show up in the remainder spectrum unless you used Add Remainder Afterw
169. e is organized into the following regions as shown in Figure 80 Search Parameter Display Scores Box Fragments Explained Box Mass Diagram Interactive Fragment Map Amino Acid Information Box Fixed Modifications Box Matching Fragments Table Non Matching Fragments Table Figure 80 Sequence Gazer window Search parameter display Grid Display Preferences Report repository12 Experiment 42 Sequence Gazd Sequence Gazer Interactive fragment map k Sequence Gazer Mass diagram Scores box 6 Using the Sequence Gazer to Search for Single Proteins NOTE text denotes current selection Precursor Mass Type Mono or Avg Scores A m On Off Fragment Tolerance 25 Da ppm Mass Type Mono or Avg P Score 5 72E 71 Expectation 5 72E 71 PDE 8 8500 le Difference 4 7415 ba 601 8500 ppm gt l 42 Fragments Explained Rescore Save Cancel Observed 7878 2800 Theoretical 7883 0200 H o r s r vto P 11g L A R viT K LQFCLTLQ V RLVLELFEM DIDLT S RLS LRLE GLD V L T LTLLELSLEFRTEFATREREL R P Show Matching Fragments Total 62 fragments fragments table Yshow Non Matching Fragments Total 84 fragments ID m z Monoisotopic Mass Monoisotopic Intensity 6p 789 4226 788 4154 8 086638E 05 9p 859 4683 858 4610 8 493902E 06 11i 870 4611 869 4538 5 419794E 07 586 8399 171 6652 2 830307E 05
170. e selecting top filters represent the most commonly used Click an operator to change its value Custom Filters 4 can be defined and used below V Pending Search Successful Search E Search Type F Best Expectation lt V Marked 7 Matching Forms E Pending Search X Matching Forms gt Exp Comment V Best Eqectation Marked A Best PDE A Search Comment Best P Score 7 Search Type Best PDE E Only Experiments where At Least One search has Best lt First Precursor Mono E Highest Total lons First Precursor Avg E b c lons nit Largest Precursor Mono y z lons Use f Is Value Then Otherwise Largest Precursor Avg Fragments First m z Mono E Precursors F First m z Avg E First Abundance Largest m z Mono Largest Abundance Largest m z Avg Best Seq Score fase Boa e Using Filters in the Show Columns Area e Using the Filters in the Quick Filters Area e Using the Filters in the Custom Filters Section Using Filters in the Show Columns Area Thermo Scientific Use the Show Columns area to display or hide columns in the data grid Each of the parameters shown in Table 73 controls the appearance of a column in the data grid To access the Grid Display Preferences page e Click the Grid Display Preferences tab To set the default columns displayed in the data grid 1 Choose Tools gt Options gt Grid Columns to open the Grid Columns page of the Options dialog box shown in Figure 115 ProSightPC User Guide 267
171. earch type selected in the adjacent list absolute mass biomarker sequence tag single protein gene restricted absolute mass GRAM or gene restricted biomarker mass GRBM Thermo Scientific ProSightPC User Guide 177 5 Viewing Search Results Viewing the Results in a Repository Report E value confident match Displays all the experiments whose e value is less than the value that you entered in the box The default value 1E 4 is recommended for a confident match For more information on the e value see Expectation Value e value on page 194 e PTMs Displays all the experiments with PTMs when you select Show e Mass Difference Displays all the experiments whose mass difference is less than the value that you entered in the box e Category Displays all the experiments whose category is the same as that selected in the adjacent list e Unique Identifications Displays all the experiments except for those with redundant accession numbers Figure 72 Fixed Filters section Filters Fixed Filters Search Type x m Evalue lt 1E 4 confident Hit 7 PTMs Y Mass O Difi lt 50 Da L Category X r Unique Identifications filters redundant accession number 2 Click Apply Filters To apply existing custom filters 1 In the Custom Filters section of the Actions area see Figure 70 on page 171 select the Show Custom Filters check box The Custom Filters section
172. earching for Gene Restricted Absolute Masses on page 146 Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Describes the database that you want to search Precursor Mass Type Specifies the type of precursor ion mass to use Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Precursor Tolerance Specifies the tolerance within which your sliding window must fall when you test all protein forms for biomarker peptides and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Fragment Mass Type Thermo Scientific Specifies the mass type of the fragment ions to use Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion ProSightPC User Guide 157 4 Searching Databases Performing Gene Restricted Searches 158 ProSightPC User Guide Table 31 New Search in Experiment X dialog box parameters for biomarkers Sheet 2 of 2 Parameter Fragment Tolerance Description
173. ection 271 R RAW files analyzing scans in 54 assigning neutral mass to 79 contents of 9 fragmentation scans in 54 59 importing into ProSightPC 7 27 importing targeted 73 246 Post Xtract 74 THRASH 79 80 input to High Throughput Wizard 29 34 input to ProSightPC 9 removing 34 targeted 28 reagent ions 58 65 Refresh icon 215 Remove Database icon 215 Remove Repository icon 216 Remove Selected Search icon 88 103 105 110 repositories adding 250 creating 7 36 67 creating in the High Throughput Wizard 36 deleting 69 description 66 editing 68 250 exporting 219 exporting data to 246 exporting experiments from data grid to 71 exporting experiments to 176 exporting from proteome warehouse 216 218 importing 218 importing experiments from 69 175 246 importing into proteome warehouse 216 217 removing from proteome warehouse 216 219 reports See repository reports selecting in the High Throughput Wizard 36 specifying name of 49 Repository Report dialog box 169 174 250 261 Repository Report icon 169 261 repository reports changing the order of columns in 172 columns in 173 contents 165 example 170 171 Thermo Scientific Index Q exporting experiments to Excel spreadsheets 177 filtering data in 177 generating 168 181 250 252 261 opening 43 RESID button 164 database 12 198 221 235 designation 199 237 identification number 12 237 number 173 RESID annotated sequence 164 reverse databases 222 Reve
174. ed Minimum RL value Specifies the minimum confidence level Maximum Charge Specifies the maximum charge to be used by the THRASH algorithm Last m z Start Scan Number Specifies the highest mass to charge ratio m z value considered Specifies the first scan number scanned End Scan Number Specifies the last scan number scanned Precursor Mass Specifies the mass of the precursor ion Type m z Specifies the type of precursor ion mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Calculates the intact mass if only the mass to charge ratio and the charge are known It opens the Intact Mass Calculator dialog box shown in Figure 38 on page 75 Fragmentation Method Specifies one of the following fragmentation methods e CID e ECD e IRMPD e HCD e ETD For information on these methods see Fragmentation Methods on page 9 Thermo Scientific 2 Getting Started Entering Data Manually Table 18 Build Experiment from Profile RAW Data dialog box parameters Sheet 2 of 2 Parameter Description Predefined Search Displays the predefined searches set for an experiment Check All Selects all predefined searches Uncheck All Does no
175. ee Viewing the Results in a Repository Report on page 168 To generate a status report 1 Open the desired PUF file 2 Choose Tools gt Reports gt Status Report A summary of all experiments and searches contained in the PUF file appears in a new window as a text document as shown in Figure 66 This text document is organized by experiment number and is subdivided into the types of searches Thermo Scientific ProSightPC User Guide 165 5 Viewing Search Results Viewing the Results in a Search Report Figure 66 Status report 3 Absolute Mass Searches Biomarker Searches Sequence Tag Searches GRAM Searches GRBM_ Searches Single Protein 1 Total number of hits with an expected score no greater than 2 2 Total number of hits with an expected score no greater than 0 5 3 Total number of defined searches that have been run 4 Total number of defined searches Source C Program Files ProSightPc Test See Te Res raw Absolute Mass Searches 0 Biomarker Searches Sequence Tag Searches GRAM Searches GRBM_Searches Single Protein Source C Program Files Prosight Absolute Mass Searches Biomarker Searches Sequence Tag Searches GRAM Searches GRBM_ Searches Single Protein Test Data ETDfraction03 raw o o Source C Program Files ProSightPc Test Absolute Mass Searches Biomarker Searches Sequence Tag Searches GRAM Searches GRBM_ Searches Single Protein ata ETDFraction03
176. eee 110 Performing Searches in Batch Mode 5 2040 spear baraecre Dawes 111 Searching for Absolute Mass s i4 s 00 advo paces hed eae ote ade eA 113 Setting Absolute Mass Search Preferences 0 0c cece eee eee 115 Searching for Absolute Mass y os 00b 3 Soe e vent veces by Same a 119 Thermo Scientific Contents Searching for Bioharkersss Sastieate a daG auton Baan bn atginbo dal ota Dewees 125 Setting Biomarker Search Preterences vc 4 2 casa ide ceeded wasecartsh alae 127 Searching for Bigmarkerse iso dao ae bawn tee eee ue ane ee ee 130 Searching for Sequence Tags casa d alsa dake e rs bal dies Mike Beg a 136 Setting Sequence Tag Search Preferencess os baited eens ae eed ees 136 Searching for Sequence Tags wis sede deisel eh baal sa Ree eS 139 Searching for Single Proteins 43a ae eae eee ee aNd 143 Setting Single Protein Search Preferences hoi a waka nae ahi wes 143 Performing Gene Restricted Searches nuunuu domed eee aes ae eee 146 Searching for Gene Restricted Absolute Masses 000 eee aes 146 Searching for Gene Restricted Biomarkers 000 0000 eee eee 152 Performing MS Hybrid Searches 2 o 4 ceiaegaledaga seadan detains 159 Analyzing MS MS Experiments vies sao enka ton a eke s Rete ees 159 Chapter5 Viewing Search Results 0 00 ee cece eee eee e eee ee eeee 161 Viewing the Results in the Tab Controller 0 000008 161 Viewing the Results in a Search Report j2i
177. een run In the Data Manager click Run Search To view the results of the search see Viewing Search Results on page 161 Identifying a Protein and Accessing the Sequence Gazer Thermo Scientific To access the Sequence Gazer from any protein identified in a completed search Select the desired search and click its corresponding side arrow in the Data Manager Locate the desired protein identification in the search results and click its corresponding side arrow Click Take to Sequence Gazer circled in Figure 79 ProSightPC User Guide 187 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer Figure 79 Take To Sequence Gazer button Grid Display Preferences Experiment 1 ID Length Mass Mass Diff PPM Diff C Ions Z Ions Total Ions PDE Score Expectation 674572 146 16406 900000 372 647000 22208 400000 11 1 12 89 300000 7 83E 11 Take to Sequence Gazer gt gt CALM_HUMAN P62158 Calmodulin CaM Type predicted Signal Peptide false Propep false A D Q L T E E Q I A E F Kt E A F S L F D Kt D Gt D G T I T T K E L G T V M R S L G QHtN P T E A E L QtD M I N E V D A D G N 6 7 2 D F PEF LTT ABARKA DT DS E E ET R E A FT Re V FIDIKID G N G Y 1 S A A E L R H V HN TIN L G E K L T D E E N D E A I R E A D r D G D G O WN Y E E F G f 8 T A K ID Mass Diff PPM Diff C Ions Z Ions Total Ions PDE Score Expectation 41 695800 2484 9
178. eferenced for each search contains recommendations for running the search Table 2 Types of searches available in the ProSightPC application Type of search Location of information Absolute mass searches Searching for Absolute Mass on page 113 Biomarker searches Searching for Biomarkers on page 125 Sequence tag searches Searching for Sequence Tags on page 136 Single protein searches Using the Sequence Gazer to Search for Single Proteins on page 183 Gene restricted absolute mass Searching for Gene Restricted Absolute Masses on searches page 146 Gene restricted biomarker searches Searching for Gene Restricted Biomarkers on page 152 MS hybrid searches Performing MS Hybrid Searches on page 159 Iterative Searching 4 ProSightPC User Guide You can build an automatic iterative score based search tree in the ProSightPC application You select a predefined search specify a condition select an action and select a category All experiments pass through a first level of search logic and the action taken next depends on the results of the search for each experiment If the experiment results pass the condition that you set for example if at least one of the matching protein forms received an expectation value e value less than 1E 4 you can either load the experiment to the category selected or indicate that a second level of searching be performed Figure 4 illustrates this
179. elta m Am mode Performing Searches in Delta m Mode on page 110 explains this mode Disulfide Indicates whether a protein s cysteines are oxidized Min of Matching Determines whether the search algorithm finds only proteins Fragments containing a minimum number of matching ion fragments The Min of Matching Fragments Min Score box to the right specifies the minimum number of matching ion fragments Determines whether the search algorithm finds only proteins containing a minimum percentage of matching ion fragments The box to the right specifies the percentage of matching ion fragments Determines whether the search algorithm finds only proteins with an e value that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right box e Default lt Indicates that the first value is less than or equal to the second value e gt Indicates that the first value is greater than or equal to the second value Max Proteins to Return Specifies the maximum number of proteins to return in the search Fixed Modifications Specifies the chemical modifications present on all instances of a given type of amino acid in the observed protein PTM Handling Specifies the PTMs that you want queried Terminal Mods Specifies the fixed terminal modification for each terminus e N Terminal Mod Specifies the fixed terminal modificat
180. ensity of 1 is assigned to each fragment ion 6 Optional In the Please Check Any Predefined Analyses That You Would Like Included with Your Experiment box select any predefined searches to add Click Check All to add all listed predefined searches Click Uncheck All to clear all listed predefined searches 7 Click Create The ProSightPC application creates a new experiment from all the values entered for intact masses and fragment masses and adds it to the data grid If the experiment already exists in the data grid it receives the next available experiment number Thermo Scientific ProSightPC User Guide 85 2 Getting Started Entering Data Manually Experiment Adder Dialog Box Parameters 86 ProSightPC User Guide Table 19 lists the parameters in the Experiment Adder dialog box shown in Figure 42 on page 84 Table 19 Experiment Adder dialog box parameters Sheet 1 of 2 Parameter Fragmentation Methods Description Specifies one of the following fragmentation methods e CID e ECD e IRMPD e HCD e ETD For information on these methods see Fragmentation Methods on page 9 Precursor Ion Data Type Specifies the method of inputting the precursor ion data You can select Manual or Upload from the Type list e Manual Inputs the precursor ion data e Upload Loads the precursor ion data from an ASCII text file or files Precursor Ion Data m z Precursor Ion Data Mass Type
181. ent Ion Data Type box select the mass type of the fragment ion in the Mass Type area Thermo Scientific 2 Getting Started Entering Data Manually Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Intensities Specifies the intensity of the fragment mass If you select Upload in the Fragment Ion Data Type box enter the path and name of the ASCII text file or files containing the precursor ion data in the Text File box or click Browse to browse for them ASCII text files must be formatted with five columns of numbers separated by white space Each row represents a separate ion mass The columns must be arranged as follows Monoisotopic m z Specifies the monoisotopic mass to charge ratio m z value corresponding to the fragment ion Average m z Specifies the average mass to charge ratio m z value corresponding to the fragment ion Monoisotopic Mass Displays the observed monoisotopic mass of the fragment ion measured in Da Average Mass Displays the observed average mass of the fragment ion measured in Da Intensity Specifies the abundance of the fragment ion When entering fragment ion density data manually you can leave the Intensities box empty In this case the default int
182. enter multiple sequence tags properly formatted with one sequence tag per line in the space provided The sequence tag search automatically searches for the entered sequence tag and its reverse 11 Click Save The new search appears in the data grid with yes appearing in the Pending Search column 12 To execute the search from the data grid right click the pending search and then choose Run Search number To view the results of the search see Viewing Search Results on page 161 Thermo Scientific ProSightPC User Guide 141 4 Searching Databases Searching for Sequence Tags New Search in Experiment X Dialog Box Parameters for Sequence Tags Table 28 lists the parameters in the New Search in Experiment X dialog box for sequence tags shown in Figure 58 on page 140 Table 28 New Search in Experiment X dialog box parameters for sequence tags Sheet 1 of 2 Parameter Search Type Database Description Description Specifies the type of search to perform Absolute Mass See Searching for Absolute Mass on page 113 Biomarker See Searching for Biomarkers on page 125 Sequence Tag See Searching for Sequence Tags on page 136 Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 Gene Restricted BioMarker See Searching for Gene Restricted
183. ents Sheet 1 of 2 System Requirements Computer e 1 GHz processor 2 GHz dual core recommended e 1 GB 666 MHz RAM 2 GB recommended e CD ROM drive e 32 MB graphics card 64 MB or greater e 75 GB available on the C drive e Video card and monitor capable of 1280 x 1024 resolution XGA e NTFS format X ProSightPC User Guide Thermo Scientific Preface Table 1 System requirements Sheet 2 of 2 System Requirements Thermo Scientific All LTQ FT based instruments Panne e All Thermo Orbitrap based instruments supported Software Thermo Xcalibur 2 1 recommended Note The ProSightPC application operates in the Microsoft Windows XP and Windows 7 environments and is not guaranteed to function on any other platform Installing ProSightPC To install the ProSightPC software 1 Open the PSPC 3 0 folder on the CD DVD 2 In the setup folder double click setup exe The InstallShield Wizard opens 3 On the first page of the wizard shown in Figure 1 click Install to allow the prerequisites to be installed Figure 1 First page of the InstallShield Wizard ProSightPC InstallSh l hield d Wizard ProSightPC requires the following items to be installed on your computer Click Install to begin installing these requirements Status Requirement Pending Microsoft Visual C 2010 Redistributable Package x86 4 On the Welcome to the InstallShield Wizard for ProSightPC pa
184. epository list on the Running Highthroughput Logic page shown in Figure 20 select the name of the repository To create a new repository 1 Click New Repository 2 Enter the new repository name in the New Repository dialog box shown in Figure 33 on page 67 36 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files 3 Click OK The Edit Add Repositories dialog box appears as shown in Figure 34 on page 68 4 If you do not want to edit the repository click Save If you want to edit the repository follow the instructions in Editing a Repository on page 68 and click Save in the Edit Add Repositories dialog box The name of the repository appears in the Repository box of the Running Highthroughput Logic page of the High Throughput Wizard For more information on creating a repository see Creating a Repository on page 67 Selecting an Existing Search Tree To select an existing search tree e From the Search Tree Name list on the Running Highthroughput Logic page of the High Throughput Wizard dialog box shown in Figure 20 on page 36 select the name of the search tree Creating a Search Tree Thermo Scientific The ProSightPC application automates searches through an iterative search tree to make the best use of your time If you find a match during the first search you do not have to run the second longer search Each experiment created by the ProSightPC application
185. er it is possible regardless of whether the input includes it N terminal acetylation is a very common PTM Apply N Terminal Formylation Adds N terminal formulation Select this option if you are building a prokaryotic database Prokaryotes use N formylmethionine for initiation 10 Click Next The Complexity page of the Create New Database Wizard appears as shown in Figure 98 Thermo Scientific Thermo Scientific 11 12 13 8 Using Proteome Databases Creating a Proteome Database Figure 98 Complexity page of the Create New Database Wizard amp Create New Database Complexity Rarely a protein has so many known modifications that it s not feasible to store all possible forms These settings specify howto control this combinatorial expansion V Consider SNPs V Consider PTMs Maximum features per sequence 13 Maximum mass Da 70000 All PTMs As indicated on the Complexity page a protein might have so many known modifications that it is not feasible to store all possible forms On this page you can set options to specify how to control this combinatorial expansion If you are uncertain about the values to set use the default values If you want to include potential genetic variation as annotated in the UniProt database select the Consider SNPs check box This option enables you to incorporate sequence polymorphisms into the database If you wan
186. erime Fa Gxperiment 11 Ea Eqperiment 39 FA Bperime Fi Experiment 12 EA Experiment 40 Fd Eperime FG Experiment 13 EA Emerment 41 FA perme DHK i lt EE r Adding Experiments to PUF Files There are several different ways to add experiments to the PUF file To add an experiment to the PUF file and to the data grid e Import RAW files with the Post Xtract option See Importing a Targeted RAW File with the Post Xtract Option on page 74 e Import RAW files with the Profile option See Importing a Targeted RAW File with the Profile Option on page 79 e Use the Experiment Adder See Entering Data Manually on page 84 e Import data from a repository See Importing Experiments from a Repository on page 69 and Using the Repository Report To Import Experiments from a Repository into the PUF File on page 175 Copying Experiments from One PUF File to Another You can copy experiments from a source PUF file to a destination PUF file using the two panes in the Experiment Manager To copy an experiment from one PUF file to another 1 Choose Tools gt Experiment Manager 2 In the source left pane of the Experiment Manager shown in Figure 43 on page 92 select an experiment to be copied 3 Drag the experiment from the source left pane to the destination right pane or click the green arrow to send a copy of the experiment to t
187. ers Parameter Description Rescore Recalculates all scores and matching fragment information Save Adds a new completed single protein mode search to the experiment Cancel Discards the changes that you have made and returns to the Data Manager ProSightPC User Guide 197 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Mass Diagram The mass diagram displays the difference between the observed and theoretical mass expressed in daltons and parts per million It contains the boxes or lists described in Table 40 Table 40 Mass diagram parameters Parameter Description Observed Contains a list that displays all the precursor masses detected by the ProSightPC application Theoretical Displays the experimental precursor mass including all user input changes as of the last score Difference Displays the difference between the figure in the Observed list and the figure in the Theoretical box Interactive Fragment Map The interactive fragment map shown in Figure 82 is an interactive display of the protein sequence along with any PTMs and the matching fragment information Figure 82 Interactive fragment map Graphical Fragment Mapper GSS HH alL t F LN S LN FKSVR H HHS 8 G LY PRGSHMAT O TREODI S5 a A EEE EEL EREE The theoretical protein sequence taken from the proteome warehouse is listed from left to right and from top to bottom Depending on the ion ty
188. es menu Table 61 Databases menu commands Command Databases gt Database Manager Description Opens the Database Manager window shown in Figure 90 on page 215 The Database Manager handles all proteome warehouse management and manipulation functions Databases gt Create a Custom Database Opens the Welcome to the New Database Wizard page of the Create New Database Wizard shown in Figure 94 on page 221 so you can manually create a custom database Databases gt Download ProSightPC Databases Databases gt Link to UniProt Downloads databases from the ProSightPC application FIP Web site Connects you to the UniProt database which is an international repository of organisms It contains all the proteins and genes that are known for a specific organism ProSightPC User Guide 249 A ProSightPC Reference ProSightHT Menu ProSightHT Menu Table 62 lists the commands on the ProSightHT menu 250 ProSightPC User Guide Table 62 ProSightHT menu commands Command ProSightHT gt HighThroughput Wizard ProSightHT gt Edit Add Repository Description Opens the Process a Dataset page of the High Throughput Wizard shown in Figure 18 on page 29 so you can import data from a RAW or PUF file specify a repository in which to store the results of the search and create a search tree Opens the Edit Add Repositories dialog box shown in Figure 34 on page 68 so you can edit an existing reposit
189. es the repository data displayed in the bottom half of the Database Manager window Changes the view of the repositories listed in the bottom half of the Database Manager window Repository Name Displays the name of the repository Repository Description Displays a brief description of the repository of Projects Displays the number of project categories in a repository of Files Displays the number of files included in a repository of Experiments Displays the number of experiments included in a repository Thermo Scientific 8 Using Proteome Databases Importing a Proteome Database or Repository Importing a Proteome Database or Repository Use the following procedures to load an existing proteome database or repository respectively into the ProSightPC application proteome warehouse To import a proteome database 1 Choose Databases gt Database Manager to open the Database Manager window 2 Click the Import Proteome Database icon Qi in the toolbar at the top of the Database Manager window The Open dialog box appears so that you can enter or browse for the name of the PWE 3 Select the PWF that you want and click Open The Import Databases dialog box appears as shown in Figure 91 Figure 91 Import Databases dialog box mpor oles Databases F saccharomyces_cerevisiae_2012_06_top_down_complex impot J _Cancet _ 4 Select the check box next to the
190. esults in the tab controller 1 When the job queue indicates that a search has finished running double click the corresponding experiment identification Exp ID line in the data grid to open the Data Manager for the experiment The ProSightPC application automatically highlights this line 2 In the Data Manager click the side arrow that precedes the precursor ion that you are interested in Search results open that are similar to those shown in Figure 63 Thermo Scientific ProSightPC User Guide 161 5 Viewing Search Results Viewing the Results in the Tab Controller Figure 63 Typical search results in the tab controller Data Management meea o Sj Experiment 93 Source C Data TopDownClass ETDfraction03 raw 1021 0567 ETD fragmentation for precursor at m z 1022 06 from retention time min 32 45 240 32 71 242 with FT detection Exper i ment defi n iti on E gt Fragmentation Method ETD Ton Type CZ An gt Precursor Mass List gt Fragment Mass List b Search 1 Absolute Mass Search w Search 2 BioMarker Search Edit Comment Search Parameters Fragment Tolerance 15ppm Fragment Type Monoisotopic Precursor Tolerance 10ppm Precursor Type Monoisotopic Database Saccharomyces cerevisiae m Mode Off Neuro Peptide On Disulfide Off 2012_06 Top Down Complex Matching Proteins to Return aa Matches 4 Sa Percent 0 Sea rch pa ram eters gt PTM List Formylation FAD Geranyl gerany
191. ew File Changes the PUF or RAW file on whose data the experiments were based In the File box that opens select the name of the file from the list or type the name of the file Importing Targeted RAW Files 72 ProSightPC User Guide If you want to import a targeted RAW file as input or if you want to enter data manually into the ProSightPC application you cannot use the High Throughput Wizard as you can with LC MS MS RAW files You must use the procedures in this section to import targeted RAW files or use the instructions in Entering Data Manually on page 84 if you want to enter data manually In order for the ProSightPC application to identify and characterize proteins mass spectral data must be converted to neutral mass values An analysis to infer mass AIM is an operation in which high resolution mass spectral data from proteins or large peptides is converted into neutral monoisotopic or average masses IMPORTANT The ProSightPC application works with neutral masses only The relative advantages of different AIMs are beyond the scope of this manual For more information refer to the XTRACT Manual or Horn et al 2000 The ProSightPC application supports three different AIMs e Post Xtract Takes the small file generated by the Xtract algorithm within Qual Browser and uses it as the neutral mass data This algorithm has a better mass accuracy than THRASH but is a little slower This option is the default l Horn
192. ext box originally Specifies the text that is used to search for files in the list If files in blank this list match the search text they are selected for inclusion in the report Search Searches for the text specified in the text box Unselect Clears a particular search Export Directly to File Exports a file without displaying the report Report Only Best Hit Attempts to break ties in e values or p scores by examining the Per Search intact mass differences and choosing the one with the smallest mass difference Generate Generates the repository report and displays it in the tab controller section of the graphical user interface as shown in Figure 70 on page 171 Using the Repository Report To Import Experiments from a Repository into the PUF File The repository report page displays the desired data from the repository but the data is only for viewing To manipulate the data you must import the data into the PUF file perform any desired operations such as adding or changing searches and export it back to the repository to save the changes that you have made You can import experiments from a repository by using the following procedure or by using the procedure outlined in Importing Experiments from a Repository on page 69 To use the repository report to import experiments from a repository 1 In the repository report page select the experiments that you want to import into the ProSightPC application
193. file files for future conversion on your computer processing Browse Enables you to browse to the directory in which to save the PUF file Skip search tree logic Does not search the data against a proteome database Process Puf files Processes PUF files Remove Removes the selected PUF file displayed in the box Add Opens a dialog box so that you can choose the PUF file to import Thermo Scientific ProSightPC User Guide 35 2 Getting Started Processing LC MS MS Data Files Selecting or Creating a Repository When you click Next in the Process a Dataset page of the High Throughput Wizard the Running Highthroughput Logic page of the High Throughput Wizard appears as shown in Figure 20 so that you can select or create a repository and define a search tree Figure 20 Initial Running Highthroughput Logic page of the High Throughput Wizard High Throughput Wizard ACEA Running Highthroughput Logic Select a repository to load results to and select create a search tree with Repository Edit Repository New Repository Search Tree Name New search tree x Save V Experiment Filter Min fragments 10 Max fragments 500 4 Min Intact Mass 750 _ lt Prev_ _Net gt For information about the parameters on this page of the dialog box see Table 7 on page 49 To select a repository e From the R
194. frared laser is directed at the ions in the vacuum of the mass spectrometer The target ions absorb multiple infrared photons until they reach more energetic states and begin to break bonds resulting in fragmentation e HCD With the high energy collision induced dissociation HCD method of fragmentation the projectile ion has laboratory frame translation energy higher than 1 keV e ETD With the electron transfer dissociation ETD method of fragmentation singly charged reagent anions transfer an electron to multiply protonated peptides within an ion trap mass analyzer to induce fragmentation ETD cleaves randomly along the peptide backbone while side chains and modifications such as phosphorylation are left intact This method is used to fragment peptides and proteins lon Types The ProSightPC application supports both c z and b y ion types which are shown in Figure 6 Figure 6 c z and b y ion types y i Z H RIH A N gt C terminus N terminus 8 em Az O H R c introduction to Proteomics The ProSightPC application works with mass values inferred from mass spectral data from middle down bottom up and top down proteomics MS MS experiments 10 ProSightPC User Guide Thermo Scientific 1 Introduction to the ProSightPC Application Introduction to Proteomics Middle Down Bottom Up Proteomics Middle down bottom up proteomics uses two methods to prepare samples for introduction into the mass analyzer depending
195. g 1 Start a search by following the instructions in Creating a Predefined Search on page 101 2 In the Search Type list in the New Predefined Search dialog box shown in Figure 53 on page 120 select Sequence Tag The appearance of the dialog box changes as shown in Figure 58 Thermo Scientific ProSightPC User Guide 139 140 4 Searching Databases Searching for Sequence Tags ProSightPC User Guide Figure 58 New Search in Experiment X dialog box for sequence tags ew Search in Experim eA Search Type Sequence Tag Search Database Demo Database for ProSightPC Minimum Tag Score 2 Compile Sequence Compiler Tolerance in ppm 10 Minimum Tag Size 4 Fragment Mass Monoisotopic Fixed Modifications Cysteine E F Methionine Lysine Isoleucine H E Leucine G4 Arginine Valine ch Tl Dentinn D Manually Enter Example R V P IJL 5 Select either the Compile Sequence or the Manually Enter option 3 In the Database list select a description of the database that you want to search 4 In the Minimum Tag Score box enter the lowest acceptable sequence tag score reported as a match Thermo Scientific 4 Searching Databases Searching for Sequence Tags e Compile Sequence Determines the sequence tags and compiles them before searching them If you select this option complete step 6 through step 9 and step 11 This o
196. g organisms not the observed form but real nonetheless Finally the red balls represent the forms that you actually observed in the mass spectrometer Because you generate all potential protein forms in shotgun annotation a large number are not going to exist in nature Using shotgun annotation you can detect protein forms that you previously were not aware of or could not observe Figure 9 shows an example of a shotgun annotated sequence Figure 9 Shotgun annotated sequence Amino Acid Sequence TKDSSELVQS TKDSSELVQS Ac P Ac P Protein Annotated T055 KD 037SSELVQS T 055 KDSSELVQ 037 S Sequence gt Search Modes and the Top Down Funnel In all but the most exceptional cases top down proteomics experiments only generate partial fragment information in the MS MS phase so there is no guarantee that you will observer all the information necessary to fully characterize an unknown protein This limitation leads to what is known as the top down funnel Figure 10 shows a schematic representation of the top down proteomics funnel The top of the funnel represents the space of all possible observed combinations of MS and MS MS data A certain area at the top contains those combinations that identify the unknown protein and fully characterize any PTMs present Additional combinations allow for identifying and partially characterizing the protein In some cases only identifying the protein is possible Thermo Scientifi
197. ge Close All to close all open pages or Close All But This to close all open pages except the selected page e Right click a page and choose Refresh to re display the contents of the page For more information see Displaying Data in the Data Manager on page 205 Data Manager The Data Manager appears in the ProSightPC interface when you double click an experiment choose View gt Open Data Manager or click the Open Data Manager icon al Displaying Data in the Data Manager on page 205 describes the functions of the Data Manager in detail Grid Display Preferences Page Use the Grid Display Preferences page shown in Figure 114 to select the type of information to display in the data grid The Grid Display Preferences page automatically appears when you open a PUF file Clicking View gt Grid Preferences also displays this page The Grid Display Preferences page consists of three areas Show Columns Quick Filters and Custom Filters 266 ProSightPC User Guide Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface Figure 114 Grid Display Preferences page Grid Display Preferences Show Columns Quick Filters Use check boxes to display values in the data grid Press Refresh Use to filter shown rows in the datagrid Rows will only show if their condition is evaluated to TRUE The when don
198. ge click Next Thermo Scientific ProSightPC User Guide xi Preface 5 On the License Agreement page select I accept the terms of the license agreement and click Next The Thermo License Activation dialog box appears 6 Enter your activation code in the Activation Code boxes and any contact information in the Contact Information boxes as shown in Figure 2 and click Activate Obtain the activation code from the back of the DVD jewel case Activation code in the Thermo License Activation dialog box 3 Figure 2 Please enter the activation code and identify yourself Activation Code 5J4T AIS v72 FSGP Contact Information Name Leonardo da Vinci Street Line 1 355 River Oaks Parkway Street Line 2 City State San Jose CA Zip Postal Code County 95134 UNITED STATES E Mail Address _leo davinci thermofisher com Telephone 123 456 7890 Computer you want to activate USSJO BGIBS PC if you do not have an Intemet connection on this computer Export the license request import it on another computer that has an Internet connection to activate the license request on that computer Reimport the activated license request to continue activation If you have issues installing or licensing the software please click on Export the license request save that file and contact licenses ms thermo com with a short description and attach that file Activate Cancel 7 On the Choose Destination Location page c
199. ger shown in Figure 43 on page 92 Table 20 Experiment Manager parameters Sheet 1 of 2 Parameter Description Creates a new source PUF file left side or a new destination PUF file right side ray Opens an existing source PUF file left side or a destination PUF file right side PUF File Specifies the name of the source PUF file left side or the destination PUF file right side X Removes the selected experiment from the source PUF file left side or the destination PUF file right side Ld z Thermo Scientific Reverts to the last version of the source PUF file saved left side or the destination PUF file saved right side Any experiments removed after the last time you saved reappear in the pane Saves the source PUF file left side or the destination PUF file right side ProSightPC User Guide 97 3 Working with Experiments Deleting PUF Files Table 20 Experiment Manager parameters Sheet 2 of 2 Parameter Description Opens a popup menu so that you can change how the experiments are displayed in the Experiment Manager e Details Lists the experiments by number in a single column in the pane A comment identifying each experiment appears in an adjoining column e List Lists the experiments by number in multiple columns in the pane e Small Icons Lists the experiments from left to right in the pane using smaller icons than the Large Icons command does e
200. ger Dialog Box Parameters Table 21 lists the parameters in the Predefined Search Manager dialog box shown in Figure 45 on page 101 Table 21 Predefined Search Manager dialog box parameters Parameter Description Opens the New Predefined Search dialog box so that you can et create a new predefined search See Creating a Predefined Search on page 101 for information on this dialog box Ey Opens the Edit Predefined Search dialog box so that you can edit the parameters for the search See Editing a Predefined Search on page 105 for information on this dialog box Removes the selected predefined search from the list of predefined searches to add to an experiment Search Name Displays the name of the predefined search Type Displays the type of predefined search You can select absolute mass biomarker sequence tag single protein gene restricted absolute mass and gene restricted biomarker searches Database Displays the database on which the search is run New Predefined Search Dialog Box Parameters The parameters in the New Predefined Search dialog box shown in Figure 46 on page 102 depend on the type of search that you select in the Type list e Absolute mass search See New Search in Experiment X Dialog Box Parameters for Absolute Mass on page 123 e Biomarker search See New Search in Experiment X Dialog Box Parameters for Biomarkers on page 134 e Sequence tag searc
201. ghtPC application is a suite of tools designed to identify and characterize proteins and peptides from mass spectrometry data This chapter introduces you to the ProSightPC application and to proteomics in general To install the ProSightPC application see Installing ProSightPC on page xi Contents e Features e LC MS MS Workflow e Inputs and Outputs e Fragmentation Methods e Ion Types e Introduction to Proteomics Features The ProSightPC application is the only proteomics software suite that adequately supports high mass accuracy MS MS experiments performed on the LTQ FT and Orbitrap based mass spectrometers including the Q Exactive It operates on mass data from MS MS experiments or any MS experiment on intact and digested proteins For accurate MS MS data it produces highly confident identifications and also automatically detects and annotates post translational modifications in database files that are in the UniProtKB flat file format The application can identify more than one peptide or protein in a spectrum and includes a biomarker search mode to determine if a protein has been truncated The ProSightPC application complements the Proteome Discoverer application and is best used with it to find new or unexpected modifications To identify these unexpected modifications you can use the ProSightPC delta m Am mode with its ability to search databases in UniProtKB flat files Although you can use either tool to searc
202. ghtPC application prioritizes the protein forms storing only those forms most likely to lead to protein identification These forms are then shotgun annotated Shotgun annotation is therefore the process of generating potentially observable protein forms from the information known about a given protein For example for a given protein that has only four phosphorylation sites and no other modifications the ProSightPC application enters a record into the proteome database for the base sequence with no PTMs It also enters the following e Four records for the four protein forms each containing one modification e Six records for the possible combination of two phosphorylations e Four records for the three triphosphorylated forms e One record for the form with all four possible phosphorylations The ProSightPC application processes all of these combinations even if the phosphorylation events have only been observed separately If one of the multiphosphorylated forms occurs in nature and is observed in an MS MS experiment the ProSightPC application can readily identify it ProSightPC User Guide 13 1 Introduction to the ProSightPC Application Introduction to Proteomics Searching Databases You then search the resulting database All searches require matching observed masses to the masses stored in your database theoretical masses Matches are not exact but are within a tolerance The matches are then scored using various fragment based s
203. gments Min Score Determines whether the search algorithm finds only proteins with a p score that matches the filter with the expectation value set in the left list the operator in the middle list and an appropriate value in the right box e Default lt Indicates that the first value is less than or equal to the second value e gt Indicates that the first value is greater than or equal to the second value Max Proteins to Return Specifies the maximum number of proteins to return in the search Fixed Modifications Specifies the chemical modifications present on all instances of a P P given type of amino acid in the observed protein PTM Handling Specifies the PTMs that you want queried Terminal Mods Specifies the fixed terminal modification for each terminus e N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus Save Saves the search information ProSightPC User Guide 135 4 Searching Databases Searching for Sequence Tags Searching for Sequence Tags Tandem mass spectrometry experiments are known to create series of consecutive fragment ions from which you might infer a partial protein sequence You can then use these sequence tags to identify the protein when you search the sequence database The sequence tag search is a two step process to identify but not characterize prote
204. gure 32 on page 54 for the Xtract algorithm This table includes the parameters for both the THRASH and the Xtract algorithms Table 10 Advanced Settings dialog box parameters Sheet 1 of 8 Parameter Precursor Detection Options Description Fragmentation MSz Analysis Level Specifies the level of analysis that includes your fragmentation data in the scan in the RAW file where the ProSightPC application infers the precursor scan You can select one of the following e ms2 For data dependent LC MS MS experiments e ms3 For ion trap marching experiments Specify Start Time Specify End Time Specifies the start of the chromatographic time range in which to analyze the data This is the start of the first scan Default 10 0 minutes Specifies the end of the chromatographic time range in which to analyze the data This is the end of the first scan Default 8 0 minutes Mass Tolerance Specifies a tolerance that determines which scan filters are summed together If the mass and retention time is within the tolerance the ProSightPC application combines the scan filters Range 0 01 1 0 m z Default 0 05 m z Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 2 of 8 Parameter Description Retention Time Tolerance Determines which scan filters are summed together If the retention time and mass is within the tolerance t
205. h See New Search in Experiment X Dialog Box Parameters for Sequence Tags on page 142 Thermo Scientific ProSightPC User Guide 103 4 Searching Databases Performing Searches e Single protein search See Chapter 6 Using the Sequence Gazer to Search for Single Proteins e Gene restricted absolute mass search See Searching for Gene Restricted Absolute Masses on page 146 e Gene restricted biomarker search See Searching for Gene Restricted Biomarkers on page 152 Adding Predefined Searches to an Experiment You can append a single predefined search or multiple predefined searches to an experiment To add a single predefined search to an experiment e In the data grid right click the appropriate experiment and choose Append Predefined Search gt search_name The experiment that the search has been appended to appears in the data grid with the same experiment number in the Exp ID column and a different number in the Search ID column or Follow the next procedure To add multiple predefined searches to an experiment and select the appropriate search To add multiple predefined searches to an experiment 1 Right click the experiment in the data grid and choose Append Predefined Searches You can also choose Experiment Tools gt Append Predefined Search The Append Predefined Searches to Experiment X dialog box opens as shown in Figure 47 Figure 47 Append Predefined Searche
206. h and name of the RAW file that you want import or click Browse to browse for the file the box in the Precursor Mass area enter the mass of the precursor ion 4 Optional Click m z to have the ProSightPC application calculate the intact mass if you kn ow only the mass to charge ratio and the charge The Intact Mass Calculator dialog box appears as shown in Figure 38 Figure 38 Intact Mass Calculator dialog box Intact Mass Calculator B Precursor miz Charge State C 5 In In the Precursor m z box enter the mass to charge ratio m z value of the precursor ion In the Charge State box enter the charge state z to assign to the mass to charge m z data found in the data files Click OK the Precursor Mass area select the mass type of the precursor ions Average Mass Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 The uncharged average mass data is scan 1 in the Post Xtract file Monoisotopic Mass Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion The uncharged monoisotopic mass data is scan 2 in the Post Xtract file Thermo Scientific 2 Getting Started Importing Targeted RAW Files 6 In the Fragmentation Method list select one of the following fragmentation methods e CID e ECD e ETD e HCD e IRMPD For information on these method
207. h bottom up and top down experiments the ProSightPC application is uniquely suited to top down experiments and the Proteome Discoverer application is better suited to bottom up experiments Thermo Scientific ProSightPC User Guide 1 1 Introduction to the ProSightPC Application Features 2 ProSightPC User Guide As Figure 3 shows the ProSightPC application first creates a new proteome database Then it gathers intact protein sequences of a specific organism along with information about known modifications and loads them into a proteome warehouse 1 During loading the ProSightPC application calculates all possible combinations of known modifications and applies them along with single nucleotide polymorphisms SNPs and sequence variants to each protein sequence in a process called shotgun annotation see Shotgun Annotation on page 13 Next it imports the mass values inferred from mass spectral data from top down and middle down bottom up proteomics MS MS experiments into a ProSightPC upload format PUF file 2 The ProSightPC application then searches the appropriate proteome databases for these mass values and compares them 3 Figure 3 The ProSightPC protein and peptide identification process ProSightPC Sequence data application Proteome PUF file warehouse 2 MS data in RAW file The basic unit of analysis in the ProSightPC application is the MS MS experiment An experiment is defined as
208. he ProSightPC application combines the scan filters Range 0 1 no maximum Default 2 0 minutes Analyze Only FTMS Determines whether the ProSightPC application Fragmentation processes only high resolution fragmentation data from Thermo Scientific Fourier Transform instruments e Default Selected Processes only high resolution fragmentation data from Thermo Scientific Fourier Transform instruments e Cleared Processes both high and low resolution fragmentation data from Thermo Scientific Fourier Transform instruments You can select either the Analyze Only FTMS Fragmentation parameter or the Analyze Ion Trap and FTMS Fragmentation parameter When you select one parameter the other parameter is automatically cleared Analyze Ion Trap and FTMS Determines whether the ProSightPC application Fragmentation processes both high and low resolution fragmentation data from Thermo Scientific Fourier Transform and ion trap instruments e Default Selected Processes both high and low resolution fragmentation data from Thermo Scientific Fourier Transform instruments e Cleared Processes only high resolution fragmentation data from Thermo Scientific Fourier Transform instruments You can select either the Analyze Only FTMS Fragmentation parameter or the Analyze Ion Trap and FTMS Fragmentation parameter When you select one parameter the other parameter is automatically cleared Thermo Scientific ProSightPC User Guide 59
209. he Custom Filters section changes to the default configuration shown in Figure 116 Figure 116 Custom Filters section of the Grid Display Preferences page Custom Filters Hide Show Color This filter will hide or show rows of the datagrid If the row evaluates to TRUE the row will be shown otherwise it will be hidden Use the boxes below to specify the TRUE condition 7 v Add Canca 3 Specify the way that the data is displayed in the data grid Do one of the following e Ifyou want a search to be displayed in the data grid if it meets the specified condition and not to be displayed if it does not meet the specified condition click Show Hide if it is not already selected or e Ifyou want to apply colors to specific columns on the basis of the condition that you just set follow this procedure i Click Color Two new boxes with drop down lists appear as shown in Figure 117 Figure 117 Color selection lists Custom Filters Hide Show Color This filter applies colors to specific columns based on the condition below Best Eectation v 16d YellowGreen Orange True Color False Color Ad Canca ii In the True Color list select a color for columns containing data that meets your condition 274 ProSightPC User Guide Thermo Scientific Thermo Scientific B Using the ProSightPC Interface The ProSightPC Interface iii In the False Color list select a color for columns conta
210. he Fragmentation Analysis Options area specify the parameters for analyzing fragment ions a In the Minimum S N box enter the lowest signal to noise ratio that the algorithm considers when trying to assign neutral mass to data in your RAW files The minimum value is 1 and there is no maximum value The default is 3 0 b Inthe Minimum RL box enter the minimum confidence level THRASH only The minimum value is 0 and the maximum value is 1 0 The default is 0 90 c Inthe Minimum m z Considered box select the lowest m z to be considered for fragments THRASH only The minimum value is 1 and there is no maximum value The default is 50 d In the Maximum Charge box enter the maximum charge to be used by the algorithm The minimum value is 1 and there is no maximum value The default is 40 e Inthe Minimum Fit box enter the minimum fit parameter used by the algorithm Xtract only The minimum value is 0 and the maximum value is 100 The default is 10 f In the Maximum Mass kDa box enter the highest mass to be considered for the fragments THRASH only The minimum value is 1 and there is no maximum value The default is 60 g In the Maximum m z Considered box select the lowest m z to be considered for fragments THRASH only The minimum value is 1 and there is no maximum value The default is 50 56 ProSightPC User Guide Thermo Scientific Thermo Scientific 2 Getting Started Processing LC MS MS Da
211. he Kelleher Laboratory and distributed by Thermo Fisher Scientific You can also create your own PWF files for exchanging proteome information e RAW files contain data that you must convert to the mass domain with the Xtract or THRASH algorithms by using the ProSightPC tools Outputs As output the ProSightPC application produces the following files e A PWF file can contain any proteome databases and repositories that you have created and want to export to others e A PUF file contains experiments and searches Fragmentation Methods The ProSightPC application supports the following fragmentation types e CID With the collision induced dissociation CID method of fragmentation molecular ions are accelerated to high kinetic energy in the vacuum of a mass spectrometer and then allowed to collide with neutral gas molecules such as helium nitrogen or argon The collision breaks the bonds and fragments the molecular ions into smaller pieces Thermo Scientific ProSightPC User Guide 9 1 Introduction to the ProSightPC Application lon Types e ECD With the electron capture dissociation ECD method of fragmentation multiply protonated molecules are introduced to low energy free electrons Capture of the electrons releases electric potential energy and reduces the charge state of the ions by producing odd electron ions which easily fragment e IRMPD With the infrared multiphoton dissociation IRMPD method of fragmentation an in
212. he destination right pane 94 ProSightPC User Guide Thermo Scientific 3 Working with Experiments Removing Experiments from PUF Files 4 Click the Save This PUF icon Ld on the source left side of the dialog box to save the source PUF file Click the same icon on the destination right side to save the destination PUF file If two or more experiments share the same experiment identification number when you are copying experiments to PUF files a message informs you that the Experiment Manager will reassign the experiment number of the incoming experiment 5 Click OK to confirm You can also copy experiments from the destination right pane to the source left pane Removing Experiments from PUF Files You can remove experiments from a PUF file by using a shortcut menu or by using the Experiment Manager To remove an experiment from the PUF file and the data grid by using the shortcut menu 1 In the data grid right click the experiment that you want to delete 2 From the shortcut menu choose Remove Experiment x 3 In the Confirm Delete confirmation box click Yes The experiment is only deleted from the data grid but not from the PUF file before you choose File gt Save 4 Choose File gt Save or click the Save icon el l To delete experiments from a PUF file by using the Experiment Manager 1 Choose Tools gt Experiment Manager 2 In the Experiment Manager shown in Figure 43 on page 92 select the ex
213. he ion data When you take a protein sequence to the Sequence Gazer the ProSightPC application automatically scores the sequence on the basis of the initial search parameters Scores Box on page 193 explains the scoring system in ProSightPC You can change parameters and add or remove PTMs or fixed modifications The ProSightPC application then rescores the modified sequence Ideally changes to the sequence followed by rescoring yield more matching fragments than before narrowing the possible matching protein forms that explain the MS MS data By rescoring the ProSightPC application compares the new protein sequence configuration with all changes in place to the fragment ion data This comparison helps to determine the new number of fragments explained along with all corresponding scores Thermo Scientific ProSightPC User Guide 183 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer The Sequence Gazer is usually used for one of two purposes e MS MS data might have been gathered on a known pure protein containing one or more unknown PTMs In this case you build a single protein mode search and add it to the MS MS experiment You use the Sequence Gazer to test hypotheses regarding which PTMs are present e The result of any other search mode might identify and partially characterize a protein whereas the Sequence Gazer can fully characterize the protein Accessing the Sequence Gazer You
214. hermo Scientific 4 Searching Databases Searching for Absolute Mass Searching for Absolute Mass Thermo Scientific The absolute mass search matches MS MS data against all intact forms of proteins in a database It is the defining search mode for top down proteomics Absolute mass searches use the precursor mass to generate a subset of the proteome database to query For each protein form with a theoretical precursor mass within the window of the observed precursor ion mass plus or minus the defined search tolerance the absolute mass search compares all theoretical fragments and masses to observed fragment ion masses The ProSightPC application determines the number of observed fragment ions matching the fragment tolerance and uses this value to score the identification Figure 50 shows this methodology Figure 50 Absolute mass searches Intact precursor mass _ _ _ _ Shotgun annotated proteome database Y Top down fragments Proteins within tolerance Query proteome subset Proteome subset Identified characterized protein Although the ProSightPC application queries each protein form with a theoretical precursor mass in the window only those protein forms meeting user defined search result filtering minimum number of matching fragments minimum percentage of matching fragments or minimum score are displayed In summary the ProSightPC application performs the following steps in a search for absolute
215. hoose a Process Specifies the method for converting mass spectral data to neutral Algorithm mass values when importing the data files Thrash Uses the THRASH algorithm to process the input file e Xtract Uses the Xtract algorithm to process the input file This option reduces analysis and search time and should give better results This option is the default For more information on these algorithms see Importing Targeted RAW Files on page 73 Choose a Process Specifies the settings for the Xtract and THRASH processing Option algorithms e Middle Down See step 5 of the Using the High Throughput Wizard section for this set of default settings e Top Down MS3 See step 5 of the Using the High Throughput Wizard section for this set of default settings e Top Down M82 See step 5 of the Using the High Throughput Wizard section for this set of default settings e Custom Gives you the ability to specify your own settings by clicking on Advanced Settings and using the Advanced Settings dialog box e Advanced Settings Opens the Advanced Settings dialog box so that you can specify custom default settings for the Xtract and THRASH processing algorithms See Advanced Settings Dialog Box Parameters on page 59 for information on the settings in this dialog box The Advanced Settings option is only available when you select Custom Save a copy of the PUF Saves a physical PUF file containing the results of the RAW
216. hreshold option as a percentage determines whether a packet is further processed after an averagine pattern is subtracted This option is important if overlapping peaks are analyzed If there is an overlapping pattern of two peptides and the first pattern has been identified the first averagine pattern is subtracted The remaining pattern is only processed if its peaks the remainder have an intensity that is greater than that specified by the Remainder Threshold option Setting the Remainder Threshold option to 100 percent disables deconvolution of overlapping patterns The ProSightPC application recognizes only the first most intense pattern and ignores overlapping less intense patterns Setting Remainder Threshold to 10 percent allows the deconvolution of a peptide even if it is overlapped by a peptide pattern with 10 fold intensity Range 0 100 Default 20 Precursor Selection Criterion Specifies the type of precursor mass to use for searching e Default Highest Intensity Uses the precursor mass of the most abundant ion in the precursor scan for searching e Closest Average m z Uses the precursor mass that is closest to the mass to charge ratio m z of the data dependent scan for searching Allow Multiple Precursors Determines whether the ProSightPC application multiplexes fragmentation data that is whether it scores multiple precursors in a single experiment If two different ions are fragmented at the same tim
217. ic Marketing department To edit a search tree 1 Choose ProSightHT gt Edit Add Search Tree The Running Highthroughput Logic page appears in the High Throughput Wizard as shown in Figure 20 on page 36 2 From the Search Tree Name list select the search tree that you would like to edit 3 Follow the instructions in Creating or Editing a One Level Search Tree on page 38 and Creating a Two Level Search Tree on page 43 to edit your search tree settings 4 Click Save on the Running Highthroughput Logic page Deleting a Search Tree 46 ProSightPC User Guide You must delete a search tree from the Search Trees folder in the Data folder of your installation directory To delete a search tree 1 Close the ProSightPC application 2 Navigate to the Data Search Trees folder directory under the ProSightPC installation directory 3 Delete the XML file that shares the same name as your search tree Thermo Scientific 2 Getting Started Processing LC MS MS Data Files 4 Reopen the ProSightPC application Demonstrating the High Throughput Wizard The following demonstration shows you how to use the High Throughput Wizard Click the button below to view the demonstration To enlarge the demonstration once you start it right click and choose Full Screen Multimedia Thermo Scientific ProSightPC User Guide 47 2 Getting Started Processing LC MS MS Data Files Running Highthroughput Logic Page Parameters
218. in forms matched in a sequence tag search The ProSightPC application scores any sequence found containing one or more of the sequence tags and reports any sequence scoring above this defined minimum tag score Set the following parameters e Lower Sets the minimum value for a minimum tag score that does not trigger an out of range warning e Default Sets the default value for a minimum tag score e Upper Sets the maximum value for a minimum tag score that does not trigger an out of range warning Compiler Tolerance in Specifies the permissible error measured in ppm between two ppm fragment ion masses that are still considered as matching an amino acid Set the following parameters e Lower Sets the minimum value for a compiler tolerance that does not trigger an out of range warning e Default Sets the default value for a compiler tolerance e Upper Sets the maximum value for a compiler tolerance that does not trigger an out of range warning Minimum Tag Size Specifies the lowest acceptable sequence tag score reported as a match Set the following parameters e Lower Sets the minimum value for a minimum tag size that does not trigger an out of range warning e Default Sets the default value for a minimum tag size e Upper Sets the maximum value for a minimum tag size that does not trigger an out of range warning Searching for Sequence Tags To search for a sequence ta
219. in the Database Manager Click the Remove Repository icon mee in the toolbar in the middle of the Database Manager window ProSightPC User Guide 219 8 Using Proteome Databases Changing View 3 In the Confirm Delete dialog box click Yes to remove the repository from the proteome warehouse IMPORTANT Removing a repository from the proteome warehouse is a permanent change and cannot be undone except by reloading the data from the original source into the proteome warehouse Changing View To change the display of databases in the Database Manager 1 Click the Change View icon Gis on the Database Manager to change the display of databases 2 Choose one of the following display modes e Details Lists the names of the loaded databases and gives a brief description of each database its proteome its strain by whom it was annotated its basic sequences its protein forms its size and the date that it was created e List Vertically lists the names of the loaded databases starting in the upper left corner of the Database Manager e Small Icons Horizontally lists the names of the loaded databases starting in the upper left corner of the Database Manager Each name is preceded by a small icon e Large Icons Horizontally lists the names of the loaded databases starting in the upper left corner of the Database Manager Each name is preceded by a large icon Note You can drag files in the Database Manager e
220. inal Mod None o C Terminal Mod None 7 In the Database Description list select the proteome database to compare the entry or entries to 8 In the Precursor Mass Type list specify the type of precursor ion mass to search for e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 148 ProSightPC User Guide Thermo Scientific Thermo Scientific 10 IL 12 13 4 Searching Databases Performing Gene Restricted Searches e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Mass Type list specify the type of fragment ion mass to search for e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Tolerance box specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match From the adjacent list select the units in which to express the fragment tolerance either absolute in daltons or relative in parts per million An observed fragment ion matches a theoretical fragment ion if the observed
221. information on the p score e lt Indicates that the first value is less than or equal to the second value This setting is the default e gt Indicates that the first value is greater than or equal to the second value d Inthe Max Proteins to Return list select the maximum number of proteins to return in the search ProSightPC User Guide 149 4 Searching Databases Performing Gene Restricted Searches 150 ProSightPC User Guide 14 15 16 17 18 With this option you can truncate the results of a search because the data from all of the similar matching proteins do not need to be returned You can load the results faster In the Fixed Modifications box select no more than one fixed modification per amino acid type In the PTM Handling box select the PTMs that you want to search for The PTM Handling box displays PTMs arranged in one or more tiers based on the selected proteome database The ProSightPC application only queries theoretical protein forms containing exclusively selected PTMs Every form containing an unselected PTM is excluded from the interrogation In the Terminal Mods area select the fixed terminal modification for each terminus A fixed terminal modification is a chemical modification that is present on the terminus of the observed protein N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for
222. ingle Proteins e Gene restricted absolute mass search See Searching for Gene Restricted Absolute Masses on page 146 e Gene restricted biomarker mass search See Searching for Gene Restricted Biomarkers on page 152 3 Click Save Note Altering search parameters has no effect on searches already added to MS MS experiments Edit Predefined Search Dialog Box Parameters The parameters in the Edit Predefined Search dialog box depend on the type of search that you select in the Search Type list e Absolute Mass See New Search in Experiment X Dialog Box Parameters for Absolute Mass on page 123 e BioMarker See New Search in Experiment X Dialog Box Parameters for Biomarkers on page 134 Sequence Tag See New Search in Experiment X Dialog Box Parameters for Sequence Tags on page 142 e Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins e Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 e Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Running a Predefined Search You can run a predefined search by using either of the following procedures Torun a predefined search with the Run Search command 1 Select all the desired experiments in the data grid e To help you sort entries in the data grid you can click the title row of the column to sort entries f
223. inimum percentage of matching ion fragments Specify the percentage of matching ion fragments in the box to the right c Select the Min Score check box to determine whether the search algorithm finds only proteins with a p score that matches the filter with the expectation value set in the left list box the operator in the middle list and an appropriate value in the right box See p Score on page 193 for more information on the p score ProSightPC User Guide 155 4 Searching Databases Performing Gene Restricted Searches 156 ProSightPC User Guide 15 16 17 18 19 e lt Indicates that the first value is less than or equal to the second value This setting is the default e gt Indicates that the first value is greater than or equal to the second value d From the Max Proteins to Return list select the maximum number of proteins to return in the search With this option you can truncate the results of a search because the data from all of the similar matching proteins does not need to be returned You can load the results faster In the Fixed Modifications box select no more than one fixed modification per amino acid type In the PTM Handling box select the PTMs that you want to search for The PTM Handling box displays PTMs arranged in one or more tiers based on the selected proteome database The ProSightPC application only queries theoretical protein forms containing exclusively selected PT
224. ining data that does not meet your condition In the leftmost list select a filter For example ExpID 5 displays only the experiment whose identifier is 5 The parameters available in this list are the same as those given in Using Filters in the Show Columns Area on page 267 In the middle list select an operator The operators available in this list are the same as those given for the check box labeled Only Experiments Where number Search Has option operator value in Using the Filters in the Quick Filters Area on page 271 For information on these operators see Table 74 on page page 272 In the rightmost list type an appropriate value Here are some examples largest precursor mono gt 1000 b c ions gt 7 y z ions lt 20 Click Add The filter appears with a small check box to the left as shown in Figure 118 Figure 118 Filter added to list of custom filters Custom Filters Use f Is Value Then Otherwise Beste 164 M To apply the filter select the check box next to the filter and click Apply To remove custom filters Click a filter to highlight it Right click and choose Remove from the shortcut menu ProSightPC User Guide 275 B Using the ProSightPC Interface The ProSightPC Interface 276 ProSightPC User Guide Custom Filters Table 75 describes the filters available in the Custom Filters section of the Grid Display Preferences page
225. ino acid that has the PTM of the best search result Theoretical Mass Displays the theoretical precursor mass of the protein identified Observed Mass Da Displays the observed precursor mass of the precursor experimental protein in daltons Mass Diff Da Displays the difference between the observed precursor mass and the theoretical precursor mass of the protein identified in daltons Mass Diff ppm Displays the difference between the observed precursor mass and the theoretical precursor mass of the protein identified in parts per million ProSightPC User Guide 173 5 Viewing Search Results Viewing the Results in a Repository Report Table 35 Repository report columns Sheet 2 of 2 Parameter Protein Description Description Displays a description of the match that is the protein that was found in the search Source Displays the path of the RAW or PUF file that an experiment was based on File Name Displays the name of the RAW or PUF file that an experiment was based on Search Number Displays the number of a search in an experiment A report can contain multiple rows searches for an experiment and for each search it can have more rows if there were some intact ions Intact ID Displays the number of an intact ion in an experiment Experiment Comment Number of Best Hits Displays any comments about an experiment such as the filters that it passed
226. ins The two steps are compilation and search 1 During compilation also known as de novo sequencing the ProSightPC application analyzes the fragment ion masses and orders the mass list from largest to smallest looking for sets or ladders of mass differences exactly equal to the mass of a single amino acid or select amino acid pairs within the compiler tolerance that you defined The application always gives the compiler tolerance in parts per million ppm Compilation returns only those sequence tags equal to or longer than the defined minimum tag size Note Multiple independent sequence tags are frequently found with ECD and ETD data The ProSightPC application queries the sequence tag list against every base sequence in the proteome database for the presence of any of the sequence tags It scores any sequence found containing one or more of the sequence tags and reports any sequence scoring above the defined minimum tag score The sequence tag score is based on the negative log of the probability of the sequences existing in nature The sequence tag search automatically searches both the forward and reverse direction of every sequence tag Thermo Fisher Scientific recommends the following when you conduct sequence tag searches e If absolute mass or biomarker searches fail to identify the protein in the presence of rich fragmentation data a sequence tag search can frequently identify but not characterize the protein
227. ion Determines whether the Grid Display Preferences page is displayed in the tab controller area Using Filters in the Show Columns Area on page 267 describes the function of each of the options on the Grid Display Preferences page View gt Start Determines whether the Start page is displayed in the tab controller area View gt Job Queue Determines whether the job queue pane is visible See Figure 109 on page 258 for the location of the job queue and Job Queue on page 264 for a description of the job queue View gt Toolbar Determines whether the toolbar in the ProSightPC window is displayed See Toolbar on page 259 for a description of the icons on the toolbar View gt Close Data Tab Closes the experiment page and all tabs related to it for example the Sequence Gazer for the selected experiment View gt Close All Data Tabs Thermo Scientific Closes experiment pages and all pages related to them for example the Sequence Gazer for all the experiments ProSightPC User Guide 247 A ProSightPC Reference Experiment Tools Menu Table 59 View menu commands Sheet 2 of 2 Command Description View gt Close All Data Tabs But Selected Closes the experiment pages and all pages related to them for example the Sequence Gazer for all experiments except the one selected View gt Open Data Manager Opens the Data Manager in the tab controller for the selected expe
228. ion for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus Save Saves the search information Thermo Scientific 4 Searching Databases Searching for Biomarkers Searching for Biomarkers Thermo Scientific A biomarker search matches MS MS data against all subsequences of all forms of proteins in a database It is similar to a bottom up no enzyme search A biomarker search is a brute force search of an entire database and can take a long time It looks at every possible subsequence of every base protein form unless mentioned otherwise in the database and attempts to identify any subsequence that matches the observed intact ion mass within a tolerance For each subsequence matching the intact ion mass the biomarker search then performs an absolute mass search and reports any subsequence that matches the observed intact ion mass and is able to generate the observed fragment ion pattern In a typical top down experiment not all polypeptides identified are intact proteins A biomarker search identifies those proteins that are a product of biological degradation and cannot be logically predicted It compares the observed precursor mass to all possible entries of a particular database within a defined tolerance for example less than 10 ppm The ProSightPC application theoretically fragments those entries that fall within the defined tolerance and compares the observed fragment ions
229. itory on page 217 for details e Create databases from UniProtKB or FASTA formatted text files See Creating a Proteome Database on page 220 for details Accessing the Database Manager Use the Database Manager to handle all proteome warehouse and repository management and manipulation functions To access the Database Manager e Choose Databases gt Database Manager or click the View Database Info icon i The Database Manager window opens as shown in Figure 90 214 ProSightPC User Guide Thermo Scientific 8 Using Proteome Databases Accessing the Database Manager Figure 90 Database Manager window SEG E Database Name Database Description Proteome Strain Annotated By Basic Sequences ProteinForms Size MB Date demo Demo Database for ProSightPC Human none Kelleher Gro 42 102 0 02 10 26 2008 EB saccharomyces_cerevisiae Saccharomyces cerevisiae 2012_0 Saccha S288c Proteomics 23564 1196890 661 88 6 18 2012 eo Xe Repository Name Repository Description Hof Projects Hof Files of Experiments EB PheATE_Hibi PheATE_HiHi 2 2 129 Database Manager Window Parameters The Database Manager window contains the parameters and toolbar icons shown in Table 47 Table 47 Database Manager window parameters Sheet 1 of 2 Parameter Description Qi top toolbar Imports a proteome database Id top toolbar Exports a proteome database ne top toolbar Removes a prote
230. k below Thank you in advance for your help SURVEY Related Documentation In addition to this user guide the ProSightPC application includes Help and the ProSightPC Quick Start Guide as a PDF file To view ProSightPC manuals ProSightPC User Guide Go to Start gt All Programs gt Thermo ProSightPC gt ProSightPC User Guide ProSightPC Quick Start Guide Go to Start gt All Programs gt Thermo ProSightPC gt ProSightPC Quick Start Thermo Scientific ProSightPC User Guide ix Preface To open Help e From the main ProSightPC window choose Help gt ProSightPC application Help e If Help is available for a specific window or dialog box click Help or press F1 for information about setting parameters For more information including upcoming application notes visit www thermo com Special Notices Make sure you follow the precautionary statements presented in this guide Special notices appear in boxes 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 System Requirements The ProSightPC application requires a license In addition your system must meet the minimum system requirements shown in Table 1 Table 1 System requirem
231. l Cancel 2 Type the name of the new repository As indicated in the dialog box do not use spaces in the repository name use underscores in place of spaces 3 Click OK By default the name and the identifiers of the categories in the results appear in the Categories box of the Edit Add Repositories dialog box shown in Figure 34 4 Optional In the Add Category box of the Edit Add Repositories dialog box type any new categories that are included in the results and click Add 5 Click Save New Repository Dialog Box Parameters The New Repository dialog box shown in Figure 33 specifies a name for the repository that you created 66 ProSightPC User Guide Thermo Scientific 2 Getting Started Using Repositories Editing a Repository You can edit existing repositories as well as create new ones To edit a repository 1 Choose ProSightHT gt Edit Add Repository to open the Edit Add Repositories dialog box shown in Figure 34 Figure 34 Edit Add Repositories dialog box W Edit Add Repositories kabai Repository PheATE_HiHi M Add New Repository Categories Name ID good 1 bad 2 2 Select the repository that you would like to edit from the Repository list 3 Optional In the Add Category box type any new categories that are included in the results and click Add 4 Click Save You can also access the Edit Add Repositories dialog box in the High Throughput Wizard by goi
232. l Carboxylation Farnesyl Phosphorylation Heme Dimethylation Double oxidation Acetylation Hypusine Methylation mono Palmitate GPI anchor Trimethylation Myristate Pyruvic acid Phospho_DNA No hits returned for this search gt Results for Precursor Ion 2 Protein forms found 8 Results for Precursor Ion 3 Protein forms found 8 Click the arrow preceding the precursor ion fe Add Gene Restricted Search I Cysteine ID Gene Length Mass Mass Diff PPM Diff Clons Zlons _Total Ions PDE Score E value gt TOM1_YEAST Q03280 RecName Full E3 ubiquitin protein ligase TOM1 EC 6 3 2 AltName Full Suppressor of snRNA protein 2 AltName Full Temperature dependent organization in mitotic nucleus protein 1 Type predicted Signal Peptide false Propep false Y L D E K Y E D Y A F M D V M K V L N D Q L E N L N D F L N S P N D R S F F L E R D G E N S V R S C H S K L C R L A A I L N I V T N V Y I D L T TEL S C K R I M Q I Y S Y F D K R G F S L I K 1D Gene Length Mass C Ions Z Ions Total Ions E Value 2731370 2841 95 11226 6 E 1 3 4 k 143 Take to Sequence Gazer SEQ gt TOM1_YEAST Q03280 RecName Full E3 ubiquitin protein ligase TOM1 EC 6 3 2 AltName Full Suppressor of snRNA protein 2 AltName Full Temperature dependent organization in mitotic nucleus protein 1 Type basic Signal Peptide false Propep false C 0 E K E B A F A B V 8 K V 8 0 D G L E 8 ERF
233. l Cysteine F Methioni F Lysine Isoleucine Leucine bama gt 0 0 0 A A 4 Handling All PTMs E High priority PTMs Tier 1 v Terminal Mods N Terminal Mod Diim m C Terminal Mod None x 4 In the Search Type list select Single Protein The New Predefined Search dialog box changes its appearance as shown in Figure 78 Thermo Scientific ProSightPC User Guide 185 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer Figure 78 New Predefined Search dialog box for a single protein W New Predefined Search l Sox Search Name Search Type Single Protein Mode Precursor Mass Monoisotopic X Fragment Mass Monoisotopic X Fragment 10 ppm v Am Mode o Fixed Modifications Cysteine a Methionine Lysine E Isoleucine E Leucine E Arginine a Valine v m GG f 5 In the Precursor Mass Type list select one of the following e Monoisotopic Specifies that the mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Mass Specifies that the mass is the mass of the most abundant isotope of the protein peptide or fragment ion 6 In the Fragment Mass Type list select one of the following e Monoisotopic Specifies that the mass is monoisoto
234. l Selects all the predefined searches listed to add to an experiment Uncheck All Clears all the predefined searches O Opens the New Predefined Search dialog box so that you can create a new predefined search See Creating a Predefined Search on page 101 for information on this dialog box Eil Opens the Edit Predefined Search dialog box so that you can edit the parameters for the search See Editing a Predefined Search on page 105 for information on this dialog box Q Removes the selected predefined search from the list of predefined searches to add to an experiment Editing a Predefined Search To edit a predefined search 1 Open the Edit Predefined Search dialog box by doing one of the following e Follow this procedure i Choose Tools gt Manage Predefined Searches ii In the Predefined Search Manager dialog box shown in Figure 45 on page 101 click the name of the appropriate predefined search Thermo Scientific ProSightPC User Guide 105 4 Searching Databases Performing Searches iii Click the Edit icon in the Predefined Search Manager dialog box or right click the search name and choose Edit from the shortcut menu The Edit Predefined Search dialog box opens as shown in Figure 48 e Right click an experiment in the data grid and choose Edit Search x A dialog box similar to the one shown in Figure 48 appears although it has a slightly different header Instead of Edit Predefined Search
235. l spreadsheet you must have Excel installed To export experiments to an Excel spreadsheet 1 In the repository report page select the experiments that you want to export to the Excel spreadsheet You can select experiments in the following ways e Select the check box to the extreme left of each experiment row e Use the SHIFT key to select consecutive experiments e Use the CTRL key to select separate experiments e Right click an experiment and choose either Select All or Check Selected Rows from the shortcut menu You can also choose Unselect All or Uncheck Selected Rows to clear rows 2 Click Export to Excel in the Actions area or right click the selected experiments on the page and choose Export to Excel from the shortcut menu An Excel spreadsheet now opens showing all the experiments that you selected Applying Filters to Repository Report Data You can apply fixed filters or define custom filters by which to refine the type of data shown in the repository report The fixed filters are the most common filters that users apply You can also set a tolerance that causes the ProSightPC application to merge matches that are very similar but differ by a small amount This merging reduces the size of the data To apply fixed filters 1 In the Fixed Filters section of the Actions area shown in Figure 72 select one or more of the following filters e Search Type Displays all the experiments whose search type is the s
236. lick Next 8 On the Select Features page click Next 9 On the Ready to Install the Program page click Install xii ProSightPC User Guide Thermo Scientific Preface 10 In the Installation Qualification dialog box click Yes or No to view the Installation Qualification report 11 On the Setup Status page click Finish Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need io Xa To contact Technical Support Phone 800 532 4752 Fax 561 688 8736 E mail us techsupport analyze thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at mssupport thermo com To contact Customer Service for ordering information Phone 800 532 4752 Fax 561 688 8731 E mail us customer support analyze thermofisher com Web site www thermo com ms To get local contact information for sales or service Go to www thermoscientific com wps portal ts contactus To copy manuals from the Internet Go to mssupport thermo com agree to the Terms and Conditions and then click Customer Manuals in the left margin of the window To suggest changes to documentation or to Help e Fill out a reader survey online at www surveymonkey com s PQM6P62 e Send an e mail message to the Technical Publications Editor at techpubs lcms thermofisher com Thermo Scientific ProSightPC User Guide xiii ee E Introduction to the ProSightPC Application The ProSi
237. loaded to the categories that depend on the conditions See Creating a Two Level Search Tree on page 43 for instructions on creating a two level search Category Specifies that the searches that fail the conditions set in the search tree be stored in the repository in that specific category Edit Add Searches for HT Dialog Box Parameters Table 8 lists the parameters in the Edit Add Searches for HT dialog box shown in Figure 22 on page 39 Thermo Scientific ProSightPC User Guide 49 2 Getting Started Processing LC MS MS Data Files Table 8 Edit Add Searches for HT dialog box parameters Parameter Description Please Check Any Lists the available predefined searches Predefined Searches That You Would Like Included with Your Experiment Demo Search Searches the demonstration database included in the installation of the ProSightPC software Check All Selects all the available predefined searches Uncheck All Clears all the available predefined searches Save Saves the changes that you made to the predefined searches Adds a search oF Ey Edits a predefined search R Removes a predefined search Condition Dialog Box Parameters Table 9 lists the parameters in the Condition dialog box shown in Figure 23 on page 40 50 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Table 9 Condition dialog box parameters Parameter Description Left list
238. ltiple Precursors option Multiplexed scoring supports the detection of multiple precursors When calculating the score for each precursor in one experiment if you have multiple precursors it optimizes the scoring as if there were only one ProSightPC User Guide 55 2 Getting Started Processing LC MS MS Data Files precursor in the experiment Sometimes when an experiment contains multiple precursors and some fragments match one precursor and other fragments match different precursors a better and more accurate score results if the fragments that matched the others were removed as if there had been one precursor j Select the Add Remainder Afterwards check box if you want to add the remaining intensities to the output spectrum Xtract only If a pattern is identified during the processing of the input file with the Xtract algorithm the corresponding averagine pattern is subtracted from the input spectrum The remaining intensities or remainders are then processed again with the Xtract algorithm so that Xtract can find an overlapping low intensity pattern If there is no overlapping second pattern but a small spike in the first pattern the spike is not visible in the deconvolved spectrum but will show up in the remainder spectrum unless you used Add Remainder Afterwards When you select Add Remainder Afterwards the spike shows up in the deconvolved spectrum because unassigned remainders are added to the corresponding pattern 4 In t
239. m Charge 40 Precursor Maximum Mass kDA 35 Precursor Selection Criterion Highest Intensity Allow Multiple Precursors Selected Relative Precursor Threshold 10 Fragmentation Minimum S N 30 Fragmentation Minimum RL 0 Fragmentation Maximum Charge 25 Fragmentation Maximum Mass kDA 25 Minimum Number of Fragmentation Scans 1 Minimum Fragmentation Base Peak Intensity 100 Custom Click Advanced Settings and use the Advanced Settings dialog box to specify your own settings See Using Custom Settings in the High Throughput Wizard on page 52 for instructions 6 Optional If you selected the Process Raw files option select the Save a Copy of the Puf Files for Future Processing option to save a physical PUF file containing the results Click Browse to browse to the directory where you want to save the PUF files This option is useful for rapidly re searching the data instead of processing the RAW file again If you do not select this option the results reside in a ProSightPC repository You can always import them into the ProSightPC application and save them as a PUF file 7 Optional If you choose not to search the data against a proteome database select the Skip Search Tree Logic option The Process a Dataset page now resembles Figure 19 Thermo Scientific ProSightPC User Guide 33 2 Getting Started Processing LC MS MS Data Files Figure 19 Completed Process a Dataset page of the High Throughput Wizard Process a dataset
240. m value is 0 and the maximum value is 100 The default is 20 In the Precursor Selection Criterion list select the type of precursor mass to use for searching e Highest Intensity The precursor mass to use for searching is that of the most abundant ion in the precursor scan e Closest Average m z The precursor mass to use for searching is the closest to the mass to charge ratio m z of the data dependent scan The default is Highest Intensity If the data is intentionally multiplexed do the following e Select the Allow Multiple Precursors check box so that fragmentation data can be multiplexed If two different ions are fragmented at the same time in the mass spectrometer you can use this setting to search both precursor ions against the same set of fragment ions e In the Relative Precursor Threshold box specify a threshold for selecting the precursor intensities when there are multiple precursors within the window The ProSightPC application selects only precursors with intensities within the top x percent of the top precursor For example suppose that the precursor scan contains three ions Ion A is the major ion at 100 percent ion B is lower at 15 percent and ion C is very low at 3 percent If you set the threshold at 10 percent the ProSightPC application searches ions A and B with the fragmentation data but does not search ion C The ProSightPC application handles multiplexed scoring natively when you select the Allow Mu
241. matching ion fragments in the box to the right b Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum percentage of matching ion fragments Specify the percentage of matching ion fragments in the box to the right c Select the Min Score check box to determine whether the search algorithm finds only proteins with an e value that matches the filter with the expectation value set in the Thermo Scientific 4 Searching Databases Searching for Biomarkers left list the operator in the middle list and an appropriate value in the right box See Expectation Value e value on page 194 for more information on the e value e lt Indicates that the first value is less than or equal to the second value This setting is the default e gt Indicates that the first value is greater than or equal to the second value To return only good search results in your search select this option d In the Max Proteins to Return list select the maximum number of proteins to return in the search With this option you can truncate the results of a search because the data from all of the similar matching proteins does not need to be returned You can load the results faster 12 In the Fixed Modifications box select no more than one fixed modification per amino acid type A fixed modification is a chemical modification present on all instances of a given type of
242. ment Mass box specify the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Optional Select the Delta m Mode check box if you want to conduct the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 In the Fragment Tolerance boxes specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters Thermo Scientific 4 Searching Databases Searching for Single Proteins e Lower Sets the minimum value for a fragment tolerance that does not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that does not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm A fragment tolerance is a mass value either absolute or relative within which your observed masses must match the theoretical fragment mass For instance if you set your tolerance to 0 005 Da an absolute tolerance and your theoretical fragment ion is
243. meter Search Type Database Description Description Specifies the type of search to perform Absolute Mass See Searching for Absolute Mass on page 113 BioMarker See Searching for Biomarkers on page 125 Sequence Tag See Searching for Sequence Tags on page 136 Single Protein See Chapter 6 Using the Sequence Gazer to Search for Single Proteins Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Describes the database that you want to search Precursor Mass Type Specifies the type of precursor ion mass to use Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Precursor Tolerance Specifies the tolerance within which your sliding window must fall when you test all protein forms for biomarker peptides and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Fragment Mass Type 134 ProSightPC User Guide Specifies the mass type of the fragment ions to use Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptid
244. n page 29 click Advanced Settings If you chose the THRASH algorithm in the Choose a Process Algorithm area the Advanced Settings dialog box shown in Figure 31 opens If you chose the Xtract algorithm the Advanced Settings dialog box shown in Figure 32 opens Figure 31 Advanced Settings dialog box for THRASH Thermo Scientific Thermo Scientific 2 Getting Started Processing LC MS MS Data Files a Advanced Settings boba ia Precursor Detection Options Mass Tolerance 0 05 m z Fragmentation MSn Analysis Level g 3 oe Retention Time Tolerance 2 0 min E Specify Start Time E Specify End Time Analyze Only FTMS Fragmentation 100 80 0 Analyze lon Trap and FTMS Fragmentation Precursor Selection Options a Maximum Mimms Grane poema Siksin Giai 3 0 40 V Allow Multiple Precursors Minimum Fit Remainder Threshold Fane Pearsa Thai N10 a y 40 20 a E Add Remainder Afterwards Fragmentation Analysis Options Maximum Minimum a Minimum S N Charge m z Considered Minimum Number of i a z a0 z en 2 Fragmentation Scans Minimum Fragmentation 1000 a S Remainder Base Peak Intensity Minimum Fit Threshold mo 0 SSID z Window Size 100 2 V Add Remainder Afterwards V Remove low m z Interferences Absolute Minimum Intensity 100 2 In the Precursor Detection Options area specify the level of analysis that includes your fragmentation scans in the RAW file where the ProSightPC application infers
245. n 33 35 cysteines 164 D data grid adding new search to experiment 263 changing columns displayed in 261 columns displayed in 266 267 deleting experiments from PUF file 95 differentiating experiments 253 263 269 displaying PUF files 91 280 ProSightPC User Guide editing searches 263 filtering searches displayed 271 273 importing data into 263 menu 261 opening Data Manager from 263 purpose 261 redisplaying contents of 263 removing columns from 268 removing experiments from 110 255 264 removing results from search 110 removing search from experiment 264 selecting rows in 262 shortcut menu 253 sorting column data in 262 Data Manager adding an experiment 208 closing 207 248 displaying instrument data 206 displaying search data 206 editing an experiment 208 editing mass values 208 editing search comments 164 248 groups of information displayed in 206 icon 207 matching fragment tables displayed in 22 opening 207 248 260 263 266 performing gene restricted searches 164 purpose 6 205 running pending searches 212 Database Description page 228 232 Database Manager accessing 214 249 260 deleting repositories 69 displaying databases in 216 220 exporting a database 218 exporting a repository 218 importing a database 217 importing a repository 217 PTMs available to 235 purpose 5 refreshing view of databases 215 window 214 249 Database Type page 222 229 databases downloading 249 files created from 3 middle do
246. n Experiment X dialog box for that search type For information on how to edit a search see Editing a Predefined Search on page 105 This command is only available when the Pending Search column displays yes for the appropriate search Thermo Scientific ProSightPC User Guide 263 B Using the ProSightPC Interface The ProSightPC Interface Job Queue 264 ProSightPC User Guide Table 69 Data grid main shortcut menu Sheet 2 of 2 Parameter Description Edit Mass List Opens a new page in the tab controller showing the Precursor Mass List and the Fragment Mass List For information on displaying these two lists see Editing Mass Values on page 208 Remove Results Removes search results from a search that has already been run This command is useful if you want to rerun a search with different parameters This command is only available when the Pending Search column displays no for the appropriate search Run Search x Runs a pending predefined search This command is only available when the Pending Search column displays yes for the appropriate search For information on how to run a predefined search see Running a Predefined Search on page 108 Remove Search x Removes the specified predefined search from the experiment For more information on removing searches see Removing a Predefined Search on page 109 Remove Experiment x Removes the specified experiment from
247. n Search Opens a second level search tree if the experiment passed the condition as shown in Figure 28 on page 44 The experiment is re searched with the second level search and is later loaded to the categories that depend on the conditions set For instructions on creating a two level search see Creating a Two Level Search Tree on page 43 6 From the Failure list select Load or Run Search e Load Loads the results to the selected category if the experiment failed the condition e Run Search Opens a second level search tree if the experiment failed the condition as shown in Figure 28 on page 44 The experiment is re searched with the second level search and is later loaded to the categories that depend on the conditions set For instructions on creating a two level search see Creating a Two Level Search Tree on page 43 7 From the Category list under Success select Good to specify that searches that pass the conditions set in the search tree be stored in the repository in that specific category 8 From the Category list under Failure select Bad to specify that searches that fail the conditions set in the search tree be stored in the repository in that specific category 9 If you want to create a second level search see Creating a Two Level Search Tree on page 43 10 Click Save in the upper right corner of the High Throughput Wizard to save your search tree The Save Search Tree dialog box appears as shown
248. n about PTMs 16 Click Next If you selected the Middle Down Sample Proteolysis option on the Database Type page of the Wizard shown in Figure 95 on page 222 the Digestion page of the Create New Database Wizard opens as shown in Figure 99 Use it to specify the parameters for a sample proteolysis 226 ProSightPC User Guide Thermo Scientific 8 Using Proteome Databases Creating a Proteome Database Figure 99 Digestion page of the Create New Database Wizard W Create New Database x Digestion Specify your sample proteolysis parameters Max missed cleavages 4 Minimum peptide mass Da 500 Maximum peptide mass Da 10000 17 In the Method list select the proteolytic method used to catalyze the breakdown of proteins into peptides 18 In the Max Missed Cleavages box type the maximum number of cleavage sites found in the generated peptides No 0 missed cleavages indicates that there are no cleavage sites in the generated peptides One 1 missed cleavage indicates that each peptide has one site in it two 2 missed cleavages indicate that each peptide has two sites in it and so on The parameter in the Max missed cleavages box contains all values up to and including the set parameter For example if Max missed cleavages is set to 2 peptides with 0 1 and 2 missed cleavages are generated Here is a longer example If a peptide is AAAKAAAKAAA and the digestion method is Lys C no missed cleav
249. n forms that a given search considers When this number is exceeded the search automatically stops and the ProSightPC application Issues a warning 6 In the Scores to Display box specify the types of scores to display in the statistics table in the Data Manager You can choose from the following options E Value PDE Score P Score 7 Click OK Displays the expectation value e value which is the number of sequences in a database that are expected to have p scores equal to or better than what was observed simply by chance For more information on the e value see Expectation Value e value on page 194 Displays the PDE score also known as the McLuckey score which is a way of scoring how well a set of observed fragment ions matches a protein For more information on the PDE score see PDE McLuckey Score on page 196 Displays the p score which is the probability of obtaining at least as good a match between the observed fragment list and a sequence as by chance For more information on the p score see p Score on page 193 General Preferences Page Parameters Table 3 lists the parameters on the General Preferences page of the Options dialog box Table 3 General Preferences page parameters Sheet 1 of 2 Parameter Decimal Precision to Display Description Specifies the number of decimal places to display most numbers Maximum Hits to Display 22 ProSightPC User Guide S
250. nage Predefined Searches Enables you to modify the parameters of predefined searches Tools gt Batch Run Queues and runs a number of searches over any number of experiments Abort Running Job End the current search in the job queue This icon is not available unless a job is running x Abort All Jobs Ends all current and pending searches in the job queue This icon is not available unless multiple jobs are running Thermo Scientific Data Grid B Using the ProSightPC Interface The ProSightPC Interface Table 68 ProSightPC toolbar Sheet 3 of 3 Icon al Menu equivalent ProSightHT gt HighThroughput Opens the High Throughput wizard so that Wizard Function you can start searching T ProSightHT gt Repository Report Opens the Repository Report dialog box so that you can generate a repository report To display the toolbar e Choose View gt Toolbar The data grid shown in Figure 111 displays summary information about each search in the open PUF file organized into columns You can use the data grid to perform and modify searches Figure 111 Data grid ExpID A Search ID Marked Search Type A Pending Search Best Expectation Matching Forms Exp Comment 72 1 Absolute Mass no 1e B4 3 ETD fragmentation for precursor at m z 1115 84 from retention time min 28 35 204 28 55 206 with FT de 73 1 Absolute Mass no 4442 85 ETD fragmentation for precursor at m z
251. names of the database or databases that you want to import and click Import 5 Ifa message box appears indicating that the repository was imported successfully click OK If any internal name already exists in the proteome warehouse this message box does not appear instead the Internal Name Conflict dialog box opens as shown in Figure 92 Figure 92 Internal Database Name Conflict dialog box internal Database Name Conflict Intemal Name saccharomyces_cerevisiae_20 Database Name None The intemal name of this database conflicts with an existing database The intemal names of databases in the ProSight PC Warehouse must be unique saccharomyces_cerevisiae_2012_06_top_ Skip Skip installing this database and continue Replace Replace old database with this one Abort the import of all databases Thermo Scientific ProSightPC User Guide 217 8 Using Proteome Databases Exporting a Proteome Database or Repository Use this dialog box to rename the database skip the database replace the database or cancel changes Note Importing databases as PWF files is faster than recreating a proteome database with the Create Proteome Database option a process detailed in Creating a Proteome Database on page 220 To import a repository 1 Click the Import Repository icon Qi in the toolbar in the middle of the Database Manager window The Open dialog box appears so that you can enter or browse for the name
252. ne PTM Handling S TA PTMs High priority PTMs Tier 1 Terminal Mods N Terminal Mod None C Terminal Mod None x Save Cancel 6 In the Search Type list select Gene Restricted BioMarker 7 In the Database Description list select the proteome database to compare the entry or entries to 8 In the Precursor Mass Type list specify the type of precursor ion mass to search for e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 154 ProSightPC User Guide Thermo Scientific Thermo Scientific J 10 11 12 13 14 4 Searching Databases Performing Gene Restricted Searches e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Mass Type list specify the type of fragment ion mass to search for e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Tolerance box specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match From the adjacent list select the units in which to e
253. ng LC MS MS Data Files If you want to load an LC MS MS RAW file or a PUF file as input you can use the ProSightPC High Throughput Wizard to process the data against the database that you downloaded or created This section explains how to use this wizard and how to set custom Thermo Scientific processing options ProSightPC User Guide 27 2 Getting Started Processing LC MS MS Data Files e Using the High Throughput Wizard on page 28 e Using Custom Settings in the High Throughput Wizard on page 52 You cannot use the High Throughput Wizard to import a targeted RAW file as input or to enter data manually into the ProSightPC application Instead you must use the procedures given in Importing Targeted RAW Files on page 73 to import a targeted RAW file or the procedure given in Entering Data Manually on page 84 to enter data manually Using the High Throughput Wizard You can quickly process LC MS MS data through the ProSightPC High Throughput Wizard To load an LC MS MS RAW file or a PUF file follow the procedures in this section e Setting Processing Options on page 28 e Selecting or Creating a Repository on page 36 e Selecting an Existing Search Tree on page 37 e Creating a Search Tree on page 37 e Editing a Search Tree on page 46 e Deleting a Search Tree on page 46 To view a demonstration of these procedures see Demonstrating the High Throughput Wizard on page 47
254. ng to the Running Highthroughput Logic page of the Wizard shown in Figure 20 on page 36 and selecting Edit Repository from the Repository list Edit Add Repositories Dialog Box Parameters Table 11 lists the parameters in the Edit Add Repositories dialog box shown in Figure 34 on page 68 Thermo Scientific ProSightPC User Guide 67 2 Getting Started Using Repositories Table 11 Edit Add Repositories dialog box Parameter Description Repository Specifies the name of the repository to edit Add New Repository Opens the New Repository dialog box shown in Figure 33 so that you can add a new repository Categories Name Lists the names of the categories in the repository Categories ID Lists the identifiers of the categories in the repository Add Category Specifies the name of the category to add to the repository Add Adds the specified category to the repository Save Saves the new or edited repository Deleting a Repository Use the Database Manager to delete a repository To delete a repository Choose Databases gt Database Manager In the lower or repository window of the Database Manager select the repository that you want to delete Click X on the lower or repository toolbar In the Confirm Delete message box click Yes Importing Experiments from a Repository 68 ProSightPC User Guide You can import experiments from a repository into the ProSightPC application so th
255. ngs in the High Throughput Wizard If you do not want to use the predefined default settings for the Middle Down Top Down MS3 and Top Down MS2 process options in the Choose a Process Option area in the High Throughput Wizard you can define your own custom settings for these options Thermo Scientific ProSightPC User Guide 51 2 Getting Started Processing LC MS MS Data Files 52 ProSightPC User Guide To set custom processing options Precursor Detection Options Fragmentation MSn Analysis Level Specify Start Time Specify End Time 100 amp 80 0 Precursor Selection Options Minimum Minimum S N Minimum RL Charge State 3 0 039 i 1 S Maximum Maximum Mass kDa m a m g Fragmentation Analysis Options Minimum S N Minimum RL m z Considered 3 0 090 50 cal Maximum Maximum Maximum Charge Mass kDa m z Considered a a leo e 2000 4 V Remove low m z Interferences zos Mass Tolerance pos I mz Retention Time Tolerance 20 min Analyze Only FTMS Fragmentation Analyze lon Trap and FTMS Fragmentation Se A Highest Intensity V Allow Multiple Precursors Relative Precursor 10 H Threshold Minimum Number of 1 Fragmentation Scans Minimum Fragmentation 100 ical Base Peak Intensity Get Top X 5 Z Window Size 100 Absolute Minimum 100 ja Intensity 1 On the Process a Dataset page of the High Throughput Wizard shown on Figure 18 o
256. nly available when the Pending Search column displays yes for the appropriate search Opens a new page in the tab controller showing the Precursor Mass List and the Fragment Mass List For information on displaying these two lists see Editing Mass Values on page 208 Remove Results Removes search results from a search that has already been run This command is useful if you want to rerun a search with different parameters This command is only available when search results are present that is when the Pending Search column displays no for the appropriate search Run Search x 254 ProSightPC User Guide Runs a pending predefined search This command is only available when the Pending Search column displays yes for the appropriate search For information on how to run a predefined search see Running a Predefined Search on page 108 Thermo Scientific A ProSightPC Reference Data Grid Shortcut Menu Table 65 Data grid shortcut menu commands Sheet 3 of 3 Command Description Remove Search x Removes the specified search from the experiment For more information on removing searches see Removing a Predefined Search on page 109 Remove Experiment x Removes the specified experiment from the data grid For more information on removing experiments see Removing an Experiment from the Data Grid on page 110 Table 66 describes the command on the secondary dat
257. nt Manager bd 1 2 96 ProSightPC User Guide To change the experiment display Choose Tools gt Experiment Manager In the Experiment Manager shown in Figure 43 on page 92 click the Change View icon Fia on the left side of the dialog box to change the display of the experiments in the left pane Click the Change View icon Fis on the right side of the dialog box to change the display of the experiments in the right pane In the popup menu select one of the following Details Lists the experiments by number in a single column in the pane A comment identifying each experiment appears in an adjoining column List Lists the experiments by number in multiple columns in the pane Small Icons Lists the experiments from left to right in the pane using smaller icons than the Large Icons command does Large Icons Lists the experiments from left to right in the pane using larger icons than the Small Icons command does A heavy dot indicates the active command Thermo Scientific 3 Working with Experiments Deleting PUF Files This transaction changes the display of the experiments in both the source and destination PUF files Deleting PUF Files You cannot delete a PUF file from the ProSightPC application In Windows you can delete it as you would a regular file by right clicking and choosing Delete from the shortcut menu Experiment Manager Parameters Table 20 lists the parameters in the Experiment Mana
258. nt name 2 Right click and choose Append Predefined Searches from the shortcut menu 3 In the Append Predefined Searches to Experiment X dialog box select the predefined searches that you would like to include with your experiment and click Append 4 Choose Tools gt Batch Run or click the Batch Run icon The ProSightPC application queues and runs each pending search in turn You can also use the Batch Run command or icon to quickly run a single search Tip To save time use predefined searches as you import data and run all your predefined searches as a single batch job gt To run multiple searches 1 Select all the desired pending searches in the data grid e To select contiguous experiment names click the name of the first experiment hold down the SHIFT key and click the last experiment name that you want to select e To select noncontiguous experiment names click the name of the first experiment hold down the CTRL key and click each separate experiment name 2 Right click and choose Append Predefined Searches from the shortcut menu 3 In the Append Predefined Searches to Experiment X dialog box select the predefined searches that you would like to include with your experiment and click Append 4 In the data grid reselect the resulting pending searches that is the searches with yes in the Pending Search column 5 Right click and choose Run Searches 112 ProSightPC User Guide T
259. ntact ion mass in the selected units For intact ion masses the dimensions are always in daltons but for fragments they can be in daltons or parts per million Set the following parameters e Lower Sets the minimum value for a precursor search window that does not trigger an out of range warning which is displayed as yellow background in the text box e Default Sets the default value for a precursor search window e Upper Sets the maximum value for a precursor search window that does not trigger an out of range warning In the Fragment Tolerance boxes specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a fragment tolerance that does not trigger an out of range warning e Default Sets the default value for a fragment tolerance e Upper Sets the maximum value for a fragment tolerance that does not trigger an out of range warning The tolerance can be absolute set in daltons Da or relative set in parts per million ppm Thermo Scientific 4 Searching Databases Searching for Absolute Mass A fragment tolerance is a mass value either absolute or relative within which your observed masses must match the theoretical fragment mass For instance if you set your tolerance to 0 005 Da an absolute tolerance and your theoretical fragment i
260. ntific Index P precursor mass type 154 precursor scans 54 59 74 precursor search window 116 precursor tolerance 132 Predefined Search Manager dialog box 101 103 251 predefined searches adding multiple 104 single 104 to experiment 260 to search tree 39 49 50 cancelling 109 creating 101 default 104 105 definition 100 editing 103 105 managing 251 260 opening Append Predefined Searches to Experiment X dialog box 248 253 254 263 opening New Predefined Search dialog box 103 processing in batch mode 252 removing 103 109 running 108 selecting 263 printable search reports 165 166 167 252 Process a Dataset page 28 33 34 53 Profile AIM 74 79 246 Profile option 80 prokaryotic databases 224 proline 196 ProSightHT menu 250 259 ProSightPC application closing 21 constituent parts 2 customizing chemical modifications used to search 24 exiting 246 fragmentation methods supported 9 graphical user interface 257 importing data entering data manually 84 importing experiments from repository 89 importing targeted RAW files 73 Post Xtract 74 THRASH 79 importing or creating a proteome database 23 inputs 3 9 ion types supported 10 main window 20 opening 20 outputs 9 purpose 1 3 search types supported 4 setting default options 21 ProSightPC User Guide 285 Index P steps involved in using 2 7 types of searches available in 99 ProSightPC FTP Web site 23 proteolysis 3 222 226 proteome databases changing displ
261. nts 3 standard databases 229 Start page 247 status reports 165 252 Summary page 43 45 survey link xiii T tab controller displaying experiments in 266 viewing search results 161 212 Take to Sequence Gazer button 164 184 187 Terminal Modification Editor 24 27 terminal modifications adding 24 editing fixed 24 25 theoretical fragment mass 201 241 theoretical mass 163 THRASH algorithm converting RAW files to PUF files 29 34 cutoff point when searching for masses 80 81 83 231 importing targeted raw files 79 maximum charge used by 80 81 83 middle down default settings 30 setting default values for 79 82 settings for 35 signal to noise ratio 80 83 signal to noise ratio for precursor ions 61 top down MS2 default settings 33 top down MS3 default settings 31 used by Profile AIM 74 79 246 using in High Throughput Wizard 7 30 35 Thermo Scientific Thrash Preferences page 79 80 TMT quantification 58 65 toolbar displaying or hiding 247 261 icons on 259 Tools menu 251 259 Top Down MS2 processing option 32 35 Top Down MS3 processing option 31 35 top down databases 3 23 222 226 top down experiments 1 10 11 12 15 trans peptide bonds 238 trypsin 3 222 U UniProt database 220 225 231 232 249 UniProtKB files contents 23 contents of 1 220 creating databases from 214 220 searching with delta m mode 1 V View Database Info icon 214 260 View menu 247 259 W Welcome to the New Da
262. oSightPC application analyzes Range 1 no maximum Default 2000 Minimum Fit Xtract only Thermo Scientific Specifies the minimum fit parameter used by the algorithm while analyzing the precursor ions Range 0 100 Default 10 ProSightPC User Guide 63 2 Getting Started Processing LC MS MS Data Files Table 10 Advanced Settings dialog box parameters Sheet 7 of 8 Parameter Remainder Threshold Xtract only Description Specifies the remainder of the fit that is left in the scan during analysis of the precursor ions Range 0 100 Default 10 Add Remainder Afterwards Xtract only Determines whether the ProSightPC application adds the remaining intensities to the output spectrum during analysis of the precursor ions e Selected Adds the remaining intensities to the output spectrum during analysis of the precursor ions e Default Cleared Does not add the remaining intensities to the output spectrum during analysis of the precursor ions Remove Low m z Interferences Determines whether the ProSightPC application removes fragments arising from immonium ions and reagent ions from TMT and iTRAQ quantifications e Default Selected Removes fragments arising from immonium ions and reagent ions from TMT and iTRAQ quantifications e Cleared Does not remove fragments arising from immonium ions and reagent ions from TMT and iTRAQ quantifications Minimum Number of Fragmentation
263. of the PWF 2 Browse to the repository PWF select it and click Open 3 In the Import Databases dialog box shown in Figure 91 on page 217 select the PWF and click Import 4 Click OK in the message box indicating that the repository was imported successfully Exporting a Proteome Database or Repository You can also export one or more proteome databases or repositories from your proteome warehouse to a PWE To export a proteome database 1 Select the database to export in the Database Manager To combine multiple databases into a single export file hold down the CTRL key and select additional databases 2 Click the Export Proteome Database icon 1 in the toolbar at the top of the Database Manager window The Export dialog box opens as shown in Figure 93 Figure 93 Export dialog box amp Epor anim Output PWF File la saccharomyces_cerevisiae_2012_ Compression Level 6 0 None Fast 9 Highest Slow Epot __ Cancel 3 Type the name of a destination PWE or browse to it 4 Select a compression level by typing an integer ranging from 9 slow export but small file size to 0 fast export but large file size This number controls the final PWF size 218 ProSightPC User Guide Thermo Scientific 8 Using Proteome Databases Removing a Proteome Database or Repository Click Export to execute the procedure and create a new PWF When the Export Complete message box appears click O
264. olumns ProSightPC User Guide 261 B Using the ProSightPC Interface The ProSightPC Interface ii Select the appropriate check boxes iii Click Refresh To redisplay the default columns click Restore Defaults For information on the columns available in the data grid and the filters available to refine the data displayed see Grid Display Preferences Page on page 266 Figure 112 Choosing data grid columns from the data grid shortcut menu File Edit View ExperimentTools Databases ProSightHT Tools Help 4 gt za a ca as X H Del AAE eos Beas X Ut Exp D Search ID Marked Search Type Pending Search Best Expectation Matching Forms f Name Status Notes 1 1 Absolute Mass no n a 0 E 1 2 Biomarker no n a 0 2 1 Absolute Mass no na 0 2 2 Biomarker no na 0 l Columns vV Expld 3 1 Absolute Mass no n a o vV SearchId 3 2 Biomarker no 1 08e 20 13 Pal 4 1 Absolute Mass no n a o 4 2 Biomarker no nla 0 EE 5 ee ii aik Search Comment Gid Display Preferences v Search Type First Precursor Mono oe oe ee First Precursor Avg Use check boxes to display values in the data grid Press Refresh Use to filter shown rows in the datagrid Fires we ori Stor A ee oceans eraumed o TRUE when done selecting top filters repre
265. om database 1 Choose Databases gt Create a Custom Database and click the Create Database icon pp on the Database Manager 2 Follow the instructions in Using Proteome Databases on page 213 Editing Modifications You can customize the chemical modifications that you use to conduct a search You can use the Terminal Modification Editor to add terminal modifications which occur on the ends of the protein You can use the Fixed Modification Editor to add fixed modifications which apply the same specific mass to all occurrences of the named amino acid Both types of modifications are used more frequently in bottom up searches Specify these modifications before you process LC MS MS data through the ProSightPC High Throughput Wizard To edit terminal modifications 1 Choose Tools gt Terminal Modification Editor to open the Terminal Modification Editor shown in Figure 16 24 ProSightPC User Guide Thermo Scientific 2 Getting Started Editing Modifications Figure 16 Terminal Modification Editor 2 In the dialog box do the following a In the Modification box type the name of the modification that you want to create for example carboxymethyl_cysteine Use only letters numbers and underscores b In the Name box type the descriptive name of the terminal modification for example Carboxymethy cysteine c In the Terminus box type the terminus either N or C N applies the modification to the
266. ome database B top toolbar Refreshes the database data displayed in the top half of the Database Manager window Thermo Scientific ProSightPC User Guide 215 8 Using Proteome Databases Accessing the Database Manager 216 ProSightPC User Guide Table 47 Database Manager window parameters Sheet 2 of 2 Parameter top toolbar Description Changes the view of the databases listed in the top half of the Database Manager window W top toolbar Activates the Create New Database Wizard so that you can create a new proteome database Database Name Displays the name of the proteome database This name must be unique Database Description Displays a brief description of the proteome database Proteome Indicates the type of organism for the proteome database Strain Lists the strain information for the proteome database Annotated By Lists the source of the proteome data Basic Sequences Lists the number of unmodified protein forms in the proteome database Protein Forms Lists the total number of shotgun annotated protein forms in the proteome database Size MB Lists the physical size of the proteome database in megabytes Date Displays the date that the proteome database was created Qi middle toolbar Imports a repository ia middle toolbar Exports a repository x middle toolbar Removes a repository 2 middle toolbar middle toolbar Refresh
267. on where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Mass Type Specifies the mass type of the fragment ions to use e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Tolerance Specifies the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Thermo Scientific ProSightPC User Guide 151 4 Searching Databases Performing Gene Restricted Searches Table 30 New Search in Experiment X dialog box parameters for absolute mass Sheet 2 of 2 Parameter Am Mode Description Determines whether the ProSightPC application conducts the search in delta m Am mode Performing Searches in Delta m Mode on page 110 explains this mode Disulfide Indicates whether a protein s cysteines are oxidized Min of Matching Fragments Determines whether the search algorithm finds only proteins containing a minimum number of matching ion fragments The box to the right specifies the minimum num
268. on Value e value on page 194 e Best PDE McLuckey score For more information on the calculation of this score see PDE McLuckey Score on page 196 e Best P Score P score For more information on the calculation of this score see p Score on page 193 e Total Ions Highest total number of ions that matched the ions in the database e Matching Forms Number of matching forms Operator can be e Equal to e NOT Not equal to e lt Less than e gt Greater than e lt Less than or equal to e gt Greater than or equal to Refresh Displays the columns selected in the Show Columns area in the data grid Restore Defaults Reinstates the default settings in the Show Columns area Apply Using the Filters in the Custom Filters Section Executes the filters that you set in the Quick Filters and Custom Filters areas You can use the Custom Filters section of the Grid Display Preferences page to define completely custom conditions with which to filter the searches displayed in the data grid Select one or more of the criteria to filter hide certain data grid rows Click an operator to change its value Thermo Scientific ProSightPC User Guide 273 B Using the ProSightPC Interface The ProSightPC Interface To define custom filters for a search 1 Access the Grid Display Preferences page 2 Right click the Custom Filters section and choose New from the shortcut menu The appearance of t
269. on is at 1154 1126 Da observed fragment ions of 1154 1090 Da 0 0034 Da from theoretical and 1154 1167 0 0041 Da from theoretical fall within the tolerance but 1154 2312 0 1222 does not because the mass difference is greater than the tolerance that you set 8 In the Minimum Matches boxes specify the minimum number of matching ion fragments that you want the search algorithm to find in proteins Set the following parameters Lower Sets the minimum value for minimum matches that will not trigger an out of range warning e Default Sets the default value for minimum matches Upper Sets the maximum value for minimum matches that will not trigger an out of range warning 9 Click OK Note Gene restricted absolute mass searches draw their parameters from absolute mass searches Absolute Mass Preferences Page Parameters Table 23 lists the parameters on the Absolute Mass Preferences page of the Options dialog box shown in Figure 52 on page 115 Table 23 Absolute Mass Preferences page parameters Sheet 1 of 3 Parameter Database Description Specifies the name of the database to search Precursor Mass Thermo Scientific Specifies the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the
270. opriate UniProt address ProSightPC User Guide 233 ee B Using ProSightPC Tools This chapter describes the utilities included in the ProSightPC application Contents e Locating and Selecting PTMs with the PTM Tier Editor e Viewing Fragments Ions with the Fragment Predictor e Converting Text to ProSightPC Font with the Font Converter Locating and Selecting PTMs with the PTM Tier Editor You can use the PTM Tier Editor to view and to change the tier assignment of PTMs PTMs The ProSightPC application groups all PTMs in a multi tier structure enabling you to find and select PTMs quickly Assigning PTMs to tiers is intended to help you locate and select PTMs quickly and efficiently in your searches All of the PTMs in the PTM Tier Editor come from the RESID database The PTM Tier Editor has two functions e To permit you to reassign PTMs in the tier system The ProSightPC application automatically assigns many PTMs to Tier 1 and Tier 2 Tier 1 PTMs represent the most common PTMs and rarer PTMs are assigned to Tier 2 e To display which PTMs are currently included or excluded the ProSightPC application comes with a preset list of included PTMs Any PTM not listed is excluded You select which PTMs are available to be included in or excluded from analyses conducted by the ProSightPC application Use the Tier Editor to include or exclude PTMs Included PTMs are available to the Sequence Gazer the Database Manager and all sear
271. or You can use the Fragment Predictor to view all possible fragment ions for a specific sequence You can also use it to add post translational modifications PTMs or arbitrary custom masses to any amino acid in a known protein sequence and see the predicted b y c and z fragment ion masses Before data collection you might want to have a list of all theoretical fragment ion masses particularly for modified protein sequences The Fragment Predictor does not directly handle cross linked proteins such as trans peptide bonds or cyclized species like disulfides Compute these by using the Enter Custom Mass function 1 To view the fragment ions for a sequence 1 Choose Tools gt Fragment Predictor The Fragment Predictor window opens as shown in Figure 102 238 ProSightPC User Guide Thermo Scientific 9 Using ProSightPC Tools Viewing Fragments lons with the Fragment Predictor Figure 102 Fragment Predictor window Grid Display Preferences I Experiment 1 Fragment Predictor Fragment Predictor Please Enter Your Sequence Continue Enter your sequence using single letter abbreviations 2 Enter a protein sequence in the protein sequence box as shown in Figure 103 Figure 103 Entering a sequence in the Fragment Predictor window Grid Display Preferences Experiment 1 Fragment Predictor Fragment Predictor Please Enter Your Sequence ADQLTEEQIAEFKEASLFDKDGDGT
272. ort a targeted RAW file with the Profile option 1 Choose File gt Import raw gt Profile or click the Import Profile icon fa The Build Experiment from Profile RAW Data dialog box opens as shown in Figure 40 Thermo Scientific ProSightPC User Guide 79 2 Getting Started Importing Targeted RAW Files Figure 40 Build Experiment from Profile RAW Data dialog box _ pees peos W Build Experiment from Profile RAW Data c Fragment Masses RAW file to be THRASHed C Program Files ProSightPC Test Data ETDfraction03 ETDfraction03 raw Thrash Options Minimum Signalto Noise Ratio 3 Minimum RL value 9 Maximum Mass 60000 Maximum Charge 25 First m z 600 Last m z 1200 Summing Options Start Scan Number 1 End Scan Number 534 Precursor Mass Predefined Search Type Monoisotopic Please check any predefined searches E Demo Search 7 M Fragmentation Method Check All Uncheck All x CID X ok Cancel 2 To specify a RAW file to import type the full path name in the box labeled RAW File to Be THRASHed or click Browse to select the file name from the list 3 In the Minimum Signal to Noise Ratio box type the lowest signal to noise ratio that the Profile algorithm will consider when trying to assign neutral mass to data in your RAW files Values less than 5 1 significantly slow down the analysis but can result in a greater number of both real and spurious identified isotopi
273. ory or add a new one ProSightHT gt Edit Add Search Tree ProSightHT gt Repository Report Opens the Running Highthroughput Logic page of the High Throughput Wizard shown in Figure 20 on page 36 For information on the options on this page see Selecting or Creating a Repository on page 36 Generates a repository report that lists all the experiments that a repository contains Figure 70 on page 171 shows an example of this report This command opens the Repository Report dialog box shown in Figure 69 on page 169 Thermo Scientific Tools Menu A ProSightPC Reference Tools Menu Table 63 lists the commands on the Tools menu Table 63 Tools menu commands Sheet 1 of 2 Command Tools gt Experiment Adder Description Imports experiment data into the ProSightPC application and displays it in the data grid It opens the Experiment Adder dialog box shown in Figure 42 on page 84 Tools gt Manage Predefined Searches Assigns a name to a set of parameters that you can then add to any experiment You can also use this command to set up defaults for frequently run searches This command opens the Predefined Search Manager dialog box shown in Figure 45 on page 101 Tools gt PTM Tier Editor Opens the PTM Tier Editor dialog box as shown in Figure 101 on page 236 so you can manually reassign a PTM to another tier Tools gt Terminal Modification Editor Opens the Terminal Modification
274. ository You can select experiments in the following ways e Select the box to the extreme left of each experiment e Use the SHIFT key to select consecutive experiments e Use the CTRL key to select separate experiments e Right click an experiment and choose either Select All or Check Selected Rows from the shortcut menu You can also choose Unselect All or Uncheck Selected Rows to clear rows 2 Click Export to Repository in the Actions area or right click the selected experiments on the page and choose Export to Repository from the shortcut menu You can also right click the experiment in the data grid and choose Export Experiment to Repository The Export to Repository option is available only if the experiment was imported from the repository 3 When prompted if you want to replace the current experiments in the data grid click Yes No Yes to All or No to All 176 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report You can also export experiments to a repository by choosing File gt Export Data to Repository Exporting Experiments to an Excel Spreadsheet You can export experiments to an Excel spreadsheet so that you can print sort manipulate copy and paste the data to other applications The Excel spreadsheet opens with all the experiments that are selected in all the columns that are visible in the graphical user interface To export experiments to an Exce
275. otgun annotation process provides a method for rapid and accurate protein definition Top down proteomics is an emerging solution to the problem of protein identification and characterization In contrast to other proteomic techniques the unknown proteins in top down proteomics are not digested into peptides before tandem mass spectrometry This technique guarantees full sequence coverage on every protein allowing easy characterization of complex combinations of PTMs Separation and ionization of intact proteins present many challenges beyond the scope of this manual The bioinformatics of top down proteomics though has some challenges that the ProSightPC application addresses First because the precursor ions are large they are almost always multiply charged This complicates spectral comparison techniques used in certain other proteomic strategies Fortunately this issue is avoided by comparison with neutral masses Once you collect MS and MS MS spectra you sum the relevant scans and then run through an automated analysis to infer mass using the resulting mass values for protein identification and characterization Second because the precursor mass can represent either a highly modified protein or an internal fragment of the intact protein no single strategy of comparing the observed mass values to a proteome database is guaranteed to identify the protein For this reason the ProSightPC application provides the search modes described in
276. pe used in the experiment blue bars with a serif at the top going to the left represent the termination of either b or c ions A serif at the right bottom is the start of either a y or z ion A black box around an amino acid indicates the amino acid selected Choosing a PTM from the amino acid box affixes that PTM to the selected amino acid A colored background behind an amino acid indicates that the matching PTM is currently assigned to that amino acid Tip Click an amino acid to access all available PTMs contained in the RESID database that can be applied to that amino acid 198 ProSightPC User Guide Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Amino Acid Information Box Table 41 lists the parameters in the Amino Acid Information box which refers to the selected amino acid in the interactive fragment map Table 41 Amino Acid Information box parameters Parameter Description Position Displays the relation of the selected amino acid to the N and C terminals Amino Acid Displays the IUPAC single letter designation of the selected amino acid RESID Displays the RESID designation of the selected PTM Start PTM Displays the PTM attached to the amino acid as of the last score PTM Choices Adds virtual PTMs to each amino acid which changes the sequences of the protein and therefore the score e None Removes the applied PTM from the amino acid e C
277. pecifies the number of matching fragment tables that are displayed in the Data Manager Thermo Scientific 2 Getting Started Importing or Creating a Proteome Database Table 3 General Preferences page parameters Sheet 2 of 2 Parameter Description Maximum Hits to Specifies the maximum number of protein forms that a given Calculate search considers Scores to Display Specifies the types of scores to display in the statistics table in the Data Manager You can select from the following options e E Value Displays the expectation value e value which is the number of sequences in a database that are expected to have p scores equal or better than what was observed simply by chance See Expectation Value e value on page 194 for information on this score e PDE Score Displays the PDE or McLuckey score which is a way of scoring how well a set of observed fragment ions matches a protein See PDE McLuckey Score on page 196 for information on this score e P Score Displays the p score which is the probability of obtaining at least as good a match between the observed fragment list and a sequence as by chance See p Score on page 193 for information on this score For more information about setting general preferences see Setting Default Options on page 21 Importing or Creating a Proteome Database You can add proteome databases with multiple formats e FASTA databases e UniProtKB flat file datab
278. periments that you want to delete 3 Click the Delete Selected Experiments icon xX on the left side of the dialog box to remove the experiments from the source PUF file Click the same icon on the right side to remove the experiments from the destination PUF file Saving a Changed PUF File You might want to save a PUF file once you have added deleted or copied experiments You can also revert to the last saved version of the PUF file Thermo Scientific ProSightPC User Guide 95 3 Working with Experiments Changing the Experiment Display 1M kod To save a changed PUF file Choose Tools gt Experiment Manager In the Experiment Manager shown in Figure 43 on page 92 click the Save This PUF icon Ld on the left side of the dialog box to save the source pdf file Click the same icon on the right side to save the destination PUF file To revert to the last version of the PUF file saved Choose Tools gt Experiment Manager In the Experiment Manager shown in Figure 43 on page 92 click the Revert to Last Saved icon on the left side of the dialog box to revert to the last version of the source PUF file saved Click the same icon on the right side to revert to the last version of the destination PUF file that you saved Any experiments removed after the last time you saved reappear in the pane Changing the Experiment Display You can change how the experiments in the PUF files are displayed in the Experime
279. pic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Mass Specifies that the mass is the mass of the most abundant isotope of the protein peptide or fragment ion 7 In the Fragment Tolerance box specify the tolerance that determines whether comparing an observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicate whether it is expressed as absolute measured in Da or relative measured in ppm 186 ProSightPC User Guide Thermo Scientific 10 11 12 13 6 Using the Sequence Gazer to Search for Single Proteins Accessing the Sequence Gazer Optional Select the Am Mode box to perform the search in delta m Am mode For more information on delta m Am mode see Performing Searches in Delta m Mode on page 110 In the Fixed Modifications box select no more than one fixed modification per amino acid type A fixed modification is a chemical modification present on all instances of a given type of amino acid in the observed protein In the Sequence box either type the sequence or copy and paste a sequence from another source Click Save The search appears in the Append Predefined Searches to Experiment X dialog box Click Append Note After step 11 the ProSightPC application adds a new search to the Data Manager It appears in blue highlighted text to indicate that a new search is pending and has not yet b
280. ple Proteolysis Middle Down Bottom Up Sample Proteolysis Direction E Shuffled Database Shuffled databases are used to estimate false positive rates in certain types of experiments Do not create a shuffled database unless you are absolutely certain you need to a shuffled database will be useless for any other kind of experiment 3 In the Database area select the type of database to build e Top Down No Sample Proteolysis Builds a database around whole intact protein sequences and everything that could potentially happen to them in a biological system e Middle Down Sample Proteolysis Builds a database around peptide sequences that arose from ex vivo proteolysis If anything in your sample preparation protocol involves trypsin or Lys C or any other proteolytic agent select this setting 4 In the Direction area select the direction of the database to build You can select either of the following options e Standard Database Creates a database consisting of correct masses and forward sequences A standard database is a typical protein database Shuffled Database Creates a nonsense database consisting of correct masses but sequences with randomized letters Do not select this option unless it is absolutely necessary You cannot use a reverse database in any other kind of experiment 5 Click Next The Input File page of the Create New Database Wizard appears as shown in Figure 96 222 ProSightPC
281. pository Exports the experiments in the ProSightPC application data grid into a repository It opens the Export Data to Repository dialog box shown in Figure 36 on page 72 You must have experiments listed in the data grid before you can use the Export Data from Repository command File gt Import raw Imports a targeted RAW file using one of the following analyses to infer mass AIMs e Default Post Xtract Takes a small file generated by the Xtract algorithm within Qual Browser and uses it as the neutral mass data It opens the Build Experiment from Post Xtract RAW Data dialog box shown in Figure 37 on page 75 Profile Uses the THRASH algorithm to process the input file This algorithm takes raw mass to charge m z data and finds the neutral mass values It opens the Build Experiment from Profile RAW Data dialog box shown in Figure 40 on page 81 Four most recently opened PUF files Click the file name to open the file File gt Exit 246 ProSightPC User Guide Closes the ProSightPC application Thermo Scientific Edit Menu Table 58 lists the commands on the Edit menu Table 58 Edit menu commands Command Edit gt Copy A ProSightPC Reference Edit Menu Description Copies text File gt Paste View Menu Pastes text Table 59 lists the commands on the View menu Table 59 View menu commands Sheet 1 of 2 Command View gt Grid Display Preferences Descript
282. ption is the default e Manually Enter Enables you to enter sequence tags that you have determined possibly from manually analyzing a spectrum and searches them If you select this option complete step 10 and step 11 6 In the Compiler Tolerance box enter the permissible error measured in ppm between two fragment ion masses that are still considered matching an amino acid 7 In the Minimum Tag Size box enter the lowest acceptable sequence tag score reported as a match 8 In the Fragment Mass Type list specify the type of ion mass fragment type to use e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion 9 In the Fixed Modifications box select no more than one fixed modification per amino acid type A fixed modification is a chemical modification present on all instances of a given type of amino acid in the observed protein 10 If you selected the Manually Enter option enter into the box below it any sequence tags that you want to manually enter instead of compile Each sequence tag consists of the single letter designation of the amino acid separated by a space You can enter isobaric amino acids as a pipe separated list enclosed in square brackets for example I L You can
283. ptions Pata 27 General 7 Grid Columns Thrash Preferences E TE EKAN aah ets RIPE ee 27 Search Parameters se parameters are used wher Minimum S N Ratio 2 Minimum RL Value 9 Maximum Mass 60000 Maximum Charge 29 3 In the Minimum S N Ratio box enter the lowest signal to noise ratio that the THRASH algorithm will consider when trying to assign neutral mass to the data in your RAW files 4 In the Minimum RL Value box enter the minimum confidence level 78 ProSightPC User Guide Thermo Scientific 2 Getting Started Importing Targeted RAW Files 5 In the Maximum Mass box enter the cutoff point for the THRASH algorithm when searching for masses 6 In the Maximum Charge box enter the maximum charge to be used by the THRASH algorithm 7 Click OK Thrash Preferences Page Parameters Table 17 lists the parameters on the Thrash Preferences page of the Options dialog box Table 17 Thrash Preferences page parameters Parameter Description Minimum S N Ratio Specifies the lowest signal to noise ratio that the THRASH algorithm considers when trying to assign neutral mass to the data in your RAW files Minimum RL Value Specifies the minimum confidence level Maximum Mass Specifies the cutoff point for the THRASH algorithm when searching for masses Maximum Charge Specifies the maximum charge to be used by the THRASH algorithm Importing the Targeted RAW File To imp
284. r Consider SNPs Description Includes potential genetic variation as annotated in the UniProt database Consider PTMs Annotates known post translational modifications PTMs onto a protein Maximum Features Per Sequence Specifies the maximum number of features per input sequence Maximum mass Da Specifies the cutoff point for the THRASH algorithm when searching for masses PTM Selection area Thermo Scientific Specifies which PTMs should be considered for inclusion in the database ProSightPC User Guide 231 8 Using Proteome Databases Linking to the UniProt Database Digestion Page Parameters Table 52 lists the parameters on the Digestion page of the Create New Database dialog box shown in Figure 99 on page 227 Table 52 Digestion page parameters Parameter Description Method Specifies the proteolytic method used to catalyze the breakdown of proteins into peptides Max Missed Cleavages Specifies the maximum number of cleavage sites found in the generated peptides Minimum Peptide Specifies the minimum mass that a peptide must have in daltons Mass Da before it is allowed to be put into the database No peptide less than the minimum peptide mass is put into the database Maximum Peptide Specifies select the maximum mass that a peptide can have in Mass Da daltons before it is allowed to be put into the database No peptide greater than the maximum peptide mass is put into
285. r in the Included PTMs list Note Reincluded PTMs are automatically designated as Tier 1 Excluding PTMs You can also exclude PTMs from the database To exclude a PTM from the database 1 In the Included PTMs list of the PTM Tier Editor dialog box click the number in the Tier column of the row of the PTM that you want to exclude 2 Change the number in the Tier column to 1 Thermo Scientific ProSightPC User Guide 237 9 Using ProSightPC Tools Viewing Fragments lons with the Fragment Predictor 3 Click Update The PTMs now appear in the Excluded PTMs list Moving PTMs Between Tiers Use the Tier Editor to manually reassign a PTM to another tier You can enter tier assignments greater than 2 The Tier Editor does not append PTM information to databases The PTM information must be present in the proteome database before the ProSightPC application analyzes the MS data If the information for a given PTM is not in the proteome database that PTM is not available for selection in database searches even if the PTM is considered included by the tier editor To manually reassign a PTM to a tier in the Included PTMs list 1 Click the number in the Tier column for the row of the PTM that you want to include 2 Type a new positive integer in the Tier column 3 Click Update Note Once a tier is updated any excluded PT Ms reappear in the Excluded PTMs list Viewing Fragments lons with the Fragment Predict
286. raw o o OOSSSOR ooooocog ooooce eoocoo0o0o OoOo0o0oo0oo0o0 O00000 Source C Program evs Test patajernfracetono3 raw Absolute Mass Searches 10 0 To generate a printable search report 1 Select a search in the data grid 2 Choose Tools gt Reports gt Printable Report The report appears in a Web browser window Figure 67 shows an excerpt of this report 166 ProSightPC User Guide Thermo Scientific Figure 67 Printable search report Data Management for Experiment 26 Source C Program Files ProSightPC data Example Raw Files PheATE_126kDa_Trypsin_Hi_Hi_Top3 raw 1247 6183 Precursor at m z 624 82 from retention time min 27 88 33 28 06 36 with FT detectionPuf Filter This file passed the following filters 750 20 Min Intact Mass Search 1 Absolute Mass Search Search Parameters Cysteine Modification Intact Mass Type Monoisotopic PTM List Formylation Results for Intact Ion 1 Protein forms found 1 ID Length Mass Fragment Tolerance 15ppm ProSight PC Acetylation Mass Diff Database demoDemo database for Fragment Mass Type Monoisotopic PPM Diff m Mode Off B Ions YIons Total Ions PDE Score Expectation 5 Viewing Search Results Viewing the Results in a Search Report Intact Search Window 2 2Da Minimum Number Of Matches gt PheATE unknown Trypsin peptide from AA 396 406 in with 0 missed cleavage sites Type basic Signal Peptide faise Propep
287. report You can change the columns that are displayed in the repository report and the order of the columns 170 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report Figure 70 Repository report in the tab controller Experiment Tools Databases ProSightHT Tools Help Jair eo s BBS XK YE Marked Search Type Pending Search Best Expectation Matching Forms Se apy fs Sit Poet Pen Repository Name Category Name i Search Type Accession Number EValue Sequence Number of Matching Fra PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi PheATE_HiHi absolute_mass Q3E814 5060 TQLSQSNV absolute_mass 094742 51 STDVA absolute_mass P31787 16500 VSQLFEEK absolute_mass P05319 523 MKYLAAYL absolute _mass P05319 432 MKYLAAYL absolute_mass P22803 1590 49 MVTQL absolute_mass P22803 49 MVTQL absolute_mass P05745 30400 TVKTGIAIG absolute_mass P22803 VTQLKSAS absolute_mass P22803 VTQLKSAS absolute_mass P10 3420 SDSIISFAA absolute_mass 4890 51 STESAL absolute_mass 1 3E 77 MQIFVKTLT absolute_mass 3 01
288. rid searches 159 in e value calculation 22 23 194 in experiments 12 91 in McLuckey score 23 196 in p score 22 23 193 input method 85 88 interactive fragment map 163 198 282 ProSightPC User Guide listing mass values 9 136 210 264 mass type absolute mass searches 116 118 121 123 adding experiments 85 88 biomarker searches 128 129 132 134 gene restricted absolute mass searches 149 151 gene restricted biomarker searches 155 157 Sequence Gazer 192 sequence tag searches 137 138 141 142 single protein searches 144 145 186 189 mass to charge ratio 201 202 matching fragments table 200 minimum matching 119 monoisotopic mass 86 monoisotopic mass to charge ratio 86 multiplexing multiple 56 non matching fragments table 201 N terminal 7 163 241 251 observed mass 125 201 203 observed mass versus theoretical mass absolute mass searches 113 114 121 biomarker searches 125 132 delta m searches 110 111 gene restricted absolute mass searches 149 gene restricted biomarker searches 155 matching fragments table 201 search parameter display 192 permissible error in mass comparison 141 142 predicted 6 238 251 scoring 183 scoring match to proteins 22 Sequence Gazer 190 theoretical mass 114 201 THRASH parameters for analyzing 57 using MS MS data as 159 using MS data as 159 fragment maps 241 Fragment Mass List 210 254 264 Fragment Predictor 6 238 251 fragment tolerance definition 117 128 145 in absolute ma
289. right click the search in the job queue and choose Abort or click the Abort Running Job icon x in the toolbar The search ends and the status changes to Failed e Ifyou want to cancel all running searches you can click the Abort All Jobs icon E7 f Removing a Predefined Search You can remove a predefined search by using a shortcut command in the data grid or by using the Predefined Search Manager To remove a predefined search from an experiment by using a data grid shortcut command e In the data grid right click the search number of an experiment and choose Remove Search x where x is the name of the search that you want to remove To remove a predefined search from an experiment by using the Predefined Search Manager 1 Choose Tools gt Manage Predefined Searches 2 In the Predefined Search Manager dialog box select a predefined search name from the list see Figure 45 on page 101 Thermo Scientific ProSightPC User Guide 109 4 Searching Databases Performing Searches 3 Click the Remove Selected Search icon o in the Predefined Search Manager dialog box or right click the search name and choose Remove from the shortcut menu 4 Confirm the removal by clicking Yes or No You can return to the Predefined Search Manager without removing the selected search by clicking No Removing Search Results from a Search You might want to remove search results from a search that has already been run if you want to rer
290. riment If you do not select an experiment it opens the Data Manager for the first experiment listed in the data grid See Displaying Data in the Data Manager on page 205 for detailed information on the Data Manager View gt Close Data Manager Closes the Data Manager and all pages related to it for example the Sequence Gazer for the selected experiment This command is not available unless an experiment page is selected Experiment Tools Menu Table 60 lists the commands on the Experiment Tools menu which is only available when an experiment is open in the Data Manager Table 60 Experiment Tools menu commands Command Description Experiment Tools gt Append Predefined Opens the Append Predefined Searches to Search Experiment X dialog box shown in Figure 47 on page 104 so you can add a new predefined search to an experiment For information on how to select options in this dialog box see Adding Predefined Searches to an Experiment on page 104 Experiment Tools gt Edit Masses Opens a new page in the tab controller shown in Figure 88 on page 209 so you can review and edit mass values in an experiment Experiment Tools gt Edit Comment Opens a box in the Data Manager so that you can type a comment about an experiment or a search 248 ProSightPC User Guide Thermo Scientific Databases Menu Thermo Scientific A ProSightPC Reference Databases Menu Table 61 lists the commands on the Databas
291. riment Tools Menu slic eyes 4 chenlhctet Head haldredlaye ly inka dwinadtcd 248 Databases Men mirant lit alr Ses une eer aie eae cane 249 Prosiek HT Menun howe de waspde kad N a bee aR ae eta 250 Tools Meni ocean Pace eh ahah Weer es eRe Oe ee AE eR G 251 Help Menti ieee ei r e ia EE E E A E i e 253 Data Grid Shortcut Metitia 4s pare walang we ee aie Bad Ba eee 253 Thermo Scientific Thermo Scientific Contents Appendix B Using the ProSightPC Interface 0c cece eee eee eee eens 257 he Prosighte Interface eccidi ase ete cevhed ae eA ait he ah hd te ba a 257 Memi Bar izaan ats eena a e agieloter nae e e Wie site we Mi 258 Toolbar rea ma A E ane a a a aa N e ie 259 Data Giid onsker k etna ee PE E EEr D nE e E E EA 261 Job Quet s rieme na mage fianna Sa ae Sea e a e Sak E eee 264 Tab Controller roer win amen Manan E rere A e tie lence NG 266 Data Manag t erorte e E T E a eens a a e a a a iS 266 Grid Display Preferences Pages ci cs c Fe te 4 Bow ie mean Shee eee 266 Setting Default Options 4 hi sa0 noret ie eni wees tue ate hee bike eis 277 Indek naered rera e a a a E e ERE 279 ProSightPC User Guide vii Y Preface This guide describes how to use the Thermo ProSightPC 3 0 application to identify and characterize proteins Contents e Related Documentation e Special Notices e System Requirements e Installing ProSightPC e Contacting Us To provide us with comments about this document click the lin
292. rked with an asterisk Zeros appear in all the columns of this row Replace the zeros with the desired values Click Ld to save the changes In the Save Masses Confirmation box click Yes The ProSightPC application returns you to the Data Manager To remove a row from a mass list Click the margin to the left of the mass list to select an entire row Click Delete to remove the entire row from the mass list Click lH to save the changes In the Save Masses Confirmation box click Yes The ProSightPC application returns you to the Data Manager To copy mass values to an external application Copy the mass values e To select contiguous rows hold the SHIFT key down click in the leftmost column of the first desired row then click in the leftmost column in the last desired row ProSightPC User Guide 211 7 Displaying Data in the Data Manager Running a Pending Search e To select disparate rows hold the CTRL key down and click in the leftmost column of each desired row 2 Press CTRL C for copy and select the external application to paste To change the fragmentation method 1 In the Fragmentation Method list select the new fragmentation method For more information on fragmentation methods see Fragmentation Methods on page 9 2 Click lH to save the changes 3 In the Save Masses Confirmation box click Yes The ProSightPC application returns you to the Data Manager To return
293. rmation on the expectation value see Expectation Value e value on page 194 This setting is the default b From the middle list select the operator that indicates the relationship between the values in the left and right boxes e lt Indicates that the first value is less than or equal to the second value This setting is the default e gt Indicates that the first value is greater than or equal to the second value c From the right list enter the desired value The default value is 1e 4 If you enter an illogical value the background of the box becomes bright red d Select one of the following operators e AND e OR e End Condition If you only want to conduct a search with one condition select the End Condition option If you want to add another condition select AND or OR When you select AND or OR the Condition dialog box expands as shown in Figure 24 so that you can add a second condition 40 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files Figure 24 Expanded Condition dialog box Condition kabaod E Value MIE x 164 AND OR End Condition e When you have set the conditions for all searches click Save to return to the Running Highthroughput Logic page 5 From the Success list of the Level 1 search select Load or Run Search e Load Loads the results to the selected category if the experiment passed the condition e Ru
294. rom lowest to highest value or highest to lowest e To select contiguous experiment names click the name of the first experiment hold down the SHIFT key and click the last experiment name that you want to select e To select noncontiguous experiment names click the name of the first experiment hold down the CTRL key and click each separate experiment name 108 ProSightPC User Guide Thermo Scientific 4 Searching Databases Performing Searches 2 Right click and choose Append Predefined Searches from the shortcut menu 3 In the Append Predefined Searches to Experiment X dialog box select the predefined searches that you would like to include with your experiment and click Append 4 Reselect the experiments that you selected in step 1 5 Right click and choose Run Search x where x is the number that appears in the Search ID column The Run Search x command is only available when the Pending Search command displays yes for the appropriate search Torun a predefined search with the Batch Run command 1 Perform step 1 through step 3 in the previous procedure 2 Choose Tools gt Batch Run or click the Batch Run icon f The ProSightPC application queues and runs each pending search in turn You can use the Batch Run command or icon to run a single search Cancelling a Predefined Search You can cancel a predefined search that has started running To cancel a search in the job queue e Ifa search is running
295. rowse to the PUF file that you want to open select it and click Open If a PUF file is already open and you have made changes to it a prompt box appears a Click Yes to save the open PUF file Click No to discard the changes b If you clicked Yes specify the name of the file in the Save As dialog box and click Save c Inthe Opena PUF File dialog box that appears browse to the file that you want to open or enter its path and name and click Open The experiments from the opened PUF file appear in the data grid To open an existing PUF file from the Experiment Manager 1 Choose Tools gt Experiment Manager 2 In the Experiment Manager shown in Figure 43 on page 92 click the Open Existing PUF File icon on the left to open an existing source PUF file Click the same icon on the right to open an existing destination PUF file 3 Select the PUF file from those listed 4 Click Open The experiments in the PUF file are now displayed in the appropriate pane of the Experiment Manager as shown in Figure 44 Thermo Scientific ProSightPC User Guide 93 3 Working with Experiments Adding Experiments to PUF Files Figure 44 Experiments listed in Experiment Manager amp Experiment Manager be aa PUF File PUF File C Program Files ProSightPC Test Data ETDA tual Fa Experiment 1 Fai periment 29 Fa Experime Fa Fa Boperime Fa Eqperime Fd Eperme fa Boperime Fa Experime fa Exqperime fa Experime fa Bperime g FA Bp
296. rt to Last Saved icon 96 Run Search button 212 Running Highthroughput Logic page 36 49 250 Running status 265 S Save Before Closing prompt box 93 Save icon 95 260 Save Masses Confirmation box 211 212 Save Search Tree dialog box 41 49 52 Save This PUF icon 96 Scores box 193 scoring in absolute mass searches 114 in Sequence Gazer 183 in sequence tag searches 140 multiplexed 56 systems used by ProSightPC 193 search reports 165 search results viewing in repository report 168 viewing in search reports 165 viewing in tab controller 161 212 search tree See iterative search tree searches absolute mass See absolute mass searches biomarker searches See biomarker searches definition of 2 delta m See delta m searches editing 263 gene restricted 146 gene restricted absolute mass See gene restricted absolute mass GRAM searches gene restricted biomarker See gene restricted biomarker GRBM searches iterative 4 maximum protein forms considered 23 methodology to use 100 MS hybrid 159 performing 266 ProSightPC User Guide 287 Index T performing in batch mode 111 performing multiple 112 predefined See predefined searches removing from experiments 109 255 removing results from 110 reports 165 running pending 212 sequence tag See sequence tag searches single protein searches See single protein searches 143 status in job queue 265 stopping 266 types supported 4 viewing results in repository report 168 SEQ button 1
297. ry dialog box 70 71 246 Import Databases dialog box 217 218 Import Profile icon 80 260 Import Proteome Database icon 215 217 Import Repository icon 216 218 Import Xtract icon 74 260 Thermo Scientific Index G importing a database 217 importing a repository 217 infrared multiphoton dissociation See IRMPD initial methionines 224 Initial Methionines page 223 230 Input File page 222 230 Intact Mass Calculator dialog box 75 77 82 83 85 intact proteins 12 interactive fragment map 164 198 interactive sequence map 239 241 Internal Name Conflict dialog box 217 ion types 10 ion trap instruments 55 60 ion trap marching experiments 54 59 IRMPD 10 207 isobaric amino acids 141 isoleucine 136 iterative search tree adding conditions 39 40 adding predefined searches to 39 49 50 creating 36 37 creating one level 38 creating three level 45 creating two level 41 43 deleting 46 description 4 37 editing 46 naming 52 saving 41 selecting 37 skipping 33 35 specifying conditions for 49 specifying name of 49 using with LC MS MS data 7 iTRAQ quantification 58 65 IUPAC designation 199 J job queue areas of 265 cancelling searches 260 265 displaying 247 266 menu 265 purpose 264 running a job 265 L large icons 96 largest precursor ions 269 LC MS MS experiments 3 7 27 28 54 ProSightPC User Guide 283 Index M leucine 136 LTQ FT mass spectrometers 1 Lys C 3 222 lysine 196 Manage Predefined
298. s Searching for Biomarkers e Lower Sets the minimum value for minimum matches that does not trigger an out of range warning e Default Sets the default value for minimum matches e Upper Sets the maximum value for minimum matches that does not trigger an out of range warning 9 Click OK Note Gene restricted biomarker searches draw their default parameters from biomarker searches Biomarker Preferences Page Parameters Table 25 lists the parameters on the Biomarker Preferences page of the Options dialog box shown in Figure 55 on page 127 Table 25 Biomarker Preferences page parameters Sheet 1 of 2 Parameter Description Database Specifies the name of the database to search Precursor Mass Specifies the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Fragment Mass Specifies the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Delta m Mode Determines whether the ProSight
299. s see Fragmentation Methods on page 9 7 In the Fragmentation Ion Data area select the mass type of the fragment ions e Average Mass Data Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion e Monoisotopic Mass Data Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 8 Optional Select a predefined search in the Predefined Search area Select Check All to select all of the searches or Uncheck All to clear all searches 9 Click OK Build Experiment from Post Xtract RAW Data Dialog Box Parameters Table 15 lists the parameters in the Build Experiment from Post Xtract RAW Data dialog box shown in Figure 37 on page 75 Table 15 Build Experiment from Post Xtract Raw Data dialog box parameters Sheet 1 of 2 Parameter Description Post Xtract RAW File Specifies the path and name of the RAW file that you want to import You can also click Browse to find the file Precursor Mass Specifies the mass of the precursor ion e m z Calculates the intact mass if only the mass to charge ratio and the charge are known It opens the Intact Mass Calculator dialog box shown in Figure 38 on page 75 e Average Mass Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 The uncharged average m
300. s 2 Ions Total Ions 39 4337 2 0005 1071 24 16 40 gt 39 RS28_HUMAN P62857 40S ribosomal protein 28 Type conflict Signal Peptide false Propep false Length Mass Mass Diff PPM Diff C Ions Z Ions Total Ions 39 4337 2 0005 4071 24 16 Viewing the Results in a Repository Report You can generate a repository report that lists all the experiments that a repository contains Use the repository report to focus on certain experiments that you want to investigate You can control the display of many categories of information in this report You can also set fixed and custom filters by which to refine the report data Furthermore you can import specified experiments into the ProSightPC application manipulate them and export the experiments back to the repository or to a Microsoft Excel spreadsheet The ProSightPC High Throughput Wizard automatically generates a repository report when it finishes its processing However you can also manually generate a repository report The procedures in this section describe how to generate a repository report and manipulate its data See Demonstrating Repository Report Generation on page 181 for a demonstration showing you how to generate a repository report 168 ProSightPC User Guide Thermo Scientific 5 Viewing Search Results Viewing the Results in a Repository Report To generate a repository report 1 To generate a repository report choose ProSightHT gt Repository Report
301. s from 255 saving 252 Export Data from Repository dialog box 246 Export Data to Repository dialog box 71 73 Export Database s dialog box 219 Export dialog box 218 Export Proteome Database icon 215 218 Export Repository icon 216 219 ProSightPC User Guide 281 Index F exporting a proteome database 218 exporting a repository 218 F Failed status 265 FASTA database 23 FASTA files contents of 221 creating databases from 3 214 220 File Download dialog box 24 File menu 245 259 filters custom 178 273 fixed 177 quick 271 repository report data 177 178 search displays 271 first precursor ions 269 fixed filters 177 Fixed Filters section 177 Fixed Modification Editor 25 27 fixed modifications in absolute mass searches 122 124 in biomarker searches 133 135 in gene restricted absolute mass searches 150 152 in gene restricted biomarker searches 156 158 in sequence tag searches 141 142 in single protein searches 187 190 199 Fixed Modifications box 199 fixed terminal modifications in absolute mass searches 122 124 in biomarker searches 133 135 in gene restricted absolute mass searches 150 152 in gene restricted biomarker searches 156 158 Font Converter 7 241 Font Converter dialog box 242 244 251 forward databases 222 Fourier Transform instruments 29 34 55 60 fragment ions abundance 86 88 203 average mass 86 average mass to charge ratio 86 C terminal 7 163 241 251 editing mass values 208 254 260 hyb
302. s into a multi tier structure enabling you to find and select PTMs quickly Use the PTM Tier Editor to include or exclude PTMs and to view and change the tier assignment of PTMs Locating and Selecting PTMs with the PTM Tier Editor on page 235 gives detailed information about the PTM Tier Editor Fragment Predictor 6 ProSightPC User Guide The Fragment Predictor takes a known protein sequence and returns all possible b y c and z fragment ion masses You can use it to add PTMs or arbitrary custom masses to any amino acid in the protein sequence and see the predicted fragment ion masses For information on the functionality of the Fragment Predictor see Viewing Fragments Ions with the Fragment Predictor on page 238 Thermo Scientific 1 Introduction to the ProSightPC Application LC MS MS Workflow Font Converter The Font Converter converts text into the ProSightPC fragment map font used to display N terminal and C terminal fragments For more details on this feature see Converting Text to ProSightPC Font with the Font Converter on page 241 LC MS MS Workflow Following are the general steps involved in using the ProSightPC application with LC MS MS data 1 Load the proteome warehouse The ProSightPC application uses shotgun annotation to apply sequence and PTM information to a proteome database in the proteome warehouse This procedure is only performed once per proteome For information on this procedure see
303. s is the mass of the most abundant isotope of the protein peptide or fragment ion 192 ProSightPC User Guide Thermo Scientific Scores Box p Score Thermo Scientific 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer The ProSightPC application uses a number of different scoring systems to give you a greater degree of freedom when interpreting your results The Scores box in the Sequence Gazer displays the following three scores e P score as noted by Meng et al e Expectation value e value as noted by LeDuc et al e PDE McLuckey as noted by Reid et al These scores are described in the following sections A p score is the probability of obtaining at least as good a match between the observed fragment list and a sequence as by chance It is a measure of confidence in the validity of a match A low p score means that the probability of obtaining at least this many fragments matching a sequence is low so it is unlikely that random chance is the cause of the association The ProSightPC application calculates a p score as follows an ee p n 1 y e i 0 where e nis the number of matching fragments e x is the probability of an observed fragment ion matching a random theoretical fragment ion by chance e fis the total number of fragment ions observed Since the Poisson distribution allows 7 to go to infinity 1 7 is calculated to determine the probability of get
304. s that the ProSightPC application considers This parameter works with the Get Top X parameter to filter the deisotoped or decharged data Range 1 no maximum Default 100 The ProSightPC application places the output of its searches in repositories A repository can store millions of matches You can have an arbitrary number of repositories on any ProSightPC installation one per project for example Each experiment is classified in a category For information on creating and viewing repository reports see Viewing the Results in a Repository Report on page 168 Thermo Scientific ProSightPC User Guide 65 2 Getting Started Using Repositories e Creating a Repository on page 67 e Editing a Repository on page 68 e Deleting a Repository on page 69 e Importing Experiments from a Repository on page 69 e Exporting Experiments to a Repository on page 71 Creating a Repository You can create repositories for storing search results To create a repository 1 Choose ProSightHT gt Edit Add Repository to open the Edit Add Repositories dialog box shown in Figure 34 on page 68 and click Add New Repository or click New Repository on the Running HighThroughput page of the High Throughput Wizard The New Repository dialog box shown in Figure 33 opens Figure 33 New Repository dialog box New Repository x J Enter repository name Use underscores _ in place of spaces cance
305. s to Experiment X dialog box io 1 W Append Predefined Searches to Experiment 2 Collo a Please check any predefined searches that you would like included with your experiment A E Demo Search E yeast absolute mass E yeast absolute mass delta m i Check All Uncheck All Apend Cancel The default predefined search is Demo Search which searches the demonstration database included in the installation of the ProSightPC software 104 ProSightPC User Guide Thermo Scientific 4 Searching Databases Performing Searches 2 Select the predefined searches to append to the experiment and click Append To select all of the searches listed click Check All You can also execute the search from the Data Manager by clicking Run Search Tip To process several predefined searches automatically see Performing Searches in Batch Mode on page 111 Append Predefined Searches to Experiment X Dialog Box Parameters Table 22 lists the icons and parameters in the Append Predefined Searches to Experiment X dialog box shown in Figure 47 on page 104 Table 22 Append Predefined Searches to Experiment X dialog box parameters Parameter Description Please Check Any Lists the predefined searches available to add to an experiment Seca ar ae e Demo Search Searches the demonstration database included Included with Your in the installation of the ProSightPC software Experiment Check Al
306. s used to run a sequence tag search 2 Create an MS MS experiment using the MS data as precursor ions and the MS MS data as fragment ions 3 Add a sequence tag search to the experiment from step 2 and manually enter the sequence tags from the first experiment When you run this search the ProSightPC application creates a gene list for all proteins containing sequences consistent with the MS fragmentation data 4 Add a gene restricted absolute mass search to the second experiment The ProSightPC application uses this search to identify and characterize the observed protein For additional information on MS top down proteomics data see Zabrouskov 2005 l Zabrouskov V Senko M W Du Y LeDuc R D Kelleher N L New and Automated MS Approaches for Top Down Identification of Modified Proteins J Am Soc Mass Spectrom 2005 16 12 2027 2038 Thermo Scientific ProSightPC User Guide 159 ee lt a Viewing Search Results You can view the results of a ProSightPC search in the tab controller in a search report or in a repository report Contents e Viewing the Results in the Tab Controller e Viewing the Results in a Search Report e Viewing the Results in a Repository Report Viewing the Results in the Tab Controller Using the tab controller is the fastest way to see the results of your search e To display results in the tab controller e To enter and save information specific to the search To display r
307. se a gene restricted absolute mass GRAM search to perform an absolute mass search on every protein form of each gene in the gene list regardless of theoretical precursor mass Only those protein forms meeting the minimum matches parameter are reported To perform a gene restricted absolute mass search 1 Perform any search Thermo Scientific 4 Searching Databases Performing Gene Restricted Searches 2 Double click an experiment in the Data Manager to view it 3 Click the arrow next to Search x 4 Click Results for Precursor Ion 1 to view its results 5 From the results list view click the Add Gene Restricted Search icon fe circled in Figure 60 Figure 60 Performing a gene restricted search Grid Display Preferences Experiment 1 A r Search Parameters Precursor Search Window 500Da Precursor Type Monoisotopic Fragment Tolerance 0 25Da Fragment Type Monoisotopic Database Human UniProt m Mode Off Minimum Matches 5 PTM List Dimethylation Seleno cysteine Trimethylation Acetylation Hypusine Formylation Phosphorylation Methylation mono w Results for Precursor Ion 1 Protein forms found 4 B fz Add Gene Restricted Search E Acetylation E Phosphorylation E Cysteine ID Length Mass Mass Diff PPM Diff C ons Z ons Total Ions nEs Expectation gt gt Q9BRL5_HUMAN Q9BRL5 CALM3 protein Type predicted Signal Peptide false Propep false A D Q L T E E Q I A E F KtE A F S L
308. search though some search strategies conduct a gene restricted search For descriptions of these search modes see Chapter 4 Searching Databases 16 ProSightPC User Guide Thermo Scientific 1 Introduction to the ProSightPC Application Introduction to Proteomics Figure 11 Multiple searches used for the identification and characterization of an unknown protein Identified protein Partially characterized protein Identified and fully characterized protein In Figure 12 the fragmentation data is insufficient to distinguish between two or more possible protein forms In this case full identification or partial characterization is the best possible result When this occurs rerun the MS MS experiment to obtain better fragmentation data Figure 12 Results of identification partial characterization or both Identified protein Partially characterized protein Identified and fully characterized protein Thermo Scientific ProSightPC User Guide 17 es i Getting Started This chapter explains how to start using the ProSightPC application in three different ways according to the type of input data that you have e Ifyou have an LC MS MS RAW file or a PUF file as input you can use the ProSightPC High Throughput Wizard e Ifyou have a targeted RAW file as input you can use the Post Xtract or THRASH algorithm to import it e Ifyou want to enter data manually you can use the ProSightPC Experiment
309. selecting the File option is useful if you want to view the results of this run Thermo Scientific ProSightPC User Guide 169 5 Viewing Search Results Viewing the Results in a Repository Report If you do not select a specific file the ProSightPC application generates a report on the experiments in all files e To select all the listed files click Select All e To clear all the listed files click Unselect All e To clear a particular file click it e To select files that contain a certain text string type the text string in the box above the Search button 5 If you want to export the repository report data to a text file instead of to the repository report select the Export Directly to File check box Figure 71 on page 172 gives a partial example of a report in a Microsoft Notepad file 6 If you want to generate a report on only the best result per precursor ion select the Report Only Best Hit Per Search check box The ProSightPC application attempts to discriminate between very close e values or 8 PP P y p scores by examining the intact mass differences and choosing the one with the smallest mass difference 7 Click Generate The repository report automatically appears in the tab controller section of the interface as shown in Figure 70 Each row in the repository report represents the best search result per intact ion in a search in the experiment Table 35 on page 173 describes the columns displayed in the repository
310. sent the most commonly used Click an operator to change its value Custom Rite Largest Precursor Mono Exp ID V Pending Search Sn bedin el ee ee y Largest Precursor Avg Search ID Successful Search Search Type ad Eee OS First m z Mono V Marked 7 Matching Forms Pending Search X E Matching Forms gt e First m z Avg Bp Comat E pea psan Marked 5 Best PDE gt EEIN Search Comment Best P Score gest m z Mono V Search Type Best PDE E Only Experiments where At Least One search has Best lt Largest m z Avg o ee ENS Highest Total lons SS Pending Search oO recursor Avg b c lons Sie ka Sa E Largest Precursor Mono y 2 lons Use Li Is Value Then Otherwise i E Largest Pr Avg F BA Matching Forms First m z Mono Precursors v Best Expectation First m z Avg First Abundance Best P Score Largest m z Mono Largest Abundance Best PDE FAs pes ee Soe Highest Total Ions b c lons y z lons Fragments Ts Oa RET First Abundance Largest Abundance Best Seq Score To sort column data in the data grid e Click the appropriate column title to sort the data from lowest value to highest value or highest to low To select rows in the data grid 262 ProSightPC User Guide To select contiguous rows click the name of the first experiment hold down the SHIFT key and click the last row that you want to select To select noncontiguous rows click the name of the first experiment hold down the CTRL key and click each separate row Thermo Scientific
311. shown in Figure 116 on page 274 so that you can set a new custom filter Remove Removes the selected custom filter Check All Selects all the defined custom filters Uncheck All Clears all the defined custom filters Setting Default Options Use the Options dialog box to set default values for most of the interface elements in the ProSightPC application In the Options dialog box you can set the preferences shown in able 77 Table 77 Options dialog box parameters Options dialog box parameter Location of information General Setting Default Options on page 21 Grid Columns Grid Display Preferences Page on page 266 THRASH Setting THRASH Preferences on page 79 Search Parameters Absolute Mass Setting Absolute Mass Search Preferences on page 115 Biomarker Setting Biomarker Search Preferences on page 127 Sequence Tag Setting Sequence Tag Search Preferences on page 136 Single Protein Setting Single Protein Search Preferences on page 143 To access the Options dialog box e Choose Tools gt Options Thermo Scientific ProSightPC User Guide 277 ee Index A Abort All Jobs icon 109 260 265 Abort Running Job icon 109 260 265 Absolute Mass Preferences page 115 117 absolute mass searches methodology 113 parameters for 123 performing 119 results list 162 setting default values for 115 steps performed in 113 strategies used in 114 Add Experiment icon 26
312. ss searches 113 116 119 121 124 in biomarker searches 128 130 132 135 in gene restricted absolute mass searches 149 151 in gene restricted biomarker searches 155 158 in search parameter display 192 in single protein searches 144 146 186 189 fragmentation methods changing 212 importing experiments manually 85 types 9 fragmentation scans 54 59 Fragments Explained box 197 Thermo Scientific G gene identifier 163 General Preferences page 21 gene restricted absolute mass GRAM searches adding when analyzing MS MS experiment 159 methodology 146 parameters for 117 performing 146 gene restricted biomarker GRBM searches methodology 152 origin of default parameters 129 parameters for 157 performing 153 gene restricted searches 146 glutamic acid 196 Grid Display Preferences page controlling display of 247 Custom Filters section 273 purpose 266 Quick Filters section 271 Show Columns section 267 H HCD 10 207 Help menu 253 259 High Throughput Wizard demonstration of 47 generating repository reports 43 168 opening 28 250 261 place in workflow 7 Process a Dataset page 28 33 34 53 processing LC MS MS data 28 purpose 27 Running Highthroughput Logic page 36 49 250 setting custom processing options 53 setting processing options 28 Summary page 43 45 high energy collision induced dissociation See HCD HT Wizard icon 28 261 hybrid searches 136 icons See toolbar immonium ions 58 Import Data from Reposito
313. t RAW Data dialog box 74 76 246 Build Experiment from Profile RAW Data dialog box 80 82 246 C c fragment ions displayed in Show Columns section 270 in ECD and ETD analysis 207 in Font Converter 243 244 in interactive fragment map 198 returned by Fragment Predictor 6 238 241 ProSightPC User Guide 279 Index D C terminal applying modifications to 25 27 cleavage to an aspartic acid 196 fragment marks in Font Converter 243 in Amino Acid Information box 199 in delta m searches 110 Change View icon 96 216 220 chromatographic time scale 54 CID 9 207 collision induced dissociation See CID compiler tolerance 138 139 Completed status 265 Complexity page 224 231 Condition dialog box 39 40 49 51 contacting us xiii converting text into fragment map font 241 copying text 247 Create Database icon 216 221 Create New Database Wizard Complexity page 224 231 Database Description page 228 232 Database Type page 222 229 Digestion page 226 232 Initial Methionines page 223 230 Input File page 222 230 Welcome to the New Database Wizard page 221 249 Create New PUF File icon 92 Create New Search icon 88 101 103 105 creating proteome databases 220 cross linked proteins 238 cSNPs 3 C terminal fragment ions 7 163 241 251 custom filters adding 179 applying 178 merging matches with similar values 180 removing 180 repository report data 178 search displays 273 Custom Filters section 178 273 Custom processing optio
314. t can create automated iterative searches for batch processing including search trees with decision points to help create useful searches It supports ultra high resolution MS MS data for example top down and middle down bottom up LC MS MS data The ProSightPC application operates on a single PUF file that when opened is uploaded into memory and made available to a variety of search and data visualization tools Additionally the ProSightPC application includes several tools for importing LC MS MS and tandem MS data from Thermo Fisher Scientific RAW files identifying and removing chemical noise peaks and performing other utility functions It can handle and store data in RAW format in ProSightPC upload format PUF or in a repository Proteome Warehouse Thermo Scientific The ProSightPC application creates proteome warehouses which are collections of databases that it uses to identify and characterize protein data It contains all the protein forms for a specific organism based on its sequenced genome It stores many types of information including known and predicted protein sequences post translational modifications PTMs alternate splice forms and coding SNPs cSNPs The proteome warehouse contains both monoisotopic and average mass information and is organized to facilitate both protein identification and characterization Each organism in the proteome warehouse receives its own database You can create databases from UniProtKB fla
315. t of predefined searches to add to an experiment Check All Selects all predefined searches listed to add to an experiment Uncheck All Clears all predefined searches listed to add to an experiment ProSightPC User Guide 87 2 Getting Started Importing Experiments Importing Experiments Another way to import data into the ProSightPC application is to import experiments from a repository See Importing Experiments from a Repository on page 69 and Using the Repository Report To Import Experiments from a Repository into the PUF File on page 175 for instructions on this procedure For more information on handling experiments see Working with Experiments on page 91 Searching the Proteome Warehouse for Matches ProSightPC User Guide After you import your data you might want to search the proteome warehouse for matches The ProSightPC application supports six different search modes Each search mode represents a specific method used to query a proteome database within the proteome warehouse You can add a predefined search by following the instructions in Searching Databases on page 99 Thermo Scientific 2 Getting Started Searching the Proteome Warehouse for Matches Thermo Scientific ProSightPC User Guide 89 ee ie Working with Experiments This chapter explains how to work with the experiments in PUF files Contents e Experiments in PUF Files e Creating a New PUF File e Opening an
316. t or FASTA formatted text files to create your own custom databases The databases in the warehouse are MySQL relational databases which you can view by using other third party applications In addition you can export them and move them between computers The ProSightPC application searches these databases to try to find a match to the mass values inferred from mass spectral data from top down and middle down bottom up proteomics MS experiments The ProSightPC application supports the creation of top down and middle down bottom up databases e Top down no sample proteolysis databases are built around whole intact protein sequences and everything that could potentially happen to them in a biological system e Middle down bottom up sample proteolysis databases are built around peptide sequences that arose from proteolysis outside living organisms Select this setting if anything in your sample preparation protocol involved trypsin or Lys C or any other proteolysis agent For more information on top down and middle down bottom up databases see Top Down Proteomics on page 12 and Middle Down Bottom Up Proteomics on page 11 Chapter 8 Using Proteome Databases tells you how to create and manage databases in the ProSightPC application ProSightPC User Guide 3 1 Introduction to the ProSightPC Application Features Search Types The ProSightPC application supports the types of searches shown in Table 2 The section r
317. t protein sequences and Proteolysis everything that could potentially happen to them in a biological system Middle Down Builds a database around peptide sequences that arose from ex Bottom Up Sample vivo proteolysis Proteolysis Direction Standard Database Creates a database consisting of correct masses and forward sequences A standard database is a typical protein database Shuffled Database Creates a nonsense database consisting of correct masses but sequences with randomized letters Do not select this option unless it is absolutely necessary You cannot use a reverse database in any other kind of experiment ProSightPC User Guide 229 8 Using Proteome Databases Creating a Proteome Database Input File Page Parameters Table 49 describes the sole parameter on the Input File page of the Create New Database Wizard shown in Figure 96 on page 223 Table 49 Input File page parameters Parameter Description File Location Specifies the name and path of the file containing the sequence information The Browse Folder icon opens the Open dialog box so that you can browse for the input file For the file type you can select a FASTA file or a UniProtKB flat file For a description of the file types see the beginning of Creating a Proteome Database on page 220 Initial Methionines Page Parameters Table 50 lists the parameters on the Initial Methionines page of the Create New Database Wizard shown in Figure
318. t select any predefined searches Entering Data Manually As a third option you can import MS MS experiment data by manually entering the data from the ProSightPC application through the Tools menu The data is then displayed in the data grid You can also manually delete experiments To manually import MS MS experiment data gt 1 Choose Tools gt Experiment Adder or click the Add Experiment icon The Experiment Adder dialog box opens as shown in Figure 42 Figure 42 Experiment Adder dialog box W Experiment Adder fea Fragmentation Methods Experiment Comments cD 5 ECD IRMPD Gede Omo oe Precursor lon Data Fragment lon Data Type Manual mez Type Manual Mass Type Mass Type Monoisotopic Average Monoisotopic 5 Average Intensities Please check any predefined analyses that you would like included with your experiment E Demo Search P Dl ProSightPC User Guide 83 Thermo Scientific 2 Getting Started Entering Data Manually 84 ProSightPC User Guide 2 In the Fragmentation Methods area select one of the following fragmentation methods CID ECD IRMPD HCD ETD For information on these methods see Fragmentation Methods on page 9 3 In the Precursor Ion Data area select the method of inputting the precursor ion data a b In the Type list select Manual or Upload If you select Manual in the Precursor Ion
319. t to annotate known post translational modifications PTMs onto a protein select the Consider PTMs check box In the Maximum Features Per Sequence box type the maximum number of features per input sequence This option sets the maximum number of database forms produced from a particular entry If a protein has for example four PTMs the ProSightPC application puts 24 or 16 forms into the database The default value is 16384 Here is an example A short peptide SSS has a phosphorylation feature on each residue A PTM might or might not be present The total number of forms that can be produced from this sequence is 23 8 SSS S P SS SS P S SSS P S P S P S S P SS P SS P S P S P S P S P Forms are selected on the basis of which ones are most likely to be observed in the instrument for the input sequence just given if the restriction is set to 2 4 forms the forms selected are SSS S P SS SS P S P SS P ProSightPC User Guide 225 8 Using Proteome Databases Creating a Proteome Database Here is an example of an entry in the input with potential variation such as polymorphisms and PTMs MAAAVAAAPAAAA PTM 3 This protein might have a PTM at A3 A3 is a known site of modification A form is in the database It has no variation and is matched directly against the data MA PTM AAVAAAPAAAA This protein as a PTM at A3 MAAAVAAAPAAAA This protein does not have a PTM at A3 This example also demonstrates shotgun anno
320. ta Files If you want to add the remaining intensities to the output spectrum select the Add Remainder Afterwards check box Xtract only If you want the ProSightPC application to remove fragments arising from immonium ions and reagent ions from TMT and iTRAQ quantifications select the Remove Low m z Interferences check box In the Minimum Number of Fragmentation Scans box enter a value to filter out low quality spectra The minimum value is 1 and there is no maximum value The default is 1 In some cases you might want to only consider precursors that have been fragmented twice or more In the Minimum Fragmentation Base Peak Intensity box enter a value that will filter out noise and poor quality data during analysis of the fragment ions You can enter a minimum value of 1 and there is no maximum The default for THRASH is 100 The default for Xtract is 1000 A value of 500 corresponds to an NL value of 5e2 When a mass spectrometer is trying to fragment precursors the data quality is often poor for some of them If the fragmentation scan s base peak is below an intensity of 500 the ProSightPC application skips the scan and discards the precursor mass This step eliminates the processing of bad MS MS experiments and prevents the analysis of noise so it makes the application more efficient and increases the speed and performance of the searching If you are not certain what to select for this option use the default setting
321. tabase Demo database for ProSight PC Single Protein Precursor Mass Monoisotopic X Fragment Mass Monoisotopic X Delta m Mode E Lower Default Upper Precursor Search 1 io 2000 Dav Fragment Tolerance 1 15 100 ppm Minimum Matches 1 4 100 2 In the Database list select the name of the database to search ProSightPC User Guide 115 4 Searching Databases Searching for Absolute Mass 116 ProSightPC User Guide In the Precursor Mass list select the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion In the Fragment Mass list select the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion Optional Select the Delta m Mode check box if you want to conduct the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 In the Precursor Search boxes specify the dimensions of the precursor search window of the observed i
322. tabase Wizard page 221 249 X XML file 45 Xtract algorithm converting LC MS MS RAW files to PUF files 29 34 importing targeted raw files 74 middle down default settings 30 settings for 35 subtracting averagine pattern from input spectrum 57 63 top down MS2 default settings 32 top down MS3 default settings 31 used by Post Xtract AIM 73 74 246 using in High Throughput Wizard 7 30 35 Y y fragment ions displayed in Show Columns section 270 in CID HD and IRMP analysis 207 in Font Converter 243 244 in interactive fragment map 198 returned by Fragment Predictor 6 238 241 yz fragment ions 244 Thermo Scientific Z z fragment ions displayed in Show Columns field 270 in ECD and ETD analysis 207 in Font Converter 243 244 in interactive fragment map 198 returned by Fragment Predictor 6 238 241 ProSightPC User Guide Index U 289
323. tation from information about a known site that can be modified in an input entry two database forms are produced one where the site s modified and one where the site is not modified If more known sites were known database forms would be produced with all combinations 14 In the Maximum Mass Da box enter the upper limit for which PTMs are included in the database The default for top down databases is 70000 Da This option is not seen in middle down databases where it is hardcoded to 50000 Da If the mass of just the amino acids in your entry PTM masses not considered exceeds the cutoff the optimizer does not determine which PTMs to pick instead it marks all PTMs as inactive This option sets the mass cutoff for complexity management any entry exceeding the maximum mass will have variation both polymorphisms and PTMs discarded Your instrument will probably not see anything beyond a certain size and because bigger proteins typically have more PTMs polymorphisms or both they will have a disproportionate impact on database size This option can help resolve that problem 15 In the PTM Selection area specify which PTMs should be considered for inclusion in the database Ifa PTM or PTM category is clear those PTMs are not put into the database whether or not they are present in the input data This option is only available for UniProtKB flat formatted input data because the standard FASTA format cannot encode informatio
324. te Mass Search Database Description Demo Database for ProSightPC Precursor Mass Type Monoisotopic M Precursor Search Window 1000 Da Fragment Mass Type Monoisotopic M Fragment Tolerance 15 ppm v Am Mod Disulfide Hit Filtering v Min of Matching Fragments 4 Min of Matching Fragments 0 Min Score 0 Max Proteins to Return 25 X Fixed Modifications Cysteine TF Methioni Lysine Isoleucine F Leuci gt am 0 0 6006 4 EAI PTMs E E High priority PTMs Tier 1 PTM Handling Terminal Mods N Terminal Mod None 3 In the Search Name box type a name for the new predefined search 4 From the Search Type list select the search type and follow the procedure for your selection e Absolute Mass See Searching for Absolute Mass on page 119 102 ProSightPC User Guide Thermo Scientific 4 Searching Databases Performing Searches e BioMarker See Searching for Biomarkers on page 130 e Sequence Tag See Searching for Sequence Tags on page 139 e Single Protein See Searching for Single Proteins and Accessing the Sequence Gazer on page 184 e Gene Restricted Absolute Mass See Searching for Gene Restricted Absolute Masses on page 146 e Gene Restricted BioMarker See Searching for Gene Restricted Biomarkers on page 152 Predefined Search Mana
325. the box to the right of the option enter the minimum intact mass number 38 ProSightPC User Guide Thermo Scientific 2 Getting Started Processing LC MS MS Data Files The default is 750 Da indicating that experiments whose intact mass is less than 750 Da are ignored d From the list beneath the Min Intact Mass option specify the mass type e Monoisotopic Specifies that the mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the mass is the mass of the most abundant isotope of the protein peptide or fragment ion 3 To define the first level search click Add Search in the Level 1 search area The ProSightPC application opens the Edit Add Searches for HT dialog box shown in Figure 22 Figure 22 Edit Add Searches for HT dialog box Edit Add Searches for HT cees Please check any predefined searches that you would like included with your experiment A F Demo Search E yeast absolute mass E yeast absolute mass delta m E Check Al Uncheck ai Save Cancel a Select a predefined search to use by selecting the appropriate check box A predefined search enables you to assign a name to a set of parameters that you can then add to any experiment It reduces the repetition of identical searches on different sets of MS MS data For more information on predefined searches see Performing
326. the database Database Description Page Parameters Table 53 lists the parameters on the Database Description page of the Create New Database Wizard shown in Figure 100 on page 228 Table 53 Database Description page parameters Parameter Description Database Name Specifies the name of the database that you want to create Description Describes the database that you want to create Organism Specifies the name of the organism for the proteome database that you want to create Strain Specifies the strain designation for the proteome database that you want to create Owner Specifies the name of the data source Last Update Specifies the date when the database was last updated Linking to the UniProt Database 232 UniProt is an international repository of organisms containing all the proteins and genes that are known for a specific organism When you create a custom database you must have a FASTA or flat text file that contains all the known proteins of interest You can download those files from UniProt ProSightPC User Guide Thermo Scientific Thermo Scientific 8 Using Proteome Databases Linking to the UniProt Database For example suppose that you want to create a custom database for a fly You would download a flat file from UniProt and use it in the Create New Database Wizard To link to the UniProt database e Choose Databases gt Link To Uniprot This command opens a Web browser with the appr
327. the Amino Acid information box to the right as shown in Figure 105 Figure 105 Amino Acid information box Alanine Information Position N 1 C 146 Amino Acid A RESID 41 Start PTM None PTM Choices 0 None Custom 0 Tier 1 B acetylation E Methylation mono Trimethylation PTMs are arranged in tiers The PTM listed in red text is the current selection for the amino acid You can customize the PTM tier assignment by using the PTM Tier Editor described in Locating and Selecting PTMs with the PTM Tier Editor on page 235 5 Click the name of the desired PTM The designated amino acid changes to match the color of the PTM selected If desired you can enter a custom mass in daltons in the box provided 6 Click Get Fragments when you have selected all the mass changes 240 ProSightPC User Guide Thermo Scientific 9 Using ProSightPC Tools Converting Text to ProSightPC Font with the Font Converter The Fragment Predictor displays the theoretical fragment masses in four columns in the results window as shown in Figure 106 All theoretical fragment ion masses are arranged in ascending order and are classified as either b y c or z Figure 106 Results window Grid Display Preferences Experiment 1 Fragment Predictor Fragment Predictor A D Q L L 6 T B Ions 271 03711 186 06405 314 12263 2427 20669 2528 25437 2657 29696 786 33955 914 39813
328. the Database list select the name of the database to search 3 In the Precursor Mass list select the type of precursor mass e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Thermo Scientific ProSightPC User Guide 127 4 Searching Databases Searching for Biomarkers 4 In the Fragment Mass list select the type of fragment mass e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion 5 Optional Select the Delta m Mode check box if you want to conduct the search in delta m Am mode For more information on this mode see Performing Searches in Delta m Mode on page 110 6 In the Precursor Tolerance boxes specify the tolerance that determines whether comparing an observed precursor ion mass to a theoretical precursor ion mass is considered a match Set the following parameters e Lower Sets the minimum value for a precursor search window that does not trigger an out of range warning e Default Sets the default value for a precursor search window e
329. the precursor ion Charge State Specifies the charge state z to assign to the mass to charge m z data found in the data files 76 ProSightPC User Guide Thermo Scientific 2 Getting Started Importing Targeted RAW Files Demonstrating Targeted Raw File Importation with Post Xtract The following demonstration shows you how to import a targeted RAW file with the Post Xtract option Click the button below to view the demonstration To enlarge the demonstration once you start it right click and choose Full Screen Multimedia Thermo Scientific ProSightPC User Guide 77 2 Getting Started Importing Targeted RAW Files Importing a Targeted RAW File with the Profile Option The Profile option applies the THRASH algorithm to the importation of mass values The THRASH algorithm is an AIM operation that converts high resolution mass spectral data from proteins or large peptides into neutral monoisotopic or average masses Setting THRASH Preferences Before you import a targeted RAW file you might want to set the default values for the THRASH algorithm Use the Thrash Preferences page of the Options dialog box To set THRASH preferences 1 Choose Tools gt Options gt Thrash The Thrash Preferences page of the Options dialog box opens 2 From the left pane of the Options dialog box click the Thrash folder The Thrash Preferences page opens as shown in Figure 39 Figure 39 Thrash Preferences page of the Options dialog box O
330. ting at least this good of a result l Meng EB J Cargile Miller L H Forbes A J Johnson J R Kelleher N L Informatics and multiplexing of intact protein identification in bacteria and the archaea Nat Biotechno 2001 19 952 957 2 LeDuc R D Taylor G K Kim Y B Januszyk T E Bynum L H Sola J V Garavelli J S Kelleher N L ProSight PTM an integrated environment for protein identification and characterization by top down mass spectrometry Nucleic Acids Res 2004 32 W340 W345 3 Reid G E Shang H Hogan J M Lee G U McLuckey S A Gas phase concentration purification and identification of whole proteins from complex mixtures J Am Chem Soc 2002 124 7353 7362 ProSightPC User Guide 193 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Expectation Value e value Sample Calculation 194 ProSightPC User Guide The expectation value e value is the number of sequences in a database that are expected to have p scores equal to or better than what was observed simply by chance Low e values represent better matches less likely to be false positives than high e values Since the p score represents the probability of the v out of f fragments matching by chance and if it is assumed that all sequences in the database are independent the e value of a sequence fragment set association is simply the association s p value times the number of
331. tion resembles the illustration shown in Figure 75 ProSightPC User Guide 179 5 Viewing Search Results Viewing the Results in a Repository Report Figure 75 Completed Custom Filters section Custom Filters V Show Custom Filters Search Type sequence tag Add Cance oa m Merge Hits Add Custom Fitter Apply Fitters 7 To add another filter click Add Custom Filter again or right click and choose New 8 In the Use column select the filter that you want to apply 9 Click Apply Filters lt To remove a custom filter e In the Custom Filters area see Figure 70 on page 171 select the filters that you want to delete right click and choose Remove To merge a set of matches with similar values 1 In the Custom Filters area see Figure 70 on page 171 click Merge Hits The Merge Hits dialog box appears as shown in Figure 76 Figure 76 Merge Hits dialog box Merge Hits es Merge hits if they differ by less than 0 ions with tolerance 0 ppm 2 In the box to the left of Ions type the number of fragment ions by which the two matches can differ 3 In the box to the left of Ppm type a tolerance that the mass of the fragment ions must fall within for the sets of matches to be merged together 4 Click OK To reduce redundancy the ProSightPC application merges together a set of matches if the difference between the matches is fewer than the number of fragment ions
332. to the Data Manager without applying any changes to the mass values e Click The ProSightPC application does not save any of the edits that you made to the mass values It closes the Edit Masses experiment_number page and returns you to the Data Manager Running a Pending Search To run a pending search in the Data Manager 1 Click the side arrow of a pending search to reveal the search parameters and a Run Search button 2 Click Run Search to run the search For additional information on search parameters see Searching Databases on page 99 A completed search generates a results list in the tab controller as shown in Figure 63 on page 162 For information on these results see Viewing the Results in the Tab Controller on page 161 212 ProSightPC User Guide Thermo Scientific ee i Using Proteome Databases This chapter describes the proteome warehouse and how to create manipulate and modify proteome databases Contents e Proteome Warehouse e Importing Data into the Proteome Warehouse e Accessing the Database Manager e Importing a Proteome Database or Repository e Exporting a Proteome Database or Repository e Removing a Proteome Database or Repository e Changing View e Creating a Proteome Database e Linking to the UniProt Database Proteome Warehouse The ProSightPC application searches require sequence information to identify and characterize proteins This sequence information and modific
333. to use e Monoisotopic Specifies that the precursor mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the precursor mass is the mass of the most abundant isotope of the protein peptide or fragment ion Precursor Search Window Specifies a range around the observed precursor mass in daltons The ProSightPC application queries all protein forms with a theoretical mass within this range Fragment Mass Type Thermo Scientific Specifies the mass type of the fragment ions to use e Monoisotopic Specifies that the fragment mass is monoisotopic which is the mass of the protein peptide or fragment ion where all carbons are carbon 12 e Average Specifies that the fragment mass is the mass of the most abundant isotope of the protein peptide or fragment ion ProSightPC User Guide 123 4 Searching Databases Searching for Absolute Mass 124 ProSightPC User Guide Table 24 New Search in Experiment X dialog box parameters for absolute mass Sheet 2 of 2 Parameter Fragment Tolerance Description Specifies the tolerance that determines whether comparing observed fragment ion mass to a theoretical fragment ion mass is considered a match and indicates whether it is expressed as absolute measured in Da or relative measured in ppm Am Mode Determines whether the ProSightPC application conducts the search in d
334. u want to add Sequence post translational modifications PTMs or arbitrary custom masses Continue Displays a new window showing the protein sequences in an interactive sequence map Converting Text to ProSightPC Font with the Font Converter Thermo Scientific You can use the ProSightPC Font Converter to convert text into the ProSightPC fragment map font used to display N terminal and C terminal fragments You can also use it to generate fragment maps to include in publications and presentations ProSightPC User Guide 241 9 Using ProSightPC Tools Converting Text to ProSightPC Font with the Font Converter To convert text to ProSightPC fragment map font 1 Choose Tools gt Font Converter The Font Converter dialog box opens as shown in Figure 107 Figure 107 Font Converter dialog box Font Converter Sequence ProSightPC Font Equivalent Sarelesraienies b clon yz lon 2 In the Sequence box enter the amino acid sequence to be converted as shown in Figure 108 You can either type the amino acid sequence in the box or paste it from another source 242 ProSightPC User Guide Thermo Scientific 9 Using ProSightPC Tools Converting Text to ProSightPC Font with the Font Converter ADQLTE EQIAEFKEAF SLFDKDGDGTITTKELGTVMRSLGQNPTEA ELQD MINEVD ProSightPC Font Equivalent AD QL T ELE Q 1 A E F K E AFIS L F D K D G D G T I T T K E L G T V M R S L G Q N P T E A E L
335. ue Max Proteins to Return Specifies the maximum number of proteins to return in the search Fixed Modifications Specifies the chemical modifications present on all instances of a given type of amino acid in the observed protein Terminal Mods Specifies the fixed terminal modification for each terminus e N Terminal Mod Specifies the fixed terminal modification for the N terminus e C Terminal Mod Specifies the fixed terminal modification for the C terminus Save Saves the search information Thermo Scientific 4 Searching Databases Performing MS Hybrid Searches Performing MS Hybrid Searches In some cases you might need to use a sequence tag search to reduce the search space before performing an absolute mass search MS hybrid searches perform this function A hybrid search first compiles a list of all possible sequence tags consistent with the observed fragment ions and then the ProSightPC application uses these tags to identify all protein forms in the database that are consistent with the tags The list of protein forms that match the sequence tags functions as input into an absolute mass search Analyzing MS MS Experiments The ProSightPC application is built on the concept of the MS MS experiment Analyzing an MS gt experiment requires the following steps 1 Construct an MS MS experiment with the MS MS data as precursor masses and the MS data as fragment ion masses This MS MS experiment i
336. un the search with different parameters To remove search results from a search 1 Select the experiment in the data grid 2 Right click and choose Remove Results This command is only available when search results are present that is when the Pending Search column displays no for the appropriate search 3 In the confirmation box click Yes Removing an Experiment from the Data Grid To remove an experiment 1 Right click an experiment in the data grid and choose Remove Experiment x 2 In the Confirm Delete dialog box click Yes The ProSightPC application deletes the experiment from the data grid Performing Searches in Delta m Mode Delta m Am mode is a technique for identifying protein forms containing unknown PTMs The delta is the difference between the observed precursor mass and the theoretical precursor mass When you perform a search in delta m Am mode the ProSightPC application concurrently performs three queries per sequence to compare the following The theoretical fragment ion masses of the protein sequence to the observed fragment ion list as usual The theoretical fragment ion masses derived from the sequence and the delta m applied to the N terminal to the observed fragment ion mass list The theoretical fragment ion masses derived from the sequence and the delta m applied to the C terminal to the observed fragment mass list A delta m search takes approximately two times longer than the
337. ursors Relative Precursor Threshold As the dialog box itself notes Xtract and THRASH are both algorithms that interpret resolved isotopic distributions and output neutral mass values For more information on these algorithms see Importing Targeted RAW Files on page 73 5 If you choose a RAW file select a processing option in the Choose a Process Option area for importing the data files e Middle Down Specifies the following default settings for the Xtract and THRASH 7 0 25 40 20 Highest Intensity Selected 10 Cleared 3 0 25 10 10 100 Selected 7 0 0 90 25 25 Highest Intensity Selected 10 Thermo Scientific Thermo Scientific Fragmentation Minimum S N Fragmentation Minimum RL Fragmentation Maximum Charge Fragmentation Maximum Mass kDA Minimum Number of Fragmentation Scans Minimum Fragmentation Base Peak Intensity 2 Getting Started Processing LC MS MS Data Files 3 0 0 90 25 25 1 100 Top Down MS3 Specifies the following default settings for the Xtract and THRASH processing algorithms Xtract Precursor Minimum S N Precursor Maximum Charge Precursor Minimum Fit Precursor Remainder Threshold Precursor Selection Criterion Allow Multiple Precursors Relative Precursor Threshold Precursor Add Remainder Afterwards Fragmentation Minimum S N Fragmentation Maximum Charge Fragmentation Minimum Fit Minimum Fragmentation Base Peak Intensity Fragmentation Add Rem
338. ustom Adds a custom mass shift a PTM is a fixed mass shift e PTM tiers Reflects the priority of the PTMs The PTMs in tier 1 are more common To add virtual PTMs to an amino acid 1 Select an amino acid in the Sequence Gazer 2 Select the desired PTM from the Tier x box Observe that the amino acid changes color Each amino acid has its own PTMs 3 Click Rescore Fixed Modifications Box The Fixed Modifications box lists each fixed modification supported by the ProSightPC application by amino acid You can select fixed modifications in the dialog box by choosing Tools gt Fixed Modification Editor see Editing Modifications on page 24 or you can change them during rescoring To indicate that no fixed modifications are presently selected for that type of amino acid and will not be included in the next rescoring select None Each amino acid can have no more than one fixed modification Thermo Scientific ProSightPC User Guide 199 6 Using the Sequence Gazer to Search for Single Proteins Navigating the Sequence Gazer Matching Fragments Table The matching fragments table shown in Figure 83 contains a summary of all fragment ions matching the protein Figure 83 Matching fragments table HUM tYtrUsterttUatviUstvd adn YShow Matching Fragments Total 19 fragments ID Name m z Monoisotopic Mass Monoisotopic Theoretical Mass a 6 14 20 30 42 a4 60 66 72 4 8 15 19 al 47 99 67 73
339. with FT di 84 1 Absolute Mass no na 0 ETD fragmentation for precursor at m z 1096 40 from retention time min 30 52 225 30 81 227 with FT di 85 1 Absolute Mass no na 0 ETD fragmentation for precursor at m z 1023 06 from retention time min 30 52 225 31 57 233 with FT di 86 1 Absolute Mass no 1 81e 63 1 ETD fragmentation for precursor at m z 777 69 from retention time min 31 00 228 31 22 230 with FT de 87 1 Absolute Mass no 1 61e 31 1 ETD fragmentation for precursor at m z 654 81 from retention time min 31 00 228 31 22 230 with FT de 88 1 Absolute Mass no n a 0 ETD fragmentation for precursor at m z 1123 37 from retention time min 31 33 231 31 57 233 with FT de 89 1 Absolute Mass no 1 71e 38 1 ETD fragmentation for precursor at m z 845 32 from retention time min 31 70 234 31 96 236 with FT de 90 1 Absolute Mass no 1 13e 72 1 i ETD fragmentation for precursor at m z 774 69 from retention time min 31 70 234 31 96 236 with FT de Thermo Scientific To change the columns displayed in the data grid Do one of the following or e In the data grid right click the area to the right of the columns and choose Columns gt column_name as shown in Figure 112 e Follow this procedure Click the Grid Display Preferences tab i The Show Columns area of the Grid Display Preferences page displays check boxes whose names correspond to the names of the data grid c
340. with the Post Xtract Option 74 Importing a Targeted RAW File with the Profile Option 79 Entering Data Manially iene tne ib eratenayled naeeua 84 Experiment Adder Dialog Box Parameters 0 000000 ee eee eee 87 Importing Experiments 0 0 ccc eee eee eee 89 Searching the Proteome Warehouse for Matches 000 eee ea ee 89 Working with Experiments 0 0 ccc cece eee eee eee eens 91 Experiments in PUF Files oes ow whe dd eis bee ee a abe See Maca oe 91 Creating a New PUF File carn woud water eae meat R a 92 Opening an Existing PUF File pasate ean ty onthe ares 93 Adding Experiments to PUF Files o n io ede Seeks Sek ean wales 94 Copying Experiments from One PUF File to Another 00 94 Removing Experiments from PUF Files wick c cea scene aiea Senate tales eee 95 Saving a Changed PUF File l n Lkatrethn doctaMineerd bahargeretddaienancue doses 95 Changing the Experiments Displayas its con ints sea eee Hea ae eee 96 Deleting PUP Pilesacsouee Prag at ato Lee DASA tars CAE Ne Be 97 Experiment Manager Parameters 0 0 eee 97 Searching Databases 0 0 c cece eee eee eee een eens 99 Search Types aerei nesa sine a ives wee a a a aaa teat 6 99 Performing Searches so natrii nat u VEn a Diet a AT a E Aa 100 Performing Predefined Searches x slaiati cyeea AeA tata saw Wain ad atta hen 100 Performing Searches in Delta m Mode 0000s cee eee
341. wn bottom up 3 top down 3 Databases menu 249 259 de novo sequencing 136 deconvolution 56 62 63 default values 21 deisotoped peaks 66 Thermo Scientific Delete Selected Experiments icon 95 delta m searches 110 absolute mass searches 115 116 118 121 124 biomarker searches 126 128 129 132 135 gene restricted absolute mass searches 149 152 gene restricted biomarker searches 155 158 identifying unexpected modifications in UniProtKB files 1 matching fragments table 201 search parameter display 192 single protein searches 144 146 187 190 Demo Search 104 105 Digestion page 226 232 disulfide bonds search for absolute mass 121 126 search for biomarker 126 132 search for gene restricted absolute mass 149 search for gene restricted biomarker 155 using Enter Custom Mass function to compute 238 documentation survey xiii downloading databases 249 E e value calculating 194 in absolute mass searches 122 in biomarker searches 132 in General Preferences dialog box 22 23 in iterative searches 4 37 40 in Quick Filters section 272 273 in reference article 193 in results list 164 in Show Columns section 270 starting with specific search 100 ECD 10 207 Edit Comment icon 208 260 Edit icon 106 Edit Masses icon 208 260 Edit Masses page 210 Edit menu 247 259 Edit Predefined Search dialog box 88 103 105 106 108 Edit Search in Experiment X dialog box 254 263 Edit Selected Search icon 88 103 105 Edit Add
342. xpress the fragment tolerance either absolute in daltons or relative in parts per million An observed fragment ion matches a theoretical fragment ion if the observed fragment ion mass is within plus or minus the fragment tolerance of the theoretical fragment ion mass Select the Am Mode check box if you want to conduct the search in delta m Am mode For more information on delta m Am mode see Performing Searches in Delta m Mode on page 110 Select the Disulfide check box if you know that the protein s cysteines are oxidized The ProSightPC application looks for only one disulfide bond Select the Include Modified Forms check box if you want to include PTMs and polymorphisms when you perform a biomarker search To detect biomarkers with modifications on them select this option however processor time increases as a result In the Hit Filtering section set at least one of the following filters otherwise the ProSightPC application returns all protein forms that are searched even proteins that have no matching fragments a Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a minimum number of matching ion fragments these protein forms are called hits Then specify the minimum number of matching ion fragments in the box to the right b Select the Min of Matching Fragments check box if you want the search algorithm to find only proteins containing a m
343. y ProSightPC User Guide 37 2 Getting Started Processing LC MS MS Data Files Figure 21 Second level searching iS Good c2 d Load Load Good Bad Creating or Editing a One Level Search Tree To create or edit a one level search tree 1 If you are creating a search tree select New Search Tree from the Search Tree Name list on the Running Highthroughput Logic page of the High Throughput Wizard This option is selected by default If you are editing an existing search tree select the name of the search tree from the Search Tree Name list on the Running Highthroughput Logic page 2 Select the Experiment Filter check box to filter out experiments that will not yield matches If you are looking for intact proteins you might want to set a minimum precursor mass of 2000 Da to eliminate peptides from being searched a Select the Min Fragments check box this option is selected by default and in the box to the right of the option enter the minimum number of fragments to search for The default is 10 indicating that experiments that contain fewer than 10 fragments are ignored b Optional Select the Max Fragments check box and in the box to the right of the option enter the maximum number of fragments to search for The default is 500 indicating that experiments that contain more than 500 fragments are ignored c Select the Min Intact Mass check box this option is selected by default and in
344. y and Paste View Use these commands to display certain interface features such as grid preferences start screen job queue and toolbar Experiment Tools Use these commands to perform operations on experiments This menu is only available when an experiment is open in the Data Manager Databases Use these commands to handle proteome databases and repositories import and export databases and repositories create a custom database and download pre built databases ProSightHT Use these commands to run the High Throughput wizard and edit and create repositories and search trees Tools Use these commands to activate tools to process your data such as Experiment Adder PTM Tier Editor and Individual Sequence Adder Help Use these commands to view information about the current software release manage licenses and access the Help The ProSightPC toolbar pictured in Figure 110 appears directly below the menu bar on the ProSightPC graphical user interface Figure 110 ProSightPC toolbar DED AAH Eae D XZ YE Table 68 describes each of these icons Table 68 ProSightPC toolbar Sheet 1 of 3 Icon Menu equivalent D File gt New Function Clears the data grid so that you can create a new PUF file za File gt Open Opens an existing PUF file ProSightPC User Guide 259 B Using the ProSightPC Interface The ProSightPC Interface 260 ProSightPC User Guide Tabl
345. you use this dialog box to copy experiments from one PUF file to another see Copying Experiments from One PUF File to Another on page 94 Figure 43 Experiment Manager W Experiment Manager ic C fa De PUF File PUF File T eX T X E B Use the left side of the dialog box to perform operations on the source PUF file and the right side to perform operations on the destination PUF file 2 At the top left in the Experiment Manager click the Create New PUF File icon to create a new source PUF file Click the same icon on the right to create a new destination PUF file 3 In the dialog box that opens select the desired directory and type the name of the new PUF file 4 Click Save 92 ProSightPC User Guide Thermo Scientific 3 Working with Experiments Opening an Existing PUF File Opening an Existing PUF File You can open an existing PUF file by using a command on the File menu or by using the Experiment Manager Only one PUF file can be open at a time To open an existing PUF file from the File menu 1 Choose File gt Open or click the Open icon wa or Choose File gt filename where filename is the name of one of the four most recently opened PUF files listed at the bottom of the File menu 2 If no other PUF file is open or if a PUF file is open but you have made no changes to it in the Open a PUF File dialog box b

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