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Proteome Discoverer 1.4 User Guide Version A

1. OE SS BOT STOOD SH LOAD g seweti KMart ALLURE AOBTABARE Administration x bai A Import Remove Apply Is Active V Modification d Abbreviation DeltaMass Delta Average Mass Substitution Leaving Group Position a a al a al Add a Modification content Mas ar ee Amino AcidName One LetterCode Classification i Cysteine c Post translational is FASTA Indexes E Histidine H Chemical derivative Ss Lysine K Multiple m Spectral Libraries al Serine S Post translational Threonine T Post translational a Chemical Modifications al Tyrosine Y Chemical derivative 3 Cleavage Reagents _ Add a Modification g Is Active Modification Abbreviation DeltaMass DeltaAverage Mass Substitution Leaving Group Position a mena CITE S Acetyl Acetyl 42 010565 42 0367 H 2 C 2 0 Protein N amp La Acetyl Acetyl 42 010565 42 0367 H 2 C 2 0 Any_N_Te E a v Amidated Amidated 0 984016 0 9848 HN O 1 Protein_C E a Amidated Amidated 0 984016 0 9848 HN O 1 Any_C_Te E mj Vv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 N O Any E il Vv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 N O Any_N_Te Ej Bl K Carbamyl Carbamyl 43 005814 43 0247 HCNO Any amp Tel v Carbamyl Carbamyl 43 005814 43 0247 HCNO Any_N_Te El Vv Carboxymethyl Carboxymethyl 58 005479 58 0361 H 2 C 2 O 2 Any_N_Te E Fj Vv Carboxymethyl Carboxymethyl 58 005479 58 0361 H
2. OH BS B OT SOD O BH BB GD RR seuethT Q Mascot AK LU dd SHBERBFARE Celegans_FT_6ITDDDT_01_01 msf_annotation msf x 4b Peptides Search Input Result Filters Peptide Confidence Search Summary Description Peptides Molecular Function Cellular Component Biological Process Pfam IDs Gene IDs GO Terms m 1 _ UNCoordinated family member unc 54 Caenorhabdits 40 sa M il C PF00038 Pro0063 259839 G0 0000226 G 19 2 7 FO7AS 7 Caenorhabditis elegans 20 28 Dl PF01576 PF03961 _ 172491 G0 0003774 G 87 E 3 ATPsynthase subunitfamily member atp 2 Caenorhab 13 2 ME iii i TMM Proooos Pfoo306 175716 GO 0000003 G 53 f 4 7 VITellogenin structural genes yolk protein genes family 23 34 l PF00094 Pf01347 180781 G0 0005319 G 155 E 5 7 MYOsinheavychain structural genesfamily member myo 26 32 F l E ii E TMM Pfo0038 Pfo0063 179676 G0 0000003 G 196 6 VITellogenin structural genes yolk protein genes family 24 30 1 I I Pf00094 Pfo1347 180630 GO 0005319 G 160 f 7 Vrellogenin structural genes yolk protein genes family 27 34 PF00094 Pf01347 177619 G0 0005319 G 165 re a ENOLase family member enol 1 Caenorhabditis elegans 11 19 M m i Pf00113 Pf03952 174423 G0 0000015 G 43 9 Protein Disulfide Isomerasefamily member pdi 1 Caen 15 25 F mn Di I E Pf00085 175472 G0
3. 4 Select one or more of the GO Slim categories 5 If you selected more than one GO Slim category select the logical And option at the top of the dialog box to indicate that the Proteome Discoverer application should filter by the combined categories or select the logical Or option to indicate that it should filter by only one category Proteome Discoverer User Guide 211 6 Protein Annotation Displaying the Annotated Protein Results 6 Click OK The Proteome Discoverer application displays the identified proteins belonging to the selected categories The names of the categories selected appear in the filter row when you expand the width of the column as shown in Figure 154 Figure 154 List of proteins filtered by Go Slim category Molecular Function Cellular Component enzyme regulator activity OR receptor activity i aaj 2 7 m NM itt s MT m 1 OO 1 7 EOD T T T 7 EM T 1 ETD DA ANAON MINIT 1 TT MITT 3 T MITTIN 3 OTC T 3 T T 6 TOM T 1 T 1 Tn m 7 T T TT HNNNUHHAN MAMMAN T T 16 UN T 3 UOT HNNNUTHNAN HUTT Displaying GO Accessions Protein categories selected Gene
4. 0 908 18 0 720 0 849 1 182 0 690 m a Isotope Shift Isotope Intensity Ready 16 16 Protein Group s 2269 2269 Merged Protein s 6959 6959 Peptide s 16551 16551 PSM s 16344 16344 Search Input s The Quantification Method Editor dialog box also includes a General page when multiple MSF files are loaded at the same time It contains one option Treat Quan Results as Replicates as shown in Figure 189 This option treats protein level quantification values with the same ratio names and the same quantification method as replicates that is the protein ratios of the individual files are averaged into a replicate ratio Thermo Scientific Proteome Discoverer User Guide 283 7 Quantification Setting Up the Quantification Method Figure 189 General page of the Quantification Method Editor dialog box Treat Quan Results as Replicates When you select Treat Quan Results as Replicates and click OK the protein quantification data looks like the data in Figure 190 284 Proteome Discoverer User Guide Thermo Scientific Figure 190 Protein quantification data in replicate mode 7 Quantification Adding a Quantification Method File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OW SES ST SGROOSH BWA KR seqeth g Mascot AL Ue RABEBTRIARB Multi Repor
5. 5 E Documents Name Date modified Type d Music ee Pictures amp Videos BSA3 raw 10 26 2012 10 30 Xcalibur Raw File BSA3 xml 10 26 2012 10 30 XML Document BSA4 raw 10 26 2012 10 30 Xcalibur Raw File BSA4 xml 10 26 2012 10 30 XML Document Pd mudPIT msf 10 26 2012 10 31 Thermo s Mass Sp LJ mudPIT mzML 10 26 2012 10 31 MZML File j Computer amp OSDisk C TechPubs ussjo sanser G amp TechPubs Archive ussj _ a 6 items MudPIT Processing Using the Run Sequence Dialog Box Running MudPIT samples using the Run Sequence dialog box is similar to the batch processing described in Batch Processing by Using a Post Acquisition Method Xcalibur Data System 2 0 7 Only on page 89 Replace the batchprocessing param file with a parameter file for MudPIT You can use the Proteome Discoverer Daemon application to export raw files to MGF MZDATA DTA MZXML and MZML files To export files use a workflow that includes the Spectrum Files Spectrum Selector and Spectrum Exporter nodes Set the appropriate file type in the Spectrum Exporter node In batch processing the Proteome Discoverer Daemon application exports all the raw files with the file name of the spectrum 96 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application on the Command Line Running the Proteome Discoverer Daemon Ap
6. Iv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 N O Any Amino Acid Name One Letter Code Classification Cysteine c Chemical derivative Aspartic Acid D Artefact Glutamic Acid E Artefact Lysine K Artefact Add a Modification An empty row appears 4 In the empty row select the amino acid from the list in the Amino Acid Name column The amino acid and the one letter abbreviation appear 5 From the list in the Classification column select the type of modification 6 To save the modifications click the Apply icon Apply When you reimport data from unimod org the Proteome Discoverer application retains the modification that you added However if you want to change the classification of an amino acid you must do so before reimporting the Unimod data After you import the Unimod data the only way to change the classification is to delete the amino acid and re add it with another classification Deleting Chemical Modifications You can remove chemical modifications from the Chemical Modifications view To delete a modification 1 Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 2 Select the row of the modification that you want to delete 3 Click the Remove icon Remove 4 In the Delete Row dialog box click Yes The row is removed from the chemical modifications table Importing Chemical Modifications
7. OF BEBO T SAD ROR Z Dam K sequethtT K Macot i A Hf ei oBGadaAanG C18 2mgPBMC Sample A 200uLbeads 01 PhosPhoNodemsf x Administration x db Apply WA Reset Factory Defaults Process Management 2 4 1 ProteinCenter Server Sa roteinCenter U F service proteincenter proxeon com ProXwi ve ProteinCenter URL http web ProXweb CH Job Queue Number of attempts to submit the annotation request 3 Time interval between attempts to submit the annotation request sec 90 A Timeout of the annotation request min 15 a FASTA Files FASTA Indexes T Spectral Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management a R Licenses Configuration R B4 Workflow Nodes amp Mascot amp MSPepSearch amp SEQUEST Sequest HT 3 SpectraST B4 Server Settings 3 Discoverer Daemon amp 3 FASTA Indexes ProteinCenter URL The URL of ProteinCenter Ready 3 In the ProteinCenter URL box type the path and name of the ProteinCenter Web server Thermo Fisher Scientific gives you this URL a user name and a password when you subscribe to ProteinCenter Changes in the URL take effect after you restart the Proteome Discoverer application If you entered an incorrect URL the ProteinCard tab of the Protein Identification Details dialog box displays an error message 4 In the Number of Attempts to Submit the Annotation Request box specify the number of times tha
8. Enzymes Reagents Cleaves after baoe Except when Enzymes for digestion AspN D Chymotrypsin EW Y or L Chymotrypsin FWY F W or Y P is after F W or Y Clostripain R Elastase A L I or V P is after A L I or V Elastase Tryp Chymo A L L V K R W E or Y P is after AI L I V K R W F or Y 346 Proteome Discoverer User Guide Thermo Scientific B Chemistry References Fragment lons Table 27 Cleavage properties of enzymes and reagents Sheet 2 of 2 Enzymes Reagents Cleaves after rey Except when GluC EorD LysC K Proline_Endopept P Staph_protease E Trypsin KorR P is after K or R Trypsin KRLNH K R L N or H Trypsin_K K P is after K Trypsin_R R P is after R Chemicals for degradation Cyanogen bromide M Iodobenzoate W Fragment lons Thermo Scientific Fragment ions of peptides are produced by several different fragmentation techniques such as ECD ETD CID higher energy C trap dissociation HCD and infrared multi photon dissociation IRMPD As an example low energy CID spectra which are sequence specific are generated by MS MS and ESI The fragment ion spectra contain peaks of the fragment ions formed by the cleavage of the peptide bond and are used to determine amino acid sequences A fragment must have at least one charge for it to be detected The fragment ions produced are identified according to where they are fragmented in the peptide A b and c f
9. l 4 4 Dynamic Modifications N Terminal Modification Fixed Value 4 C Teminal Modification 1 Dynamic Modification 2 Dynamic Modification 3 Dynamic Modification 4 Dynamic Modification 5 Dynamic Modification 6 Dynamic Modification 4 5 Static Modifications Peptide N Terminus Peptide C Terminus 1 Static Modification 2 Static Modification 3 Static Modification 4 Static Modification 5 Static Modification 6 Static Modification To troubleshoot precursor ion quantification None None Phospho 79 966 Da S T None None None None None TMT plex 229 163 Da Any N Teminus None Carbamidomethyl 57 021 Da C TMT6plex 229 163 Da K e Ifyou obtain unexpected precursor ion quantification results verify that all settings of your processing workflow are reasonable These should match your isotope labeling sample See Check the dynamic modification parameters in the Sequest HT SEQUEST or Mascot search engines Check the node parameters that you set before performing the quantification to see if they are appropriate for your sample Performing Reporter Ion Quantification on page 249 for more information Verify that your isotopic labeling is one of the following options in the protein ID search node either Sequest HT SEQUEST or Mascot SILAC 2plex Arg10 Lys6 Uses arginine 10 and lysine 6 SILAC 2plex Arg10 Lys8 Uses arginine 10 and lysine 8 SILAC 2ple
10. 2012 Thermo Fisher Scientific Inc All rights reserved Xcalibur and LTQ are registered trademarks of Thermo Fisher Scientific Inc in the United States Proteome Discoverer is a trademark of Thermo Fisher Scientific Inc in the United States SEQUEST is a registered trademark of the University of Washington in the United States iTRAQ is a registered trademark of Applera Corporation in the United States and possibly other countries NIST is a registered trademark of the National Institute of Standards and Technology in the United States Mascot is a registered service mark of Matrix Science Ltd in the United States RAR is a registered trademark of Eugene Roshal in the United States TMT is a registered trademark of Proteome Sciences plc in the United Kingdom Excel Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries 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 change without notice All technical information in this document is for reference purposes only System conf
11. 3 Move the cursor over the GO Terms column The application displays the annotated GO term and all ancestor terms associated with a protein as shown in Figure 156 It shows the term annotated to the protein in brackets followed by their ancestor terms Each annotated GO term starts on a new line If you want all proteins to have a higher level annotation that is not provided by the Molecular Function Cellular Component and Biological Process annotation columns you can filter for the GO term in this column Proteome Discoverer User Guide 213 6 Protein Annotation Displaying the Annotated Protein Results Figure 156 The complete list of GO terms associated with a protein File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OT SDOOS Blk oom KR sequeth g Mst AL LR RABEBRRBARE Celegans_FT_6ITDDDT_01_01 msf_annotation msf x 40 Peptides Search Input Result Filters Peptide Confidence Search Summary Description Peptides PSMs i Molecular Function Cellular Component i Biological Process Pfam IDs Gene IDs GO Terms 1 J7 UNCoordinated family member unc 54 Caenorhabditis 40 61 TM mat TET Pfo0038 Pfo0063 259839 GO 0000226 G 4 2 7 FO7A5
12. 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 Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need 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 Thermo Scientific Proteome Discoverer User Guide xiii Preface 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 xiv Proteome Discoverer User Guide Thermo Scienti
13. 0 0000040 22 Configuring the SEQUEST Search Engines sicss0seicods nnwkiws noes 24 Configuring the Mascot Search Engine iis6s05 ctseeee esia tas ides 25 Thermo Scientific Proteome Discoverer User Guide iii Contents Chapter 3 iv Proteome Discoverer User Guide Starting a New Search by Using the Search Wizards 000 00 000 Starting a New Seatelin ot cmsnive ga clued eiee tineo iaoi a aa Starting a New Search by Using the Workflow Editor 0 Before Creating a Workflow 4242 o iwise4 o o duiem Salem nunen eneee Creating a Search Workflow ning Mo ciw tau na dae tae ho eee Creating a Search Workflow for Multiple Raw Files from the Same Simple Walia Kar race aoe kin ETA tana aay E N Creating a Quantification Workflow 04 6s 05s canis d aeree Creating an Annotation Workflow 00 c cece eee eee eee Creating a PTM Analysis Workflow wesc sue ees d el ke eke ne Creating Parallel Workflows 5 03 cc 0h0 Ges Bo tare e ab Dales weeks Adding a Non Fragment Filter Node for High Resolution Data Opening an Existing Worktlowi s ie iene nea aha eee we Deleting an Existing Workflow Template 0 00 000000 Changing the Name and Description of a Workflow Template Importing Raw Data Files in Other Formats into a Workflow Saving a Workflow as an XML Template ve ih eden cede eee etek tee Exporting Spectrats cin oactnaiiaun tee es aceite a a eae Using the Proteome Di
14. Table 8 Peptides found for spectrum 10 m Sequest 2 Sequest 3 Mascot 4 Mascot 5 Peptide 2 1 Peptide 3 1 XCorr 12 Peptide 4 1 Peptide 5 1 XCorr 20 IonScore 33 IonScore 34 Peptide 2 2 Peptide 3 1 XCorr 12 XCorr 8 If you selected the Merge Results of Equal Search Nodes option peptides 4 1 and 4 2 which Mascot identified are ranked together Peptides 2 1 2 2 3 1 and 3 2 which Sequest identified are ranked together Thermo Scientific Proteome Discoverer User Guide 159 5 Filtering Data Filtering the Search Results If you did not select the Merge Results of Equal Search Nodes option peptides 4 1 and 4 2 which Mascot identified are ranked independently Sequest identified peptides 2 1 and 2 2 are ranked together and peptides 3 1 and 3 2 are ranked together To calculate the rank the Proteome Discoverer application sorts all peptides belonging together by their main score For Sequest the main score is XCorr For Mascot the main score is IonScore Peptides with the same main score have the same rank For example if you selected the Merge Results of Equal Search Nodes option the Proteome Discoverer application ranks the peptides shown in Table 8 as follows Sequest e Peptide 2 1 XCorr 20 Rank 1 e Peptide 3 1 XCorr 12 Rank 2 e Peptide 3 2 XCorr 12 Rank 2 e Peptide 2 2 XCorr 8 Rank 4 Mascot e Peptide 5 1 lonScore n 34 Rank 1 e Pe
15. Thermo Scientific 6 Protein Annotation GO Slim Categories Table 18 GO Slim categories for cellular components Sheet 2 of 4 GO Slim cellular component Cytoskeleton Description Any of the various filamentous elements that form the internal framework of cells and that typically remain after treatment of the cells with mild detergent to remove membrane constituents and soluble components of the cytoplasm The term embraces intermediate filaments microfilaments microtubules the microtrabecular lattice and other structures characterized by a polymeric filamentous nature and long range order within the cell The various elements of the cytoskeleton not only serve in the maintenance of cellular shape but also have roles in other cellular functions including cellular movement cell division endocytosis and movement of organelles Cytosol That part of the cytoplasm that does not contain membranous or particulate subcellular components Endosome A membrane bound organelle that carries materials newly ingested by endocytosis It passes many of the materials to lysosomes for degradation Endoplasmatic reticulum The irregular network of unit membranes visible only by electron microscopy that occurs in the cytoplasm of many eukaryotic cells The membranes form a complex meshwork of tubular channels which are often expanded into slit like cavities called cisternae The endoplasmatic reticulum takes two forms
16. 1 2 106 Proteome Discoverer User Guide To add a protein sequence and reference Choose Tools gt FASTA Database Utilities In the FASTA Database Utilities dialog box click the Add Protein References tab The Add Protein References page of the dialog box appears Click the Browse button next to the FASTA File box In the Save Add to FASTA File dialog box select the FASTA database that you want to add the protein sequence and reference to and click Save In the Enter Description box of the FASTA Database Utilities dialog box type a description of the protein sequence that you are adding In the Enter Protein Sequence box type the protein sequence that you want to add to the FASTA database The Add Protein References page should resemble the illustration in Figure 76 Thermo Scientific 4 Searching for Data Using FASTA Databases Figure 76 Add Protein References page of the FASTA Database Utilities dialog box FASTA Database Utilities Add Protein References Compile FASTA Database Find Protein References FASTA File C Program Files Proteome Discoverer source files FASTA_Files bovine fasta m Enter Description Bovine T cell receptor delta chain Enter Protein Sequence AQQVTQVQTA 7 Click Add Entry to add the protein sequence Finding Protein Sequences and References You can find a protein sequence or reference in an existing FASTA database file Thermo
17. 79 957 Da TMT2plex 225 156 Da TMT6plex 229 163 Da Trimethyl 42 047 Da Trioxidation 47 985 Da Note In the Mascot wizard the Dynamic Modifications area replaces both the Dynamic Side Chain Modifications and Dynamic Peptide Modifications areas You set these modifications on the Mascot server The Mascot wizard does not identify by delta masses the modifications that appear on the modification lists as the Sequest HT wizard does b If you are searching for static modifications select the modifications and the amino acids on which they can occur in the Static Side Chain Modifications area In the boxes on the left select the modifications In the boxes on the right select the amino acids on which the modifications occur Note In the Mascot wizard the Static Modifications area replaces both the Static Side Chain Modifications and Static Peptide Modifications areas You set these modifications on the Mascot server The modifications that appear on the modification lists in the Mascot wizard are not identified by delta masses as they are in the Sequest HT wizard c Inthe N Terminus list in the Dynamic Peptide Modifications area select the dynamic modification that occurs on the N terminus of the peptide Thermo Scientific Proteome Discoverer User Guide 39 2 Getting Started Starting a New Search by Using the Search Wizards 40 d In the C Terminus list in the Dynamic Peptide Mod
18. 8 12 I ii i il Ww Pf00573 172074 G0 0000003 G 34 30 MYOsin heavy chain structural genesfamily member myo 20 2 Ti Te I PF00038 PFO0063 181404 GO 0000166 G 194 4 31 7 Ribosomal Protein Largesubunit family member rpl 7 L 10 16 i ii I i PF00327 Pf08079 171602 G0 0000003 G 24 32 J Y41E3 10a Caenorhabditis elegans 7 13 1 ii I Pf00736 178419 G0 0003746 G 26 H 3 7 Vacuolar HATPase family member vha 11 Caenorhabdi 9 2 1 it I 1 DEI Pfe3223 177017 G0 0000003 G 38 Displaying Protein Family Pfam Annotation Results As noted in Pfam Annotation on page 203 you can retrieve Pfam annotations from the Pfam database as an alternative to GO annotations To display Protein Family Pfam annotation results 1 Open the MSF file by following the instructions in the Help 2 In the Column Chooser dialog box of the proteins page select the Pfam IDs column For information on the Column Chooser dialog box refer to the Help Figure 157 shows the Pfam IDs column on the Proteins page Displaying Entrez Gene Identifications Entrez gene identifications are unique identifications assigned to all genes stored in the Entrez gene database NCBI s database of gene specific information The Proteome Discoverer application displays these identifications in the Gene IDs column on the Pro
19. Kod 1 To filter your search results with peptide filters Open your search results Refer to the Help Click the Result Filters tab which is shown in Figure 111 on page 154 Click Add a Filter in the Peptide Filters area A list of filters now appears For a description of the filters available refer to the Help The Peptide Rank and Peptide Confidence filters are selected by default 156 Proteome Discoverer User Guide Thermo Scientific 4 5 Filtering Data Filtering the Search Results Select the filter to apply from the list of filters Options pertaining to the selected filter now appear in the Filter or Grouping Settings area as shown for the Peptide Score filter in Figure 113 The Help describes these options Figure 113 Peptide filter options File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OW BSIS SOF SBOROEH omm BR SeuethHT Q Maso AL ARE A2QEBBRBAR Celegans_FT_6ITDDDT_01 O1 msf x wl Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Filter and Grouping Set Active Filter V Peptide Confidence V Peptide Rank V Peptide Delta Cn o Cae Amaoem Based on Filter Set Filter or Grouping Settings Peptide Filters Peptide Grouping Options Show peptide groups Rex Group peptides by Mass and Sequence X Add a Filter Protein Filters Active
20. one protein group In the results report the Unique Peptides column on the Proteins page displays the number of distinct peptide sequences for a protein group When you expand an identified peptide as shown in Figure 130 the Peptides page shows only the master proteins of all protein groups that contain the peptide To display all the proteins that belong to any of the protein groups choose Search Report gt Show Protein Group Members which opens the Protein Group Members view see Figure 132 on page 178 To display all proteins that contain the peptide choose Search Report gt Show Protein References which opens the Protein References of a Peptide view see the Help The Unique Peptides column on the Proteins page displays the number of peptide sequences unique to a protein group File Search Report Quantification Processing Workflow Editor Administration Tools Window Help ua ageger aonana A A DB WG R SequesthT g Macot i A Kh Tk BL a Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf X 1000ngYeast_Top10_DE30 01 msf x Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary fz Sequence PSM Ambiguity Proteins Protein Groups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Qua_ I hPGDFGADAQGAMTk Unambiguous 5 2 494711 1942750 N Term iTRAQ8plex K15 PQD 0 1599
21. types e Electron transfer dissociation ETD e Electron capture dissociation ECD e Collision induced dissociation CID e High energy collision induced dissociation HCD e Pulsed collision induced dissociation PQD e ETD and ECD generate primarily c and z fragment ions with preferences for precursor ion charge states of 3 or higher e CID and HCD generates primarily b and y fragment ions with preferences for precursor ion charge states of 3 or lower e PQD and HCD do not exhibit a low mass cutoff and are good for reporter ion experiments Frequently peptides identified by CID PQD or HCD are not observed with ETD or ECD and vice versa so that combining results from for example CID and ETD can enhance sequence coverage Many times CID and ETD identify the same peptides often with different precursor ion charge states Combining ETD and CID results improves confidence in identifications Proteome Discoverer User Guide 3 1 Introduction Features SEQUEST Search Engine The SEQUEST search engine is specifically developed and optimized to evaluate both high mass accuracy and low mass accuracy ETD ECD CID HCD and PQD data You can use Sequest combined with automated LC MS MS and intelligent data acquisition tools to ensure the routine identification of low abundance proteins in complex mixtures The Proteome Discoverer application extracts relevant MS MS spectra from the raw file and determines the precursor charge
22. 7 Quantification Displaying Quantification Spectra Figure 196 Quantification Summary page for reporter ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 6H BE SOT onora gomm R seqetH Q Maso AL ME RABEBERBPARG Sequest SILAC Dimethylation Search 1 3 0 297 msf X Multi Reportfrom 2Reports X PQD with TMT 6 plex 10 175 min msf X db Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Search name PQD with TMT 6 plex 10 175 min Search description Search date 10 13 2008 06 15 30 Integration Window Tolerance 0 2 Da Integration Method Most Confident Centroid m 3 Scan Event Filters Mass Analyzer ITMS MS Order MS2 Activation Type PQD Quantification Method IMT plex Method Description Method for TMT 6 plex mass tags by Proteome Sciences plc g 5 3 j Residue Modification None N Terminal Modification None 126 21930 Da 127 21200 Da 128 20460 Da 129 19730 Da 130 19000 Da 131 18340 Da 126 monoisotopic m z 127 monoisotopic m z 128 monoisotopic m z 129 monoisotopic m z 130 monoisotopic m z 131 monoisotopic m z 126 12830 Da average m z 127 13160 Da average m z 128 13500 Da average m z 129 13830 Da average m z 130 14170 Da average m z 131 13870 Da average m z Purity Correction Factors in 2 1 o 1
23. Quantification Method Editor newMethod1 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Residue Modification None xj x N Terminal Modification None x i G Cancel Hep 6 To specify the parameters of the new quantification method follow the procedure given in Setting Up the Quantification Method on page 264 Changing a Quantification Method After you perform quantification you can change the quantification method of the current report You can add new quantification methods by copying an existing method and editing it You can also activate and deactivate methods that you want visible or hidden when setting up a quantification workflow However you cannot define mass tags or labels as you can when setting up the initial quantification method because they have already been measured You can access the quantification methods without loading an MSF file by using Administration gt Maintain Quantification Methods see Adding a Quantification Method on page 285 but if you want to save any changes to the quantification method in a report you must first open that report and use Quantification gt Edit Quantification Method 288 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Changing a Quantification Method To change a quantification method 1 Select the MSF report whose quantification method you want to change 2 Do one of the fol
24. The recalculation can take some time The thresholds and FDR values will be updated in the result file Do you want to continue If you set the Peptide Confidence filter on the Result Filters page when you loaded the report the warning shown in Figure 146 appears Figure 146 FDR recalculation message box for Peptide Confidence filters Warning Ex Confidence filters were applied for loading this report Reevaluating A peptides can change peptide confidences Therefore this report has to be reloaded Do you want to continue 3 In either box click Yes To save the peptide confidence and FDR settings on the Result Filters page e Choose File gt Save Report Thermo Scientific Proteome Discoverer User Guide 199 5 Filtering Data Calculating False Discovery Rates 200 Proteome Discoverer User Guide Thermo Scientific M Protein Annotation This chapter explains how the Proteome Discoverer application retrieves annotation information from ProteinCenter including GO Gene Ontology annotations Pfam Protein Families annotations Entrez gene annotations and information about post translational modifications PTMs from UniProt Contents e ProteinCenter e Gene Ontology GO Annotation e Pfam Annotation e Entrez Gene Database Annotation e Configuring the Proteome Discoverer Application for Protein Annotation e Creating a Protein Annotation Workflow e Displaying the Annotated Protein Results e Re
25. The x axis of the chart shows the quantification channels and the y axis shows the detected area for the given quantification channel defined by counts per minute 296 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart The dimethylation 3plex quantification method in SILAC has a sample labeled with the Light isotope a sample labeled with the Medium isotope and a sample labeled with the Heavy isotope Figure 199 shows the chart created by the Show Quan Channel Values command for these samples It shows the relative area of the samples labeled with the Light Medium and Heavy isotopes The sample labeled Medium is the sample with the greatest area Figure 199 Quan Channel Values chart for precursor ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OU SSBOTSDTOSH g OG K sett Kut ALAS ACEBTABARE Sequest SILAC Dimethylation Search 1 3 0 297 msf x 40 Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary f Accession Description ZCoverage Z Proteins Z UniquePeptides Z Peptides 3 PSMs Area A7 Heavy Light A7 Heavy Light Count A7 Heavy L G 1 M 1P100708398 2 Serum albumin 49 92 1 19 32 125 1 161 0 893 24 D 2 M 1P100706427 2 Cationic trypsin 20 73 2 3 4 10 3 040 0 834 4 5 3
26. Thermo Discoverer Public Files This directory might be invisible to you because the C Documents and Settings All Users Application data directory is hidden To display hidden directories choose Tools gt Folder Options gt View gt Hidden files and folders gt Show hidden files and folders in Windows Explorer 5 Optional To change this directory for easier data access open the Proteome Discoverer application choose Administration gt Configuration click Discoverer Daemon beneath Server Settings in the Configuration area on the left side of the Administration view and change the directory in the New Directory box shown in Figure 53 The settings are applied after you restart the Proteome Discoverer application Figure 53 Changing the destination directory where results from the Proteome Discoverer Daemon application are stored File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OU agogo BROODS zong G SequesstHT G Mascot 7 A HA HC RQBEBERBPARE Administration X WorkflowEditor x 4p Apply A Reset Process Management amp amp Current File Directory C ProgramData Thermo Discoverer 1 4 PublicFiles oh 3 Job Queue WAS New Directory C Users Desktop Proteome Discoverer source files Smoke_Test_Files ii Content Management a a FASTA Files is FASTA Indexes Spectral Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification
27. Thermo Scientific G34 Show Advanced Parameters RA Fa 8 Le Figure 31 Settings and filters 4 1 Input Data Protein Database c_elegans_100_021411_F w Enzyme Name Trypsin Full Maximum Missed Clea 2 2 Tolerances Precursor Mass Tolerar 10 ppm Fragment Mass Tolerar 0 8 Da Use Average Precurso False Use Average Fragment False 3 lon Series Use Neutral Loss a lon True Use Neutral Loss b lon True Use Neutral Loss y lon True Weight of a lons 0 Weight of b lons Weight of c lons Weight of x lons Weight of y lons Weight of z lons 4 Dynamic Modifications N Teminal Modificatior None C Teminal Modificatior None 1 Dynamic Modificatio None 2 Dynamic Modificatio None 3 Dynamic Modificatio None 4 Dynamic Modificatio None 5 Dynamic Modificatio None 6 Dynamic Modificatio None 5 Static Modifications None None None None None None When you click some parameters two lists appear as shown in Figure 31 EE 21 show Advanced Parameters Proteome Discoverer User Guide 49 2 Getting Started Starting a New Search by Using the Workflow Editor The list on the right gives the activation types available You can apply a filter option on the left to the setting that you select in the list on the right The list on the left consists of three options e Is Applies the setting selected in the list on the right In the examp
28. cleavage is full or partial c 36 Proteome Discoverer User Guide In the Missed Cleavages box use the increment and decrement buttons to specify the maximum number of internal cleavage sites per peptide fragment that is acceptable for an enzyme to miss when cleaving peptides during digestion Normally the digestion time is too short to enable the enzyme to cleave the protein at all allowed positions so you must specify the number of missed positions in one resulting peptide fragment where the enzyme could cleave but did not The minimum value is 0 and the maximum value is 12 The default is 2 Thermo Scientific Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards Note The following parameters are also available in the General Search parameters in Mascot e Instrument Specifies the instrument used to process the data in the raw data file e Taxonomy Specifies the category of organism in the Linnaean biological classification system from which the sample was drawn In the Search Tolerances area specify the precursor mass search tolerance Select the Use Average Precursor Mass option to use the average mass for matching the precursor In the Precursor Mass Tolerance box specify the precursor mass tolerance value used for finding peptide candidates in daltons Da milli mass units mmu or parts per million ppm For daltons the minimum value is 0 0001 and the maximum v
29. cleavage properties 346 deleting 152 displaying 151 filtering data 152 modifying 152 Cleavage Reagents view description 150 opening 151 coagulation GO Slim category 238 coisolation 310 Compact icon 103 105 Compile FASTA Database page 112 116 Completing the Wizard_name Search Wizard page 40 compressing protein databases 105 confidence indicators distribution in target false discovery rates 187 on Peptides page 188 350 Proteome Discoverer User Guide Configuration view configuring protein annotation 204 configuring search engines 21 configuring Mascot search engine 25 26 configuring protein annotation 204 configuring Sequest HT search engine 22 configuring SEQUEST search engine 24 conjugation GO Slim category 238 contacting us xiii Create Quantification Method dialog box 287 cSNPs 340 Custom Filter dialog box 111 152 171 custom parsing rule A 342 custom parsing rule B 343 custom parsing rule C 343 cytoplasm GO Slim category 234 cytoskeleton GO Slim category 235 cytosol GO Slim category 235 deconvolution 309 decoy database search calculating false discovery rates 186 defense response GO Slim category 238 Delete Methods dialog box 290 Deleted FASTA Indexes table 127 Delta Cn column 162 Delta Cn value 161 162 181 182 development GO Slim category 239 dimethylation 3plex quantification method description 243 246 selecting in Quantification Editor dialog box 266 268 troubleshooting precursor ion quantifica
30. definition 7 348 generated by CID 3 Z z fragment ions definition 7 348 generated by ETD and ECD 3 Proteome Discoverer User Guide 363
31. in the Quan Info column 6 To apply the purity correction for the detected quantification values select the Apply Quan Value Corrections check box For reporter ion quantification this option applies the correction for isotopic impurities No such correction is currently available for precursor ion quantification The application applies this purity correction after applying other settings that potentially change the quantification values This option is selected by default To avoid using quantification values from any of the channels when one or more of the quantification channels has a detected intensity of zero select the Reject All Quan Values If Not All Quan Channels Are Present check box By default this check box is clear To highlight a change in the ion intensity ratio that is the ratio of the ion intensity of the peptide in an experimental sample to the ion intensity of the peptide in the control sample larger than 7 or smaller than 1 7 in the results specify n in the Fold Change Threshold for Up Down Regulation box The default is 2 0 For example if you select 2 in the Fold Change Threshold for Up Down Regulation box the Proteome Discoverer application highlights those experimental results that are greater than twice as large up regulation or less than half as large down regulation as the control To exclude a peptide ion intensity ratio that is the ratio of the ion intensity of the pepti
32. 4 Drag the Precursor Ions Quantifier node to the workspace pane and attach it directly to the Event Detector node The Precursor Ions Quantifier node performs quantification for isotopically labeled amino acids Note You cannot use the Precursor Ions Quantifier node and the Precursor Ions Area Detector node in the same workflow You cannot use the Reporter Ions Quantifier node in the same workflow with either of these two nodes 5 Drag the appropriate search engine node for example SEQUEST to the workspace pane and attach it to the Spectrum Selector node 6 Drag the Fixed Value PSM Validator or the Percolator node to the workspace pane and attach it to the search engine node Figure 172 illustrates the workflow up to this point Figure 172 Beginning of the workflow for precursor ion quantification Spectrum BELH Selector 6 if EventDetector 9 Ss Precursor lons Quantifier 1 7 Add any other nodes that you want and connect them For general information about creating a workflow in the Workflow Editor see Starting a New Search by Using the Workflow Editor on page 42 8 In the Parameters pane of the Workflow Editor click Show Advanced Parameters Thermo Scientific Proteome Discoverer User Guide 247 7 Quantification Performing Precursor lon Quantification 9 Click the Spectrum Files node and specify the raw file s in the Parameters pane 10 Click the Event Detector node and set the paramete
33. Cell homeostasis The processes involved in the maintenance of an internal equilibrium at the level of the cell Cell motility Cell organization and biogenesis Any process involved in the controlled movement of a cell A process that is carried out at the cellular level and that results in the formation arrangement of constituent parts or disassembly of a cellular component The process includes the plasma membrane and any external encapsulating structures such as the cell wall and cell envelope Cell proliferation The multiplication or reproduction of cells resulting in the rapid expansion of a cell population Coagulation The process by which a fluid solution or part of it changes into a solid or semisolid mass Conjugation The union or introduction of genetic information from Defense response 238 Proteome Discoverer User Guide compatible mating types that results in a genetically different individual Conjugation requires direct cellular contact between the organisms Reactions triggered in response to the presence of a foreign body or the occurrence of an injury which result in restriction of damage to the organism attacked or prevention and recovery from the infection caused by the attack Thermo Scientific Thermo Scientific 6 Protein Annotation GO Slim Categories Table 19 GO Slim categories for biological processes Sheet 3 of 3 Go Slim biological process Development Descript
34. Enable Std QC Unk Other Action Program or Macro Name Sync Parameters 1 Yes Yes Yes Yes Run Program Yes Yes Yes Yes Yes Run Program Yes 6 In the added table row specify the name and location of the parameter file as follows a In the Enable column select the check box b In the Action list column select Run Program c Right click the Program or Macro Name column and choose Browse from the shortcut menu as shown in Figure 57 Figure 57 Programs view with the shortcut menu displayed Programs Enable Std QC Unk Other Action Program or Macro Name Sync Parameters 1 Yes Yes Yes Yes Yes Run Program Te Yes Yes Yes Yes Run Program Delete Rows Insert Row The Browse for Program dialog box opens d Browse to the following executable and click Open C Program Files Thermo Discoverer System Release DiscovererDaemon exe Note If the following warning appears click OK The file DiscovererDaemon does not exist on this computer Thermo Scientific Proteome Discoverer User Guide 83 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System e In the Parameters column type the location of the parameter file containing the commands that will execute the Proteome Discoverer Daemon application p path_to_parameter_file parameter_filename R IMPORTANT Ifthe name of the parameter file contains a space you must enclo
35. Search by Using the Workflow Editor on page 42 2 In the Workflow Editor drag the Spectrum Files node to the workspace 3 If you selected the Spectrum Files node as your input drag the Spectrum Selector node to the workspace and attach the Spectrum Files node to the Spectrum Selector node 4 Drag the Reporter Ions Quantifier node to the workspace pane and attach the Spectrum Files node to the Reporter Ions Quantifier node Note You cannot use the Reporter Ions Quantifier node in the same workflow with either the Precursor Ions Quantifier node or the Precursor Ions Area Detector node 5 Drag the search engine node that you want for example SEQUEST to the workspace pane and attach the Spectrum Selector node to the search engine node 6 Drag the Fixed Value PSM Validator or the Percolator node to the workspace pane and attach it to the search engine node Figure 174 illustrates the workflow up to this point Proteome Discoverer User Guide 253 7 Quantification Performing Reporter lon Quantification Figure 174 Beginning of the workflow for reporter ion quantification eo ms Reporter Ions Quantifier 12 Fixed Value PSM Validator 7 Add any other nodes that you would like and connect them For general information about creating a workflow in the Workflow Editor see Starting a New Search by Using the Workflow Editor on page 42 8 Click the Spectrum Files node and specify the raw file in th
36. See MSF files Maintain Chemical Modifications command icon 142 Maintain Cleavage Reagents command icon 151 Maintain FASTA Files icon adding FASTA files 104 listing FASTA files 101 Maintain Quantification Methods command icon adding quantification method 285 changing quantification method 289 deactivating quantification method 290 exporting quantification method 291 importing quantification method 291 removing quantification method 290 restoring quantification method template defaults 282 setting up Quantification Method Editor dialog box 265 Maintain Spectrum Libraries icon 130 132 134 MALDI 9 Mascot Generic Format files See MGF files Mascot search engine calculating peptide rank 160 configuring 26 configuring parameters for 21 25 description 3 5 directing application to server location 25 options for calculating FDR 197 output 13 quantification mode 17 searching for quantification modifications with 261 troubleshooting failed searches 28 wizard 2 5 29 Proteome Discoverer User Guide 353 Index M Completing the Wizard_name Search Wizard page 40 Mascot Search Parameters page 35 Rawfile and Scan Range Selection page 32 Scan Extraction Parameters page 33 Search Description page 40 Select Modifications page 38 141 starting 31 Welcome to the Search Wizard page 31 workflow 9 Mascot Significance Threshold peptide filter recalculating false discovery rates 197 mass tags 272 273 master proteins 174 membrane GO Slim category
37. The SpectraST node searches spectrum libraries downloaded from NIST and the PeptideAtlas home page It searches more slowly than the MSPepSearch node but automatically generates decoy libraries when you register a library You can therefore calculate the false discovery rate by using the Target Decoy PSM Validator node or the Percolator node Spectral Library Administration Mirror Plots The new Spectrum Libraries view on the Administration page lists all the spectrum libraries that you downloaded from NIST or the Peptide Atlas home page In the Peptide Details Identification view you can display a mirror plot for PSMs identified by a spectral library search to visually verify matches between measured spectra from your experiment and the reference spectra in the spectrum library New Workflow Editor Nodes Proteome Discoverer version 1 4 divides the Peptide Validator node of the 1 3 release into the Fixed Value PSM Validator node and the Target Decoy PSM Validator node Fixed Value PSM Validator Node The Fixed Value PSM Validator node assigns confidence levels according to the fixed score thresholds that you chose in preceding searches You can only connect search nodes that do not perform decoy searches such as MSPepSearch to the Fixed Value PSM Validator node The Fixed Value PSM Validator node has no parameters Target Decoy PSM Validator Node PhosphoRS 3 0 Node Thermo Scientific The Target Decoy PSM Validator node auto
38. The phosphoRS node retrieves the phosphorylation sites that were searched and the mass tolerance used for matching fragment ions directly from the attached search nodes It has two additional parameters for choosing a specific mass tolerance to use when matching fragment ions refer to the Help With these parameters you can overwrite the default mass tolerance setting used in the search node 56 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Creating Parallel Workflows Parallel workflows are workflows that search the same raw data file and the same part of the spectrum but specify different criteria different search nodes for the search or both They resemble the example workflow shown in Figure 35 You can use parallel workflows to conduct two or more searches using two or more search engines on the same raw data and to compare the results of these two searches at the same time For example you may want to search both CID and ETD data from the same raw data file to increase the chances of finding a match CID data contains b and y ions and ETD data contains b c and z ions so the two types of data are complementary You can also use a parallel workflow for quantification Figure 35 Parallel workflow File Search Report Quantification Processing Workflow Editor Administration Tools Window Help LERALERA E Spectrum amp Feature Retrieval A Event D
39. WB Add Remove eae eee 2 Name File Size kB Spectra Type Last Modified ww spectrast 03 13 oh lt Job Queue Content Management a FASTA Files is FASTA Indexes T Spectrum Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management R Licenses Configuration a 4 Workflow Nodes E Annotation amp Mascot E MSPepSearch 3 SEQUEST G3 Sequest HT amp SpectraST E 5 Server Settings 9 Discoverer Daemon amp FASTA Indexes Ready Now you are ready to search the spectrum library For more information on the SpectraST node refer to the Help To search with the SpectraST node see Searching Spectrum Libraries with the SpectraST Node on page 137 For more information on the SpectraST node refer to the Help To add a spectrum library for searching with the MSPepSearch node 1 Download the appropriate spectrum libraries from the NIST at http peptide nist gov or from Peptide Atlas at http www peptideatlas org speclib The Proteome Discoverer application recognizes the following file formats for searching spectrum libraries with the MSPepSearch node e zip gz files from NIST or PeptideAtlas You can find these files in the _nist tar gz file on the library download site at NIST or the _nist zip file on the PeptideAtlas home page The file must contain a complete spectrum library in MSPepSearch If files are missing the
40. You can import chemical modifications from a local file or obtain an updated version from unimod org a public domain database When you install the Proteome Discoverer application it automatically imports accessions from unimod org as chemical modifications 146 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 4 Searching for Data Updating Chemical Modifications To import chemical modifications from a local file To import chemical modifications from unimod org To import chemical modifications from a local file Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 Click the Import icon Import In the Import From list of the Import Modifications dialog box select Local File In the adjacent box click the Browse button to browse for your file or type the name and path of the file in the box To overwrite an existing upload select the Overwrite Existing check box Click Import A status message appears When the upload is complete click Close To import chemical modifications from unimod org Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 Click the Import icon J Import The Import Modifications dialog box appears as shown in Figure 107 Figure 107 Import Modifications dialog box I
41. You can save your selected filter settings as a group for future use You can also save your protein and peptide grouping settings as a set You can make this set the default or assign it a name These sets are saved in and loaded from external files so that you can export filter sets from one instance of the Proteome Discoverer application and import them into another instance The filter sets have an extension of filters If you want to use a filter set from one installation of the Proteome Discoverer application in another installation of the Proteome Discoverer application you must copy the filter set from the root directory of the first installation to the root directory of the other installation You can create these filter setting groups on the Result Filters page that appears during report loading or on the Result Filters page that appears after the report has already been opened You can load a previously stored filter set Loading a filter set replaces the currently set peptide and protein filters and the settings for the protein grouping with the filters and settings stored in the loaded filter set unless the filters were loaded before the MSF file was opened e To create and save a filter set e To load a filter set e To delete a filter set Thermo Scientific Proteome Discoverer User Guide 163 5 Filtering Data Filtering the Search Results To clear the default filter set To restore the default filter set in effect after inst
42. activation fragmentation types 8 Add Protein References page 106 adding and removing amino acids 145 adding chemical modifications 144 amino acids adding to chemical modifications 145 deleting from chemical modifications 148 filtering PSMs or peptides for site localization scores from phosphoRS 173 in Chemical Modifications view 142 mass values 345 number found during FASTA file processing 103 symbols 345 annotated spectra 3 Annotation node creating workflow that uses 55 retrieving protein annotation 202 retrieving protein annotations 206 Annotation view 204 annotation workflow 55 206 antioxidant activity GO Slim category 233 Any filter 50 arginine 244 Auto Layout command icon 48 autosamplers 85 86 available search engines 3 b fragment ions 347 348 b fragment ions 347 348 b fragment ions definition 7 348 generated by CID 3 Thermo Scientific base peak chromatogram Rawfile and Scan Range Selection page of search wizards 32 batch processing creating workflow 71 72 definition 72 in the Discoverer Daemon application 69 72 74 87 monitoring job execution in the Discoverer Daemon application 75 reannotating MSF files 217 using a post acquisition method 89 biological process codes 230 Biological Process column 206 biological processes 201 Biological Processes page 202 222 229 Blast searches 339 Browse for Program dialog box 83 C c fragment ions 347 348 c fragment ions abstracting proton from
43. dimethyl labeling The following default quantification methods are available for precursor ion isotopically labeled quantification e SILAC 2plex Arg10 Lys6 Uses arginine 10 and lysine 6 e SILAC 2plex Arg10 Lys8 Uses arginine 10 and lysine 8 e SILAC 2plex Ile6 Uses isoleucine 6 e SILAC 3plex Arg6 Lys4 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 4 for medium labels e SILAC 3plex Arg6 Lys6 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 6 for medium labels e Dimethylation 3plex Chemically adds isotopically labeled dimethyl groups to the N terminus and to the amino group of lysine e 180 labeling Introduces 2 or 4 Da mass tags through the enzyme catalyzed exchange reaction of C terminal oxygen atoms with 18O SILAC 2plex Methods Thermo Scientific In a typical SILAC quantification experiment two cell populations grow in media that are deficient in lysine and arginine One population grows in a medium containing normal light amino acids such as lysine 2G Nb The other population grows in a medium containing amino acids where stable heavy isotopes such as lysine 6 13 Cz 4N or lysine 8 18C PN have been substituted for normal atoms SILAC quantification usually uses Proteome Discoverer User Guide 243 7 Quantification Performing Precursor lon Quantification heavy arginine and
44. even when there is a large spread of the displayed values In binary logarithmic form a value of 1 means a two fold increase a value of 2 means a four fold increase a value of 3 means an eight fold increase and so forth Each of the separate distribution charts displays the peptide ratios in three sections The chart legend explains the meaning of these sections You can access the chart legend by right clicking the chart and choosing Show Legend The Peptide Ratio Distributions charts contain the three sections illustrated in Figure 212 The first section displays the distribution of the ratios of all peptides considered for calculating the ratio of this protein as a box and whisker plot A box and whisker plot is a convenient way of graphically depicting groups of numerical data through a five number summary 5 percent lower bound lower quartile median upper quartile 95 percent upper bound The range between the lower and upper quartile this is the range of the box is also known as the inter quartile range IQR and like the standard deviation for normally distributed data is a measure of the spread of the data e The box represents the peptide ratios between the 25th and the 75th percentiles The error bars represent the peptide ratios below the 5th and the 95th percentiles e The blue lines inside the horizontal bar represent the median of the distribution e The second section blue circles displays the distribution of the ra
45. quantification 268 Quan Info column calculating peptide ratios 313 displaying peptide classification 311 including excluded peptide in quantification results 310 Peptides page 300 Quan Usage column including peptides in quan results 310 Peptides page 297 quantification channels displaying values 295 for precursor ion quantification 268 for reporter ion quantification 271 missing 277 setting up a quantification method 264 setting up for ratio reporting 273 with only one peak 277 Quantification menu 242 quantification method adding 285 changing 288 checking the parameters set 281 deactivating 290 deleting 290 exporting 291 importing 291 restoring original template 281 setting up for multiple MSF files 282 setting up for precursor ion quantification 264 setting up for reporter ion quantification 264 Quantification Method Editor dialog box changing quantification method 289 Experimental Bias page 280 General page 283 loading multiple MSF files 282 opening 264 Protein Quantification page 278 Quan Channels page 268 271 287 Ratio Calculation page 275 300 Thermo Scientific Ratio Reporting page 273 287 setting options for multiconsensus reports 328 setting up quantification method 264 Quantification Methods view adding quantification method 286 changing quantification method 289 deactivating quantification method 290 exporting quantification method 291 importing quantification method 291 removing quantification method
46. some of the nodes in the workspace pane might exhibit a yellow warning symbol as shown in the example in Figure 38 This symbol indicates that the version of the node used when the template was created has been superseded by a later version in the current Proteome Discoverer application Delete the node from the workflow and drag the node with the same name from the Workflow Nodes pane to the workspace pane Proteome Discoverer User Guide 61 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 38 Warning symbol indicating an outdated node version Spectrum Selector A round blue warning symbol containing an exclamation point as shown in Figure 39 indicates that one or more of the parameter settings for the node are incorrect or outdated Click on the node and reset the parameters in the Parameters pane Figure 39 Warning symbol indicating incorrect parameter settings When you use a node that is outdated or has incorrect parameter settings a Workflow Failures pane opens beneath the Workflow Nodes pane as shown in Figure 40 62 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 40 Workflow Failures pane File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BROOD RH LOAD K seustH K Mascot AMAR GEBTABAR ou jiaalpler WorkflowEditor x WorkflowEditor x B
47. the peptides from different charges of the same sample for example different treatment states are modified with special isobaric labels The isobaric labels disaggregate during precursor ions fragmentation and create reporter tags that appear in the low mass region of the fragment spectra You use the intensity ratio of the observed fragment tags for relative quantification of the peptides from the different sample charges The co isolating peptides also create reporter tags that superimpose on the reporter tags of the selected peptide Because most of the proteins in a real sample are unregulated the co isolated peptides often create reporter tags with equal intensity If these superimpose on the reporter tags of a selected peptide of a regulated protein the observed ratios of the reporter tags in the fragment spectra can be false Furthermore the perturbed ratios of the selected peptides that are greatly affected by co isolation can also adversely affect the ratios that the Proteome Discoverer application calculates for the proteins that include these peptides Determining the extent to which the real reporter tag ratios of the selected peptides are perturbed is difficult It depends on the level of co isolation and the isolation characteristics of the instrument The Proteome Discoverer application flags PSMs with a high level of co isolation For newly generated MSF files it calculates and displays the percentage of interference within the
48. 0 000 32 26 14 11 5 051 2 604 3 3 0 0 0 00 2 462 2 636 1 1 0 0 0 00 1 419 2 331 1 1 0 0 0 00 1 348 3 094 1 1 0 0 0 00 1 508 2 038 2 2 0 0 0 00 2 713 3 028 1 1 0 0 0 00 9 948 2 377 2 2 0 0 0 00 4416 2 356 2 2 0 0 0 00 1 264 2 598 2 2 0 0 0 00 2 285 2 086 1 1 0 0 0 00 1 657 2 368 1 1 0 0 0 00 2 275 2 263 1 1 0 0 0 00 nu 4 Ready 220 234 Protein Group s 3238 3238 Merged Protein s 2354 2354 Peptide s 8981 8981 PSM s 20765 20765 Search Input s Identifying Isotope Patterns in Precursor lon Quantification The quantification spectra on the pages of the MSF report show the isotope pattern used for quantifying the peptides The algorithm used in precursor ion quantification finds isotope patterns by identifying target components that is known elemental compositions from event lists It identifies the peptides and searches in the event lists for the isotope patterns of these identified peptides After peptide identification the algorithm follows the steps shown in Figure 227 to identify the isotope patterns Thermo Scientific Proteome Discoverer User Guide 333 7 Quantification Identifying Isotope Patterns in Precursor lon Quantification Figure 227 Identifying isotope patterns Identified peptide Event list Y Calculate elemental composition Read events RT range around peptide RT Simulate theoretical isotope pattern f Simulate
49. 0 0000 aii ca 2 I hPGDFGADAQAAMS Unconsidered 2 494711 1942750 N Term iTRAQ8plex K15 PQD 0 1599 2 2 3 Ie Unconsidered 1 1 1942750 N Term iTRAQ8plex K15 PQD 0 3761 3 3 4 I GGSVTLPESGTDLLTHRLEK Unconsidered i 0 PQD 0 3941 4 4 5 D MAITDILSAK Unambiguous 1 1 131122 M1 Oxidation PQD 1 0000 0 0000 1 1 6 I LRAmVPDNIPVVAESGK Unambiguous 1 1 136294 M4 Oxidation PQD 1 0000 0 0000 1 1 7 D AAKPAEKK Unambiguous 3 1 104786 K3 iTRAQ8plex PQD 1 0000 0 0000 1 1 8 M ADKLIGTVTSSLK Unambiguous 1 1 7474950 PQD 1 0000 0 0000 us 1 9 LVEAVSSAK Unambiguous 2 1 143091 PQD 1 0000 0 0000 1 H 10 I VAPIQGFSAK Unambiguous 2 1 1942989 PQD 1 0000 0 0000 1 1 H it I VAPIQGFSAK Unambiguous 2 1 1942989 PQD 1 0000 0 0000 N 1 B 12 I hPGDFGADAQGAmIk Unambiguous 5 2 494711 1942750 N Term iTRAQ8plex M13 PQD 0 1968 0 0000 1 1 E Accession Description Score Coverage Proteins UniquePeptides Peptides PSMs 114 113 114 113 Count 114 113 V F i m 1942750 Myoglobin Horse Heart Mutant With Ser 92Replaced By 631 05 54 25 8 Y 173 0 980 10 aa 494711 Myoglobin Horse Heart Mutant With His 64Replaced By 567 37 54 25 5 1 158 1 102 3 A Sequence PSM Ambiguity Proteins ProteinGroups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Qua 13 hPGDFGADAQAAm amp Unconsidered 1 2 494711 1942750 N Term iTRAQ8plex M13 PQD 0 1968 2 2 14 I hPGDFGADAQGAmTk
50. 0000 3764 Unique Used 1 166 2 7 eWGSAEAGVDAASVSEFR Unambiguous OVAL_CHICK N Term TMT plex cD 1 0000 0 0000 3768 Unique Used 0 204 3 7 GAAQNIIPASTGAAk Unambiguous G3P_PIG N Term TMT plex K15 T cD 0 8687 0 0000 2968 Unique Used 0 988 4 7 QAAQNIIPASTGAAK Unambiguous G3P_PIG N Term TMT plex K15 T cD 0 8709 0 0000 2965 Unique Used 0 544 5 7 rHPYFYAPELLYYANk Unambiguous ALBU_BOVIN N Term TMT plex K16 T cD 1 0000 0 0000 4364 Unique Used 1310 We le Unambiguous ALBU_BOVIN N Term TMT6plex cD 1 0000 0 0000 3274 Unique Used 1 064 7 I kTGQAPGFTYTDANK Unambiguous CYC_HORSE N Term TMT 6plex K1 T M CID 0 8995 0 0000 2970 Unique Used 1 916 8 I nTDGSTDYGILQINSR Unambiguous LYSC_CHICK N Term TMT6plex PQD 1 0000 0 0000 3334 Unique Used 1 319 9 I IISWYDNEFGYSNR Unambiguous G3P_PIG N Term TMT plex CID 1 0000 0 0000 4433 Unique i 0 912 10 I rHPEYAVSVLIR Unambiguous ALBU_BOVIN N Term TMT6plex cD 1 0000 0 0000 3241 Unique Used 0 935 11 nTDGSTDYGILQINSR Unambiguous LYSC_CHICK N Term TMT6plex PQD 1 0000 0 0000 3367 Unique Used 0 990 12 7 eWGSAEAGVDAASVSEFR Unambiguous OVAL_CHIK N Term TMT6plex PQD 1 0000 0 0000 3766 Unique Used 1 496 13 nTDGSTDYGILQINSR Unambiguous LYSC_CHICK N Term TMT plex cD 1 0000 0 0000 3334 Redundant Not Used 1319 14 THPEYAVSVLLR Unambiguous ALBU_BOVIN N Term TMT6plex CID 1 0000 0 0000 3244 Unique Used 1 351 15 J nTDGSTDYGILQINSR Unambiguous LYSC_CHICK N Ter
51. 0003756 G 48 re 40 Malate DeHydrogenase family member mdh 1 Caenorh 13 24 M ii i I I Pf00056 PFO2866 175936 GO 0000166 G 34 11 VITellogenin structural genes yolk protein genes family 24 30 1 I TM Pfo0094 Pf01347 180647 GO 0005319 G 160 E 2 I Tudor Staphylococcal Nucleasehomolog family member t 13 14 L mm I PF00565 Pf00567 173811 G0 0003676 G 91 E 4B Heat Shock Proteinfamily member hsp 1 Caenorhabdit 17 18 E ii i l I I TE Pf00012 pro6723 178507 G0 0000003 G 64 14 7 HeatShockProteinfamily member hsp 6 Caenorhabditi 9 14 l iii I TE f00012 Pfo6723 178873 Go 0000003 G 65 E I ACTinfamily member act 4 Caenorhabditis elegans 6 17 iii Pf00022 180767 G0 0000166 G 36 16 F01G10 1 Caenorhabditis elegans 9 14 li E Pf00456 Pf00676 177906 G0 0002119 G 61 f 17 7 RibosomalProtein Smallsubunitfamily member rps 3 6 11 i ii i i i PF00189 Pf07650 175879 GO 0000003 G 24 E ETI J7 RO05G6 7 Caenorhabditis elegans 11 18 1 iii i m Pf01459 177524 G0 0005739 G 28 19 Temporarily Assigned Genename family member tag 61 10 17 I 1 m TMi i TY Pfooiss 176773 Go 0000003 G 30 El 20 7 H28016 1d Caenorhabditis elegans 13 17 li I I I PF00006 PF00306 173134 G0 0005524 G 51 21 7 HeatShockProteinfamily member hsp 60 Caenorhabdi 11 14 i Or I I TM Poos 175316 G0 0000003 G 56 2
52. 1 4 0 175 msf xX 4 Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Sequence PSM Ambiguity Proteins Protein Groups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Qua al m T EE E G a eeeceoe oo oqeoe one oe oe DTDTMLAAK Rejected TTTASSSKAAK Rejected YITEQLSAK Rejected mSSLQGKMR Rejected aLELFR Selected 1 1171661 PQD 0 0091 0 0000 1 124110 PQD 0 0091 0 PQD 0 0457 0 M1 Oxidation PQD 0 0731 2 494711 1942750 N Term iTRAQ8plex PQD 0 0868 2 K HUNYT eeeed o m n m ura wn ew u a w n a 8 4 SSeeceeeeoocccece 4429 gt Ready 41 103 Protein Group s 204 2266 Protein s 169 3286 Peptide s 617 7721 PSM s 1541 1541 Search Input s 3 To hide the filtered out rows after you have displayed them right click the page and again choose Show Filtered Out Rows Filtering Precursor Masses You can set filter criteria to display peptides that have precursor masses between certain specified values To seta precursor mass filter by using the row filter menu 1 Click gt and choose Custom from the menu The Custom Filter dialog box appears as shown in Figure 127 Thermo Scientific Proteome Discoverer User Guide 171 5 Filtering Data Filtering the Search Results Figure 127 Custom Filter dialog box Custom
53. 10 1942989 Chain D Structure Of Bovine Heart Cytochrome C Oxidase 651 680 2 1 1 3 a um PF 1177001 HYPOTHETICAL 15 9 KD PROTEIN IN BGLH WAPA INTER 431 8 78 1 1 1 2 1 229 1 12 6706933 AF058943 structural protein N bovine coronavirus MA 431 246 1 1 1 2 e B l 131122 PARVALBUMIN THYMIC AVIAN THYMIC HORMONE AT 4 09 9 17 7 1 1 2 ic 14 m 2492825 N CARBAMOYL L AMINO ACID AMIDOHYDROLASE L CA 4 03 2 20 2 1 1 2 15 283920 tensin chicken 3 11 081 3 1 1 1 1 023 1 m 16 T 2494769 GLUCOSAMINE FRUCTOSE 6 PHOSPHATE AMINOTRANS 3 07 229 1 1 1 1 1352 1 m a7 M 7522620 alphatectorin chicken 292 061 1 1 1 1 18 7 118453 DESMIN 286 324 1 1 1 1 m 19 M 136294 INDOLE 3 GLYCEROL PHOSPHATE SYNTHASE IGPS 281 692 1 1 1 1 Ai an 729648 TRANSCRIPTION ACTIVATOR GUTR 274 133 1 1 1 1 1 269 1 Ta Protein Group Members x Accession Description Score Coverage Peptides PSMs AAs Mw kDa calc pI IsMasterProtein a ri 4971 Myoglobin Horse Heart Mutant With His 64Replaced By 56737 5425 7 158 153 17 0 7 78 Vv a 2 M 999881 Myoglobin Mutant With His 64Replaced By Thr H64t 550 03 45 10 6 153 153 16 9 7 78 D a 3 M 127691 MYOGLOBIN 36041 27 45 4 7 153 171 717 r wm 4 127638 MYOGLOBIN 207 58 11 04 2 n 154 171 7 46 r ol S m 462677 MYOGLOBIN 49 55 3 92 1 23 153 173 8 32 ml ae 2I Ready 41 103 Protein Group s 204 2266 Protein s 169 3286 Peptide s 617 7721 PSM s 1541 1541 Search Input s Proteins page main Pro
54. 100 0 S 11 7 70 0 00 3 H 16 7 SLDsDEsEDEEDDYQQK 66 1 1P100013297 1 SA Phospho S7 Phospho 0 0000 S 1 0 0 S 4 100 0 S 7 7 68 0 00 2 E 17 J7 ATASPRPSSGNIPSsPTASGG 20 3 1 1P100797365 1 15 Phospho 19 Phosph 0 0092 T 2 0 0 S 4 0 0 S 8 0 7 53 0 00 3 G 18 I7 GLmAGGRPEGQYsEDEDDD 10 3 1 1P100749304 1 M3 Oxidation 13 Phosp 0 0000 Y 12 8 0 S 13 48 5 T 7 52 0 00 3 E 9 I7 ATAsPRPSSGNIPSsPTASGG 10 3 1 1P100797365 1 SA Phospho 15 Phospho 0 0000 Too manyisoforms 7 51 0 00 20 TRDDGDEEGLLtHsEEBEH 6 3 1 1P100514496 2 T12 Phospho 514 Phosp 0 0000 T 1 0 0 T 12 98 0 S 1 7 46 0 00 3 21 RPPSPDVIVLsDNEQPSSPR 16 4 1 1P100444526 1 S4 Phospho S11 Phospho 0 0000 S 4 100 0 S 11 100 03 737 0 00 3 amp 22 GLVAAYSGEsDsEEEQR 24 2 1 1P100375731 1 S10 Phospho S12 Phosph 0 0000 Y 6 0 0 S 7 0 0 S 10 7 32 0 00 2 fm 23 7 EADIDssDEsDIEDDQPSA 10 1 1 1P100419531 2 S6 Phospho S7 Phospho 0 0000 S 6 100 0 S 7 100 0 S 7 27 0 00 3 E 24 I AIHssDEGEDQAGDEDEDDE 1 1 1 1P100001545 3 SA Phospho S5 Phospho 0 0000 S 4 100 0 S 5 100 0 7 24 0 00 2 fm 25 7 EVEEDSEDEEMSEDEDDss 1 3 1 1P100183526 5 S6 Phospho S12 Phospho 0 0000 S 6 100 0 S 12 100 0 7 22 0 00 3 E 726 I DGPPSALGsREsLATLSADL 10 3 1 1P100640740 1 9 Phospho S12 Phospho 0 0000 S 5 50 0 S 9 50 0 12 7 415 0 00 3 27 7 tPSPLVLEGTIEQSsPPLS
55. 108 Proteome Discoverer User Guide 149 4 Searching for Data Customizing Cleavage Reagents Figure 108 The Qual Browser application window w e i Thermo Xcalibur Qual Browser Celegans_FT_6ITDDDT_01 RAW ro eee fiz File Edit View Display Grid Actions Tools Window Help e x SEBS SPA OKTAGA rA SHES amp CBE 2 o RR Celegans_FT_6ITDDDT_01 10 17 2008 4 48 27 AM Ateli hus RT 0 00 119 98 a fil Celegans_FT_6ITDDDT_01 NL JK no file 100 4 60E8 6 no file TIC MS JI no file 90 Celegans_ 4 2 no file 80 FT_6ITDD 6 no file DT_01 E K no file 70 FRI no file z 35 09 S 60 3 34 29 2 50 ia 2 31 06 40 amp 62 08 2 2981 ee 113 63 20 pE 93 99 112 55 66 52 69 21 404 94 107 96 28 76 87 22 89 28 arr 10 l 77 25 87 22 6 14 1236 18 59 2471 iij PNG tt hiner omens a aw T aha ddl fili has 0 10 20 30 60 80 90 100 110 Time min Celegans_FT_6ITDDDT_01 1 RT 0 01 AV 1 NL 1 24E5 E T FTMS p NSI Full ms 300 00 2000 00 sa 391 29 90 80 o 70 3 60 g 5 2 50 P Z 40 30 20 bs 684 20 758 22 890 75 997 75 112585 1259 42 1461 30 1594 24 1688 37 1834 81 1905 29 800 1000 1200 1400 1600 1800 2000 miz NUM 2 Right click the lower pane and choose Display Options from the shortcut menu 3 To automatically annotate your peaks with the elemental composition theoretical
56. 117 4 Searching for Data Using FASTA Databases To specify the location and number of the FASTA indexes stored 1 Choose Administration gt Server Settings gt FASTA Indexes The configuration view shown in Figure 85 appears Figure 85 FASTA Indexes configuration view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH aoge onnon A g BD GB 0 SequethT K Mascot A HK h 5k a SBBGRIARGE Administration x 4 A Apply A Reset Process Management amp FASTA index directory C ProgramData Thermo Discoverer Demo 1 4 FastaDatabases Sy Job Queue TRS New directory ka Maximum number of FASTA indexes 30 Content Management a New maximum number of FASTA indexes 30 H FASTA Files FASTA Indexes a PER hu Spectral Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification Methods m License Management R R Licenses Configuration a B Workflow Nodes 3 Annotation amp Mascot amp MSPepSearch a SEQUEST 3 Sequest HT amp SpectraST 4 Server Settings 4 amp 5 Discoverer Daemon 3 FASTA Indexes 2 In the New Directory box browse to the location of the folder to store the FASTA indexes in 3 In the New Maximum Number of FASTA Indexes box select the maximum number of FASTA indexes to store If you generate more FASTA indexes than the number to store in the New Maximum Number of FASTA Indexes
57. 12 Export Number of Rawiles Evo _ BSA_ETD_CID_HIGH_HIGH_pico_02 Output Filename test_filename 3 In the Number of Rawfiles box for a MudPIT search select the number of files that will appear in the Xcalibur Sequence Setup dialog box The Number of Rawfiles option is not available when you select batch processing 4 Click Export The Save a Parameter File dialog box appears 5 Specify the path and name of the parameter file and click Save The Proteome Discoverer application writes the parameter file in xml format to the specified directory To call the Proteome Discoverer Daemon application through the parameter file see Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Thermo Scientific Proteome Discoverer User Guide 81 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Creating a Processing Method That Calls the Discoverer Daemon Application The following procedure describes how to create a processing method that calls the Daemon application It assumes that you have already created an appropriate processing method for your raw data files Processing methods have a pmd file extension To add a processing method that calls the Discoverer Daemon application to a processing method 1 Choose Start gt All Programs gt Thermo Xcalibur gt Xcalibur to start the Xcalibur data system The Roa
58. 131 National Institutes of Health NIH 227 229 230 NCBI RefSeq database 340 neutral loss ions removing 59 neutral loss peaks 59 New Sequence Template dialog box 87 New Workflow icon 44 Non Fragment Filter node adding for high resolution data 58 normalization factors 280 NRL3D database 342 nucleotide binding GO Slim category 233 nucleus GO Slim category 236 Proteins column 174 Unique Peptides column 175 184 0 180 labeling quantification method description 243 246 selecting in Quantification Method Editor dialog box 266 268 troubleshooting precursor ion quantification 337 Open From Template icon 61 Open Processing Workflow Templates dialog box 61 64 65 Open QualBrowser command icon 149 Options dialog box ProteinCenter page 221 organelle lumen GO Slim category 237 outdated workflow nodes 61 outputs of Proteome Discoverer application 13 overtones 59 P parallel workflows 57 parameter file 93 creating 81 purpose 81 Parameters pane 6 48 Thermo Scientific Index N PDB database 339 peak area calculation quantification creating workflow for 259 description 7 259 performing 259 Precursor Ions Area Detector node 259 pen icon 65 Peptide Confidence page changing filter settings 197 changing target rate 197 functions 196 recalculating false discovery rate 197 viewing decoy database search results 194 peptide filters applying 156 Delta Cn 161 rank 157 161 search engine rank 163 Peptide Ra
59. 194 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates Figure 142 Peptide Confidence page with the actual relaxed and strict FDRs File Search Report Quantification Processing Workflow Editor Administration Tools Window Help ajeje vr onnna Sm CK SequestHT G Mascot AM LHe 2GSRBRRBSARE Workflow Editor helaRVD1_1_1_1 percolator x Administration helaRVD1_1_1_1 percolator msf x Celegans_FT_GITDDDT_01_decoy msf x x Ti E_coli msf x Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary f Xan Score Versus Modest Confidence Filter Setings High Confidence Filter Settings Filter settings z ORAE TEE n arba eatbiterType Apply Fitters Apply FDRs Minimal Score for charge state 2 1 765 2215 Minimal Score for charge state 3 217 246 Processing Node Name lame Short Display Name Tama Scoss for Gigs stato lt 4 3705 3705 gt T T_6l A2 Minimal Score for charge state 5 371 Minimal Score for charge state 5 371 Minimal Score for charge state 6 3715 Minimal Score for charge state 6 3715 Minimal Score for charge state 7 372 Minimal Score for charge state 7 372 Minimal Score for charge state gt 7 Minimal Score for charge state gt 7 Minimal Score for charge state 1 Peptides having a charge state of 1 and an
60. 2 C 2 0 2 Any o M Deamidated Deamidated 0 984016 0 9848 H 1 N 1 0 Any Ej iy K Deamidated Deamidated 0 984016 0 9848 H 1 N 1 O Protein N o je M Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Any Ej H v Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Any_N_Te E Wi v Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Protein_N a Ww Dimethyl 2H 4 Dimethyl 2H 4 32 056407 32 0778 2H 4 C 2 Protein N o e Table 7 Available modification categories Sheet 1 of 2 Classification Post translational Description Protein modification after translation in vivo Co translational Amino acid modified in translation for example myristyl glycine Pre translational Amino acid modified before integration into a protein for example formyl methionine Chemical derivative Chemically induced modification for example during sample preparation Artifact Modification made during sample preparation N linked glycosylation Glycosylation in vivo O linked glycosylation Glycosylation in vivo Other glycosylation Glycosylation in vivo Thermo Scientific Proteome Discoverer User Guide 143 4 Searching for Data Updating Chemical Modifications Table 7 Available modification categories Sheet 2 of 2 Classification Description Synthetic peptide Protection group used in chemical peptide synthesis for protection group example trityl triphenylmethyl Is
61. 231 External Links Pages deere ute Mes wad ain ee hae wales 231 Thermo Scientific Thermo Scientific Chapter 7 Contents GO Silin Care cones tre eana ath wants wih tartare eu E a AAA 233 GO Slim Categories for Molecular Functions 000 00000 ee 233 GO Slim Categories for Cellular Components 0 0005 234 GO Slim Categories for Biological Processes 0 1 0 0c cee ee eee 237 Quantification aiaia aE e aie den alee manne dase RS 241 Activating the Quantification Menu 3 405 0 0 0 a hsets ha a uurre 242 Proteins Included in the Quantification 0 cee cece eee 242 Performing Precursor Ion Quantification 0 eee eee eee eee ee 243 SILAC 2 plex Methods iwi dno preted cere es E eee wis 243 SILAC 3plex Methods scasicloss 6 ce ossacuceca ee gneclersooah es racen sii ieee eloopeeegnoees 246 Dimethylation 3plex Method iiins ie Min te eiea pate erreneren 246 180 Labeling Methodi srra esd 0G ices a wage er oameh oa ict 246 Creating a Workflow for Precursor Ion Quantification 246 Performing Reporter Ion Quantification c 000 0eiscus wooeiae wie ees 249 EMT Quantification c aa s aitae Akt dette ead a 249 iTRAQ Quantification 6 cect tne n eens 252 Creating a Workflow for Reporter Ion Quantification 253 Performing TMT Quantification on HCD and CID Scans 257 Demonstrating How to Create a Workflow for Reporter Ion Quantification aeni e nA eai aAa a
62. 295 Displaying Quantification Channel Values for Reporter Ion Quantification a ects Bi ant angsty Moma earn anes 295 Displaying Quantification Channel Values for Precursor Ion Qian tifa ti On aeee hane e ceed oshandivg a dy Sarg a aaa EE EEE 296 Proteome Discoverer User Guide vii Contents viii Displaying the Quantification Spectrum Chart 0 000 eee eee 297 Displaying the Quantification Spectrum Chart for Reporter Ion Qanititicario nes s yer te a a e A aara ai ahead le Nie Nak oe Ah 298 Displaying the Quantification Spectrum Chart for Precursor Ion Quantification upas pa a steels aan ahs Se Rae Eka sek De 304 Using Reporter Ion Isotopic Distribution Values To Correct for Impurities een ars aue etna a ea cheers MO al nets Sane Rolle gala TANS 308 Excluding Peptides from the Protein Quantification Results 309 Excluding Peptides with High Levels of Co Isolation 310 Classifying Peptides erun aie eas Sade AS eae eRe phe ihe hile E 311 Calculating Peptide Ratios a0 osc dba ol cakes es Sales HESS 313 Understanding the Peptide Ratio Distributions Chart 314 Handling Missing and Extreme Values in Calculating Peptide Ratios 317 Calculating Protein Ratios from Peptide Ratios 000 0000 320 Case 1 Quantification Result Associated with One Spectrum One Peptide and One Proteiti 2 120 055 tata Eo elaine See a ee 320 Case 2 Two Quantification Results Associated with Tw
63. 2H 4 Dimethyl 2H 4 32 056407 32 0778 2H 4 C 2 Protein N 2 M Dimethyl 2H 4 Dimethyl 2H 4 32 056407 32 0778 2H 4 C 2 Any_N_Te ad bagasse V Dimethyl 2H 4 Dimethyl 2H 4 32 056407 32 078 2H 4 C 2 Any S ae M Dimethyl 2H 6 13C 2 Dimethyl 2H 6 13C 2 36 07567 36 0754 H 2 2H 6 13C Any E MSPepSearch M Dimethyl 2H 6 13C 2 _ Dimethyl 2H 6 13C 2 36 07567 36 0754 H 2 2H 6 13C Any_N_Te EA RE V Formyl Formyl 27 994915 28 0101 CO Protein N a Re We M Formyl Formyl 27 994915 28 0101 CO Any Server Settings Ms Formyl Formyl 27 994915 28 0101 CO Any_N_Te E Discoverer Daemon v GIn gt pyro Glu Gin gt pyro Glu 17 026549 17 0305 H 3 N 1 Any_N_Te MEU Tee Vv ICAT C ICAT C 227 126991 227 2603 H 17 C 10 N 3 Any Vv ICAT C 13C 9 ICAT C 13C 9 236 157185 236 1942 H 17 C 13C 9 Any gt 2 Click to the left of each modification row to see the amino acids that the modification is found on the letter abbreviation of this amino acid and the modification type or 142 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Updating Chemical Modifications category Figure 104 shows an example of the information given for the Acetyl modification Table 7 lists the available modification categories Figure 104 Displaying modification information for acetyl File Search Report Quantification Processing Workflow Editor Administration Tools Window Help
64. 332 Troubleshooting Quantification ey 00 8 eu cece ake patel ee ele POG 334 Appendix A FASTA Reference 0 ecee eee eee cece eee eee eens 339 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific Appendix B Contents FASTA Dat bas ss 5 canta praien tan a antec eh hiactare katt tanh A dines 339 INGBE s cdccessectot oyats tyrbasuntedeais dod avenkcape e ansien ato botheybee a KEGY 339 MS IP Vive odie sare We teal nang ale eh a hs cede ls hate Ne ha ar Mid ME a lO 340 WE Mes hed seta ct EASE EAE AEE SE eee taro arsine ena Broce 340 Wai RehO0 occ5bs nin eh Rie ues Roe aed AMER E weed aa 341 SwissProt and ee MB Lis Gin knw Good ohio doe ba ahh ean ees 341 MSD Bio a an a E a Maes wad Rake tea aeons 342 Custom Database Support iiiuornc ities hi ha ee eee see nee 342 Custom Parsing Rule Ais s 0a Scns Soe datas Bow are keane Marden Nneascuns 342 Custom Parsing Rule Be dete Ga vee ie tee te ctu kee he 343 Custom Parsing Rule CG easels elon eae abe we ee 343 Chemistry References cccee eee ee eee e eee e eee e ee eeneaae 345 Amino Acid Mass Values onunu unese 345 Enzyme Cleavage Properties 342 tra wide ten neee Shh bee eee ees 346 Fragment Jons erenn nei e D aa See ake are aa ately Sede 347 dek o at eae E EA E A 349 Proteome Discoverer User Guide ix ee i Preface This guide describes how to use the Proteome Discoverer 1 4 application for peptide and protein mass spectr
65. 4plex Thermo Scientific Instruments Method foriTRAQ 4 plex mass tags by Applied Biosystems optimize Vv v iTRAQ 8plex Method foriTRAQ 8 plex mass tags by Applied Biosystems Iv v iTRAQ 8plex Thermo Scientific Instruments Method for iTRAQ 8 plex mass tags by Applied Biosystems optimize Iv v SILAC 2plex Arg10 Lys6 SILAC 2plex Arg10 Lys6 Method Iv v SILAC 2plex Arg10 Lys8 SILAC 2plex Arg10 Lys8 Method Vv v SILAC 2plex Ile6 SILAC 2plex Ile6 Method Vv v SILAC 3plex Arg6 Lys4 Arg10 Lys8 SILAC 3plex Arg6 Lys4 Arg10 Lys8 Method Iv v SILAC 3plex Arg6 Lys6 Arg10 Lys8 SILAC 3plex Arg6 Lys6 Argi0 Lys8 Method Iv v TMT 2plex Method for 2 plexTandem Mass Tag of Proteome Sciences plc Iv v TMT 6plex Method for 6 plexTandem Mass Tag of Proteome Sciences plc Iv The Quantification Method Editor dialog box appears as shown in Figure 181 on page 272 through Figure 186 on page 280 3 Follow the procedure in Setting Up the Quantification Method on page 264 Thermo Scientific Proteome Discoverer User Guide 289 7 Quantification Removing a Quantification Method The Proteome Discoverer application checks the parameters that you have changed to be sure that they conform to the guidelines given in Checking the Quantification Method on page 281 It does not apply the changes to a quantification method unless the method meets all these criteria The changes that you make to a quantification method only a
66. 7 AF006830 integrase Actinobadll 24 15 1 219 1 436 11 32 3 4 2 19 1321 4 4 23 7 p40 boradiseasevirus 11 62 1 202 2 2 06 7 03 2 6 0 00 459 2 2 24 J7 collagenalpha2 VI chainshort for 6 86 1 185 1 0 00 2 72 2 6 2 02 577 6 8 25 J VOLTAGE DEPENDENTL TYPE CAL 4 16 1 183 1 0 00 2 24 4 6 0 00 2 19 6 6 26 7 AF092942 fusion glycoprotein pre 17 60 1 177 1 2 70 6 10 3 6 8 85 11 50 5 21 27 gt RNAPOLYMERASESIGMA DFACT 21 26 1 151 2 2 34 16 54 4 23 0 00 4 72 1 1 4 28 hypothetical protein pxO1 59 Bad 17 40 1 137 1 0 00 7 13 4 6 3 84 10 27 5 11 29 7 AJ000005 glucose kinase Bacillu 12 65 1 125 2 3 91 5 86 2 z 232 679 2 3 30 070843 cytochromec oxidasesu 5 45 1 123 1 12 51 5 45 1 19 0 00 31 7 PROTO ONCOGENETYROSINE PR 4 48 1 114 1 0 00 2 05 1 1 0 00 2 43 3 3 32 targetofmybihomolog chicken 11 59 1 113 1 2 12 630 3 3 0 00 6 91 3 4 33 AGRIN PRECURSOR 6 96 1 092 2 4 14 2 35 5 7 0 00 4 91 11 16 34 DNA qtosine 5 methyltransferas 11 24 1 068 2 2 15 5 62 3 Td 0 00 5 62 3 35 alphatectorin chicken 3 68 1 068 4 477 170 3 i 0 00 1 98 4 36 hypothetical protein ydil Bacillus 22 81 1 034 1 0 00 22 81 2 2 0 00 H 37 7 probable magnesium chelatase EC 6 11 1 026 1 2 38 1 63 2 3 0 00 5 42 5 6 38 M AJ011849 fengycin synthetase B 6 18 1 023 1 518 233 6 11 942 397 10 10 39
67. 7 Caenorhabditis elegans 20 28 M mi Pf01576 Pf03961 172491 G0 0003774 G 3 J ATPsynthasesubunitfamily member atp 2 Caenorhab 13 28 l Mi i PF00006 PFO0306 175716 G0 0000003 G a 4 7 ViTellogenin structural genes yolk protein genes family 23 34 1 1 PF00094 PF01347 180781 G0 0005319 G 155 El G0 0000003 GO 0008150 G0 0000166 GO 0036094 GO 1901265 GO 0005488 GO 0097159 GO 0003674 _ 60 0000226 GO 0007010 GO 0007017 GO 0006996 GO 0009987 GO 0071842 GO 0008150 GO 0016043 GO 0071841 GO 0071840 G0 0000795 GO 0000794 GO 0044454 GO 0000228 GO 0000793 GO 0044427 GO 0044428 GO 0005694 GO 0031981 GO 0044446 GO 0005634 GO 0043232 GO 0070013 GO 0043229 GO 0044422 GO 0044424 GO 0043231 GO 0043228 G0 0043233 GO 0043226 GO 0005575 GO 0005622 GO 0044464 GO 0043227 GO 0031974 GO 0005623 G0 0002119 GO 0002164 GO 0009791 GO 0007275 GO 0032502 GO 0032501 GO 0008150 G0 0003774 GO 0017111 GO 0016462 GO 0016818 GO 0016817 GO 0016787 GO 0003824 GO 0003674 G0 0003779 GO 0008092 GO 0005515 GO 0005488 GO 0003674 G0 0005515 GO 0005488 GO 0003674 G0 0005524 GO 0032559 GO 0035639 GO 0030554 GO 0032555 GO 1901265 GO 0017076 GO 0032553 GO 0097159 GO 0000166 GO 0005488 GO 0036094 GO 0003674 eo 0005737 GO 0044424 GO 0005622 GO 0044464 GO 0005575 GO 0005623 G0 0005863 GO 0005859 GO 0030017 GO 0032982 GO 0016460 GO 0044449 GO 0
68. ALLERGEN GAL D 2 GAL Not CYTOCHROME C o 7115 8 6 it 228 ChainY IgG1Fab Fragment Anti LysozymeAntibody D1 61 07 34 11 Hp aj 4 0 917 Cenoke_Test Files OmixTMT_ASCE_1us RAW 5104 RT 2405 min FTM HOD Precursor med Mono min672 02185 Da MH 2014 05100 De integrationaMiost Confident Cestoid integration tolerences20 pom Intensity counts 165 165 Protein Group s 255 9007 Protein s 251 16804 Peptide s PSM s 2504 2504 Search Input i Thermo Scientific Proteome Discoverer User Guide 301 7 Quantification Displaying the Quantification Spectrum Chart Figure 202 Ratio Calculation page of the Quantification Method Editor dialog box G M i Quantification Method Editor x TMT 6plex Mi Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias F Show the Raw Quan Values Minimum Quan Value Threshold 0 0 V Replace Missing Quan Values With Minimum Intensity Use Single Peak Quan Channels V Apply Quan Value Corrections Z Reject All Quan Values If Not All Quan Channels Are Present Fold Change Threshold for Up Down Regulation 2 0 Maximum Allowed Fold Change 100 E Use Ratios Above Maximum Allowed Fold Change for Quantification Percent Co lsolation Excluding Peptides from Quantification 100 For information about the options on the Ratio Calculation page of the Quantifica
69. Annotation gt m Spectrum y Selector J Spectrum Spectrum RL Exporter 1 mL Exporter 10 Ready Thermo Scientific After starting the export process the workflow starts like any other workflow processing job After the application has finished processing the workflow you can find the output of the Spectrum Exporter node in the same folder as the raw file The Spectrum Files node specifies the location of the raw file You can also attach the Spectrum Exporter node to every node that creates modifies or outputs spectra as shown in Figure 44 For example you can add the Spectrum Exporter node to the Spectrum Selector node the Spectrum Filter node and the Spectrum Processing node You can use this type of process flow to more closely inspect different spectrum processing steps in a workflow Proteome Discoverer User Guide 67 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 44 Using the Spectrum Exporter node to export spectra from different steps of the workflow File Search Report Quantification Processing Workflow Editor Administration Tools Window Help ieu aalsler BD g Seuetht Mat ARAM KCESBTABARE 68 Proteome Discoverer User Guide Thermo Scientific ee Using the Proteome Discoverer Daemon Utility This chapter describes the Proteome Discoverer Daemon utility which you can use to monitor job execu
70. B Redundant 322 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Calculating Protein Ratios from Peptide Ratios Case 4 Quantification Result Associated with One Spectrum Two Peptides and One Protein In case 4 shown in Figure 217 the two peptides could be identified by the same search engine and have different ranks or they could be identified by different search engines and both have rank 1 It does not matter whether peptide A and B have the same sequence with different PTM states or different sequences The quantification result is unique for protein A You can use it to calculate the protein ratio but you must only use it once The Proteome Discoverer application marks the better peptide as Unique and the other as Redundant for quantification Figure 217 Case 4 Quantification result associated with one identification spectrum two peptides and one protein Peptide A Quantification result A Unique Peptide B Redundant Case 5 Quantification Result Associated with One Spectrum One Peptide Two Proteins In case 5 shown in Figure 218 the quantification result is associated with one identification spectrum and one peptide but this peptide is contained in more than one protein The quantification result is potentially shared between these proteins and you do not know how to share it If the quantification method specifies using only unique peptides for pro
71. Cellular Component Biological Process Pfam IDs Gene IDs GO Terms 40 a MW mia LEL Pf00038 Proo0es 259839 G0 0000226 G 20 28 li I ii L PF01576 PF03961 172491 G0 0003774 G 3 2 M E M Pooo06 Proo306 175716 G0 0000003 G 23 34 1 1 PF00094 PF01347 180781 G0 0005319 G 155 26 2000 A I Pfo0038 Pro0063 179676 G0 0000003 G 196 24 30 im PF00094 PF01347 180630 G0 0005319 G 160 27 34 I PF00094 PF01347 177619 G0 0005319 G 165 11 19 ait Ii wm PF00113 Pf03952 174423 GO 0000015 G 15 25 I Dou Hl Pfooos5 175472 G0 0003756 G 13 24 li T I E Pf00056 Pf02866 175936 G0O 0000166 G 24 30 l mi PF00094 PF01347 180647 G0 0005319 G 160 13 14 li i I E Pf00565 Pf00567 173811 G0 0003676 G 17 18 E E DADANT M Pfo0012 Pfo6723 178507 G0 0000003 G 3 14 Tut MME 00012 Pfo6723 178873 Go 0000003 G 6 17 I i Pf00022 180767 G0 0000166 G s 14 l E Pf00456 PF00676 177906 G0 0002119 G 6 il I a PF00189 Pf07650 175879 G0 0000003 G 11 18 1 ia PF01459 177524 G0 0005739 G 10 17 I 1 I i INI Pf00153 176773 G0 0000003 G
72. Cleavage Sites 4 3 Mass Range Settings Minimum Precursor Mass 350 Da Maximum Precursor Mass 5000 Da Use Average Precursor Mass False 4 4 Static Modifications Peptide N Terminus Peptide C Terminus 1 Static Modification 2 Static Modification 3 Static Modification 4 Static Modification 5 Static Modification 6 Static Modification N EE Auto Remove Remain FASTA index in memory or not 3 In the General section specify whether the available FASTA indexes will be removed from memory after the number of indexes reaches the specified maximum e Default True Automatically removes the FASTA indexes from memory e False Keeps the FASTA indexes in memory For information about how the Proteome Discoverer application removes FASTA indexes after the maximum has been reached see Manually Creating FASTA Indexes on page 125 For instructions on specifying the maximum number of indexes see Changing Number and Location of Stored FASTA Indexes on page 128 In the Input Data section specify the basic information that the Proteome Discoverer application needs to create the index e FASTA File Select the FASTA database to be indexed from the list e Enzyme Name Select the enzyme used in the digestion from the list on the left the enzymes on this list are set in the Cleavage Reagents window and the type of digestion from the list on the right Full Specifies a full enzymatic digestion Semi Specifies
73. Discoverer Daemon Application from the Xcalibur Data System Processing MudPIT Samples by Using a Processing Method You can process MudPIT samples by using the Quantification Method Editor To process MudPIT samples 1 Start the Proteome Discoverer Daemon application and export a parameter file for MudPIT processing For information about exporting a parameter file see Creating a Parameter File That the Discoverer Daemon Application Uses on page 81 Figure 69 shows how to configure the Export Parameter File page in the Proteome Discoverer Daemon application to export a parameter file In the following example the parameter file is saved in C Xcalibur methods Figure 69 Selecting MudPIT processing on the Start Jobs page Discoverer Daemon e Start Jobs Configuration Job Queue Spectrum Files Workflow Batch processing MudPIT export 2 k P Fil Load Files Export Parameter File Een n z local connection Number of Rawfiles 2 Output Filename MidP Tresult This example features two MudPIT samples and each one is composed of two raw data files for a total of four raw data files 2 Define a processing method see Creating a Processing Method That Calls the Discoverer Daemon Application on page 82 using the parameter file exported in step 1 and select the method as the processing method in the Proc Meth column as shown in Figure 70
74. Editor For detailed information about the wizards see Search Engines on page 3 To prepare to use the search wizards 1 Configure the search parameters for Sequest HT or Mascot See Configuring the Sequest HT Search Engine on page 22 and Configuring the Mascot Search Engine on page 25 respectively 2 Download a FASTA file if necessary if you have not already done so See Adding FASTA Files on page 128 3 Make spectrum source files available as RAW MGF MZDATA MZXML or MZML files The search wizards do not support multiple spectrum source files To process multiple spectrum source files you must use the Workflow Editor For detailed information about this process see Starting a New Search by Using the Workflow Editor on page 42 Start the appropriate search wizard See Starting a New Search by Using the Search Wizards on page 29 You can also set dynamic and static chemical modifications Figure 13 shows the general procedure for using the search engine wizards Thermo Scientific Proteome Discoverer User Guide 29 2 Getting Started Starting a New Search by Using the Search Wizards 30 Figure 13 The Proteome Discoverer search wizard process Select the wizard Select a raw data file and the scan range Select the scan extraction parameters Select the search parameters such as the FASTA database enzyme type search tolerances and ion series Select the static and
75. Figure 49 Job Queue page of the Proteome Discoverer Daemon application for batch processing Start Jobs Configuration Job Queue i Pause Resume gf Abort BM Remove Refresh X Job Queue Execution State Progress Name Spectrum Source Description Submitted at a Time Processing Node Message 11 06AM 2 Sequest HT Total search time was 13 5 s 11 06AM 2 Sequest HT Search completed 11 06AM 2 Sequest HT 2008 protein s 2083 decoy proteins scored and inserted into resultfile in 1 37 s 11 06AM 2 Sequest HT 2008 protein s scored 11 06AM 3 Target DecoyPSM Vali Start calculating relaxed False Discovery Rate 11 06AM 3 Target DecoyPSM Vali Start calculating strict False Discovery Rate 11 06AM 3 TargetDecoyPSM Vali Evaluating peptides of Sequest HT 2 started 11 06AM 2 Sequest HT Reading resultstook 0 8s 11 06AM 2 Sequest HT Storing 2510 decoy PSMs for 1429 spectra 11 06AM 2 Sequest HT Start reading results 11 06AM 2 Sequest HT Performing decoy searchcontaining 1429 DTAs took4 2s 11 06 AM 2 Sequest HT Start ISE decoy search 11 06AM 2 Sequest HT There is already an adequate decoy FASTA index 11 06AM 2 Sequest HT Looking for existing decoy FASTA index 11 06AM 2 Sequest HT Reading resultstook 0 75 11 06AM 2 Sequest HT Storing 2478 PSMs for 1429 spectra B Thermo Scientific Proteome Discoverer User Guide 75 3 Using the Proteome Discoverer Daemon Utility Logg
76. Figure 70 Sequence used for MudPIT processing Sample Type File Name Sample ID Path Inst Meth Proc Meth Unknown 1 Unknown Position Inj Vol Level C Projects Daemon C Xcalibur methods D aemo call 1 10 00 C Projects Daemon C Xcaliburmethods D aemo 2 10 00 C Projects Daemon C Xcaliburmethads D aemo 3 10 00 C Projects Daemon C caliburmethods D aemo 3 10 00 0 00 2 Unknown 3 Unknown 3 3 Start processing the MudPIT samples in the Run Sequence dialog box as shown in Figure 71 94 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 71 Starting the processing of the MudPIT samples Acquisition Options Instrument Start Instrument Simulation MS Yes Run Rows 1 4 E Priority Sequence Start when Heady Change Instruments Processing Actions Instrument Method SS asS_ eygas Quan Sat Bs G E Qual Shut Down Reports Programs 7 Programs Pre Acquisition ca C Create Quan Summary Post Acquisition Run Synchronously V Pre Acquisition Post Acquisition After Sequence Set System On Standby Off The Proteome Discoverer application processes the two samples as MudPIT as shown in Figure 72 Figure 72 Processing two MudPIT samples in the Proteome Discoverer application Pause Resume Abort l Remove Refre
77. Filter Filterbasedon All of the following conditions Reference W Does not contain x fragment Delete From the list in the center select the logic operator value for example gt Greater Than or Equal To In the box to the right type a value for example 1100 To open another row in the Custom Filter dialog box click Add From the list in the center select the logic operator value for example lt Less Than or Equal To In the box to the right type a value for example 1300 The Custom Filter dialog box should look like the example in Figure 127 Click OK to accept the filter settings In this example only peptides that have a precursor MH mass between 1100 and 1300 are displayed The filter conditions that you set appear when you move the cursor over the filters row as shown in Figure 128 Figure 128 Displaying filter conditions miz Da MH Da AM ppm 556 66772 die 719 78 556 66772 1112 32817 719 78 Filtering PSMs and Peptides for Site Localization Scores from phosphoRS You can set a row filter that allows you to filter for the following e At least one site with a localization probability equal to or above the specified value e At least one site of the specified type such as S T or Y with a localization probability equal to or above the specified value 172 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Res
78. G 2 I Seleted I S PQD MS2 4 55 103 658 86029 1316 71330 2 oan 3 J7 Unambiguous I MS PQD MS2 10 15 73 704 36084 2111 06797 3 E Ej Sequence PSM Ambiguity Proteins Protein Groups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Q 1 hPGDFGADAQGAMTk Unambiguous 5 2 494711 1942750 N Term iTRAQ8plex K15 PQD 0 1599 0 0000 1 1 2 J hPGDFGADAQAAMSk Unconsidered 1 2 494711 1942750 N Term iTRAQEplex K15 PQD 0 1599 2 2 3 J hPGNFGADAQGAMTk Unconsidered 1 1 1942750 N Term iTRAQ8plex K15 PQD 0 3761 3 3 4 7 GGSVTLPESGTDLLTHRLEK Unconsidered 1 0 PQD 0 3941 4 4 5 J7 MAAGPVEDNCISLVEMk Unconsidered 1 0 K17 iTRAQ8plex PQD 0 4437 5 5 6 J SNILDAICFVLGIASMSTVR Unconsidered 1 0 PQD 0 4752 6 6 7 7 nYVAMGAKVADPTNNK Unconsidered 1 0 N Term iTRAQ8plex PQD 0 4910 7 7 8 J7 MREGSALPGAPGAAPVPGRL Unconsidered 1 0 M1 Oxidation PQD 0 5045 8 8 o LEKASDVDMAESPSSTSSGGR Unconsidered 2 0 PQD 0 5203 9 9 10 J hGQTTELTELFVk Unconsidered 1 0 N Term iTRAQ8plex K13 PQD 0 5225 10 10 Ea JA PSM Ambiguity Mass Analyzer Activation Type MS Order Identified Peptides Isolation Interference Ion Inject Time ms Precursor m z Da Precursor MH Da Precursor Charge 4 J Selected FIMS HCD MS2 10 9 5 539 87262 1078 73796 2 5 I Selected FIMS HCD MS2 5 6 188 586 59283 2343 34951 4 6 M Seleted FIMS HCD MS2 10 180 642 35065 1925 03739 3 2 J Seleted IMs PQD MS2 10
79. HT or Q Mascot Figure 14 Two wizard options in the Processing menu File Search Report Quantification Processing Workflow Editor Administration Tools Wi TUU Start Sequest HT Search Wizard Ctrl Q W Start Mascot Search Wizard Ctrl M The Welcome to the Wizard_name Search Wizard page appears as shown in Figure 15 Figure 15 Welcome to the Wizard _name Search Wizard page Welcome to the Sequest HT Search Wizard This wizard helps you to set up a Sequest HT search 3 To use a template from a previous search select it from the Templates list To give the selected template a new name click Rename and in the Renaming Template dialog box type the new name in the New Name box and click OK To delete the selected template click Delete and in the confirmation box click OK Thermo Scientific Proteome Discoverer User Guide 31 2 Getting Started Starting a New Search by Using the Search Wizards 4 Click Next The Rawfile and Scan Range Selection page of the wizard opens as shown in Figure 16 Figure 16 Rawfile and Scan Range Selection page SS a Rawfile and Scan Range Selection Please select the rawfile and the scans you want to process Ravwfile C Program Files Proteome Discoverer source files Smoke_Test_Fi Drag with the mouse while pressing CTRL to select a scan range C Program Files Proteome Discoverer source files Smoke_Test_Files Cele
80. MYOGLOBIN y 9 6 66 2 163 4 9 0 2 163 4 9 0 2 163 a 3 ChainY IgG1 Fab Fragment Anti 28 68 3 3 36 2 167 3 73 2 167 3 73 2 167 H 4 X58989 serum albumin Bos tau 19 28 3 15 111 2 160 7 6 6 2 160 7 6 6 2 160 5 7 OVALBUMIN PLAKALBUMIN AL 18 44 2 6 75 2 253 3 48 1 2 253 3 48 1 2 253 6 J7 beta casein variant CnH bovine 17 22 3 4 39 2 266 1 2 266 1 2 266 7 GLYCERALDEHYDE 3 PHOSPHAT 14 11 2 5 42 2 204 4 3 9 2 204 4 3 9 2 204 8 7 Anionic Trypsin Wild Type 11 21 2 2 12 H 9 7 CARBONICANHYDRASE II CARB 10 04 1 3 33 1 797 2 94 1 797 94 1 797 10 I carbonate dehydratase EC4 2 1 5 02 1 2 15 2 293 2 13 8 2 293 2 13 8 2 293 11 CGMP DEPENDENTPROTEIN KIN 2 83 1 2 9 4 137 1 4 137 1 4 137 12 gramicidin Ssynthase 2 Bacillus 135 2 5 24 Kil gt Ready 12 12 Protein Group s 371 371 Merged Protein s 536 536 Peptide s 3267 3267 PSM s 17571 17571 Search Input s Calculating Protein Ratios in Multiconsensus Reports Treated as Replicates Thermo Scientific With replicates the application treats the quantification results like replicates of the same sample You can specify that quantification results be treated as replicates by selecting the Treat Quan Results as Replicates option on the Input Files page or by using the Quantification Method Editor dialog box opened by choosing Quantification gt Edit Quantification Method when the multiconsensus report is open For multiconse
81. Mascot Generic Format MGF files are mass spectral files produced during Mascot analysis They contain a list of precursor ions their fragments and the masses of the fragments Extensible Markup Language XML files contain workflow templates MZXML files are standard 2 x mass spectrometer data format files developed at the Seattle Proteome Center at the Institute for Systems Biology ISB that contain a list of precursor ions their fragments and the masses of the fragment MZDATA files are common data format files developed by the Human Proteome Organization HUPO for proteomics mass spectrometry data These files are in version 1 05 format They are exported with XML indentation enabled so that the different XML tags are broken into multiple lines instead of merged into one line MZML files are a combination of mzData and mzXML formats developed by the Human Proteome Organization Standard Initiative HUPO PSI and the Seattle Proteome Center at the Institute for Systems Biology ISB The Proteome Discoverer application supports version 1 1 0 of the MZML format Magellan Storage MSF files contain the results of the searches conducted by the search wizards or the Workflow Editor 12 Proteome Discoverer User Guide Thermo Scientific Outputs 1 Introduction Inputs and Outputs The Proteome Discoverer application creates the following file types as output Thermo Scientific DTA Archive DTA files are files containing MS d
82. Myoglobin Horse Heart Mutant Wi 84 31 1 023 4 737 78 81 05 13 354 32 08 44 44 12 17 i 40 J CYCLIC NUCLEOTIDE GATED CHAN 8 22 1 019 1 2 02 5 89 4 4 0 00 2 79 4 41 7 GLYCERALDEHYDE3 PHOSPHATE 12 20 1 012 1 2 07 6 25 2 4 173 5 95 3 42 Crystal Structure Of Calsequestrn 9 81 1 005 3 11 36 8 17 3 8 0 00 1 63 a 43 submaxillary mucin 1 bovine frag 11 33 1 001 1 492 24 453 5 32 1 60 680 10 32 44 7 hypothetical protein yheB Badllus 8 22 0 987 2 0 00 7 16 3 6 0 00 1 06 1 1 gt 2 l Ready There is only one ratio column for each specified ratio and the Ratio Counts columns show the number of peptides used from every single MSF file for calculating the individual protein ratios for each file These individual protein ratios are then averaged and the average is displayed in the ratio columns for the proteins Adding a Quantification Method You can use the following procedure to add a quantification method You can also use it to access the quantification methods without loading an MSF file To add a quantification method 1 Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g Thermo Scientific Proteome Discoverer User Guide 285 7 Quantification Adding a Quantification Method The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both
83. Note Click Reset to return to the default values 8 Restart your machine Thermo Scientific Proteome Discoverer User Guide 23 2 Getting Started Configuring Search Engine Parameters Configuring the SEQUEST Search Engine For searches with the SEQUEST search engine specify how to display the peptide confidence by default The SEQUEST Search engine scores the number of fragment ions that are common to two different peptides with the same precursor mass and calculates the cross correlation score for all candidate peptides queried from the database By default it sorts the resulting XCorr values in descending order To configure the SEQUEST search engine 1 On the Administration page click SEQUEST under Workflow Nodes in the Configuration section 2 Ifyou are using the SEQUEST search engine to search low resolution data set the XCorr confidence thresholds under the XCorr Confidence Thresholds low resolution data parameter The default values appear in Figure 8 Figure 8 XCorr confidence thresholds for the SEQUEST search engine a 1 XCorr Confidence Thresholds low resolution data 15 z 1 Medium Confidence XCorr 07 z 2 High Confidence XCorr 2 z 2 Medium Confidence XCorr 039 z 3 High Confidence XCorr 25 z 3 Medium Confidence XCorr 12 z gt 4 High Confidence XCorr 3 z gt 4 Medium Confidence XCorr 15 4 2 XCon Confidence Thresholds high resolution data z 1 High Confidence XCorr 12 z 1 Medium Confide
84. P02769 Serum albumin precursor Allergen Bos d 6 BS 547 20 93 25 1 34 142 1705 0 889 104 E 2y I G3P_PIG P00355 Glyceraldehyde 3 phosphate dehydrogenase E 393 44 66 57 1 19 58 826 0 806 87 rr 3 TRFE_LHUMAN P02787 Serotransferrin precursor Transferrin Siderop 392 53 87 82 1 35 116 1358 0 806 84 H 4 I LYSC_CHICK P00698 Lysozyme C precursor EC3 2 1 17 1 4 beta N 305 37 100 00 1 5 33 501 0 883 49 E S J OVAL_CHICK P01012 Ovalbumin Plakalbumin Allergen Gal d 2 Gal 303 68 69 09 1 14 41 494 0 836 46 ccs Weal M CYC_HORSE P00004 Cytochrome c MASS 11701 257 29 100 00 1 16 42 469 0 814 53 7 7 CAH2_BOVIN P00921 Carbonic anhydrase II EC 4 2 1 1 Carbonate 232 99 74 52 1 11 29 322 0 863 42 W 8 MYG_HORSE P68082 Myoglobin MASS 16951 79 58 88 89 1 7 28 331 0 773 15 ira eal 7 CASB_BOVIN P02666 Beta casein precursor MASS 25107 66 46 52 23 1 6 18 202 0 760 17 Ez w J LALBA_BOVN P00711 Alpha lactalbumin precursor Lactose synthase 65 01 70 42 1 6 18 262 0 804 17 X Peptide Ratio Distributions IX G3P_PIG P00355 Glyceraldehyde 3 phosphate dehydrogenase EC 1 2 1 12 GAPDH MASS 35705 131 126 130 126 129 126 128 126 127 126 oo a SO a R 031 081 s R 0 17 0 89 R 0 07 1 05 a R 020 087 a R 0 08 095 IQR 0 40 0 22 lQR 0 42 0 26 IQR 0 44 0 32 IQR 0 39 0 24 IQR 0 29 0 19 64 674 6 i o log2 Fold Change ci
85. Peak Missing Channels Allowed box specify the maximum number of single peak or missing quantification channels that are allowed for a valid peptide quantification result A single peak quantification channel is a channel that is identified with just a single peak The maximum number used will not exceed the number of specified channels The minimum value is 0 the default This value indicates that there are at least two peaks in the quantification channel used for quantification 15 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon g Performing Reporter lon Quantification In contrast to the metabolic labeling used by isotopically labeled precursor ion quantification methods such as SILAC isobarically labeled reporter ion quantification methods use external reagents or tags to enzymatically or chemically label proteins and peptides Reporter ion quantification uses tags that have the same mass A reporter ion is a fragment ion with a tag The Proteome Discoverer application supports reporter ion quantification for Tandem Mass Tag TMT and Isobaric Tag for Relative and Absolute Quantification TRAQ and any user defined tags Identification and quantification with both TMT and iTRAQ are performed in the MS MS scan You can quantify all isobarically labeled samples For iTRAQ 4plex and 8plex default methods are available For TMT 2plex and 6plex default methods are available You can also add new methods
86. Quantification 256 C Proteome Discoverer User Guide Default Most Confident Centroid Lays a Gaussian curve around the target peak the tag mass with a sigma value equal to the mass accuracy or integration window Then the Gaussian curve normalizes all peaks in the window and the largest is considered to be the most confident peak This method is also used by the Spectrum Selector node in the Workflow Editor to pick the monoisotopic peak from the survey scan The only difference is that the Spectrum Selector uses a 3 sigma interval but Most Confident Centroid uses only a 1 sigma interval This means the Most Confident Centroid is almost always the largest peak inside the integration window because of the small inclination of the Gaussian curve in the 1 sigma interval Most Intense Centroid Selects the highest peak Centroid With Smallest Delta Mass Selects the peak with the smallest deviation from the theoretical mass Centroid Sum Sums the intensity of all the peaks in the window Specify the scan event filters i In the Mass Analyzer box select the type of mass spectrometer used in the acquisition of the spectrum Ion Trap ITMS Default Fourier Transform FTMS Time of Flight TOFMS Single Quad SQMS Triple Quad TQMS Sector Field SectorMS ii In the MS Order box specify the level of tandem mass spectrum to be processed for example MS2 or MS3 The default is MS2 iii In the Activation Type li
87. Results Filters page Therefore some peptides might not belong to any protein group To investigate these cases right click the Proteins page and choose Show Filtered Out Rows to display the filtered out peptides in the results file You can also use the Protein References of a Peptide view opened by choosing Search Report gt Show Protein References to help you Number of Unique Peptides Column on the Proteins Page The value in the Unique Peptides column on the Proteins page that is listed for each protein group is the number of peptides that are only contained in this protein group The Proteome Discoverer application counts only peptides that display a status of Selected or Unambiguous in the PSM Ambiguity column because assessing the uniqueness of peptides that were not used to form protein groups has no relevance PSMs Identified by Multiple Workflow Nodes In search results where the application identifies PSMs by multiple search nodes within a single workflow the protein grouping algorithm selects one of the PSMs identified for the same spectrum for building the protein groups In search results where PSMs are identified by multiple search nodes from multiple workflows multiconsensus report the application treats PSMs and spectra from the different workflows as separate even if it searched the same raw data files and therefore the same spectra In this case determining whether the application searched the exact same spectra is d
88. TMT Quantification Thermo Scientific TMT quantification is a reproducible highly accurate quantification method that provides both comparative and absolute MS MS based quantification of proteins and peptides in biological samples TMT tagging produces data to calculate the relative abundances of proteins You can evaluate differential protein expression in one to six samples in a single experiment Proteome Discoverer User Guide 249 7 Quantification Performing Reporter lon Quantification Each sample is labeled with chemically identical tags before mixing the samples and a single MS run generates a single peak for each peptide irrespective of which tag it has been given Between the normalizer and reporter is a cleavable linker which breaks during MS MS The mass reporter ion is split off and measured by the mass spectrometer Only MS MS fragmentation can differentiate the tagged proteins The reporter ion measured by the mass spectrometer generates a different peak As a result the peak height peak integral for each reporter denotes the relative amount of protein originating from each of the labeled samples With the quantification functions in the application you can set filters to see only unique peptides so that every protein associated with the same peptide is not counted producing a best results list of peptides Filtering the number of proteins can give you a more robust final analysis of your experimental set Quantific
89. Up FDRs in the Workflow Editor You can set up FDRs through the Target Decoy PSM Validator node or the Percolator node in the workflow For information about the Target Decoy PSM Validator node refer to the Help For detailed information about the Percolator node and its processing refer to the Help To set up FDRs by using the Target Decoy PSM Validator node 1 Create a search workflow that includes at least one of the search engine nodes SEQUEST Mascot or Sequest HT and the Target Decoy PSM Validator node 190 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates For information about creating a workflow see Creating a Search Workflow on page 44 2 Click the Target Decoy PSM Validator node as shown in Figure 139 Figure 139 Setting up a decoy database search in the Workflow Editor File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE aoao onnan SAD suth Kmart AHL AGCOBERFARE Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf X Sequest TMT 6 plex Benchmark 1 4 0 175 msf Celegans_FT_6ITDDDT_01_decoy msf x Workflow Editor x 4 gt Workflow Nodes 4a Data Input i Spectrum Files A Event Detector Spectrum Selector ig Noise Peak Filter iu Non Fragment Filter ig Spectrum Grouper ij Spectrum Normalizer ig Top N Peak
90. User Guide Thermo Scientific 7 Quantification Calculating and Displaying Protein Ratios for Multiconsensus Reports Calculating and Displaying Protein Ratios for Multiconsensus Reports The Proteome Discoverer application can treat the different single quantification results in multiconsensus reports as replicates of the same sample or as different treatments of a sample In real world studies of quantitative responses of a sample to certain treatments such as a particular change in environmental condition or an administration of a drug you might be interested in the quantitative difference of the sample before and after the treatment or between different treatment states of the sample for example different points in time after application of a certain drug or application of different amounts of a certain drug Quantitative studies could also investigate the quantitative difference between samples in different states for example between similar samples from healthy and different disease states Such experimental investigations must assess the variability inherent in the different stages of the experiment For example samples from different animals or patients can vary significantly in their expression level for certain proteins or in the amount of proteins and peptides with PTMs Other sources of variability are differences in sample preparation differences in chromatographic separation or differences in measurement in the mass spectromet
91. XXXXSXXXXR XXXXXXXXXK XXXXXXXXXK LC MS MS 3cg 15No Lys and 19C6 15N4 Arg Identification F 100 XXXPSQKN0KAXXK 2 50 3 oD e 4 m z SILAC can differentiate peptides in single MS mode without requiring you to perform tandem mass spectrometry However SILAC cannot identify peptides so you must use tandem mass spectrometry for that purpose You can use several SILAC 2plex methods for example Arg10 Lys6 and Arg10 Lys8 to compare two samples Proteome Discoverer User Guide 245 7 Quantification Performing Precursor lon Quantification SILAC 3plex Methods SILAC 3plex methods are similar to SILAC 2plex methods except in addition to a heavy sample containing for example Arg10 and Lys8 they also use a medium sample containing for example Arg6 and Lys4 Protein abundance is determined from the relative MS signal intensities of the heavy sample medium sample and a control sample containing light 2C and 14N arginine and lysine Dimethylation 3plex Method The Proteome Discoverer application also includes the dimethylation 3plex method It is not metabolomic labeling in cell culture but is a form of peptide chemical labeling This method uses formaldehyde and sodium cyanoborohydride to add dimethyl groups CH3 to the N terminus and to the amino group of lysine By choosing the isotopomers of formaldehyde and sodium cyanoborohydride you can create light medium and heavy labels
92. a b c ion series and the x y and z ion series as well as the peaks from the precursor peptide It does not display labels for all fragments with a mass difference isotope peaks and peaks in the spectrum library 1M kod To generate a mirror plot 1 Open the MSF file for the results of the spectrum library search performed with the SpectraST node or the MSPepSearch node 2 If you used spectrum library nodes and other search nodes in the workflow ungroup the peptides by right clicking and clearing the Show Peptide Groups check box Ungrouping peptides is not necessary if you used only spectrum library search nodes in the workflow Thermo Scientific 4 Searching for Data Updating Chemical Modifications 3 Follow the instructions for generating a Peptide Identification Details view given in Interpreting Your Results with the Peptide Identification Details View on page 276 Updating Chemical Modifications You can update the chemical modifications that you use to conduct a peptide identification search The available modifications are defined in the Chemical Modifications view on the Administration page that is opened by choosing Administration gt Maintain Chemical Modifications Use this view to customize the chemical modifications that you use to do your search You can import a new list or the latest UNIMOD list You can also modify the chemical modification list by adding amino acids to the modifications creat
93. a reimplementation of the Sequest algorithm that increases overall performance by using modern multicore and multiprocessor systems It also uses multiple search threads It does not use the SpScore filter instead it calculates XCorr for every candidate The scores from the Sequest HT and SEQUEST search engines are not identical because the Sequest HT search engine uses a slightly changed cross correlation and exact mass differences for the flanking ions of peaks in the theoretical spectra Spectrum Library Searching The Proteome Discoverer application offers the ability to search large spectrum libraries which are libraries of measured consensus spectra from actual previous experiments Two new spectral library search nodes SpectraST and MSPepSearch use spectral libraries These search engines identify peptides by comparing the spectra to the reference spectra in the library You can search spectrum libraries downloaded from the National Institute of Standards and Technology NIST and the PeptideAtlas home page MSPepSearch Node The MSPepSearch node searches spectrum libraries downloaded from NIST It is faster than SpectraST but there are no decoy spectral libraries available that are required to estimate the false discovery rate FDR by using a target decoy false discovery rate calculation or by using Percolator 14 Proteome Discoverer User Guide Thermo Scientific SpectraST Node 1 Introduction New Features in This Release
94. box the Proteome Discoverer application discards the difference from the oldest FASTA indexes the next time that you restart the application 4 If you changed any settings click Apply A FASTA message box similar to that shown in Figure 86 appears 118 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases Figure 86 Administration message box a FASTA Index Settings E You changed the following settings Max number of FASTA indexes to 32 Choose OK to save the changes The changes are applied after a restart This can take some time 5 Click OK Note Click A Reset to return to the default values 6 Restart your machine Displaying the FASTA Indexes View You can access FASTA indexes through the FASTA Indexes view To display the FASTA Indexes view 1 Choose Administration gt Maintain FASTA Indexes or click the Maintain FASTA Indexes icon GB The FASTA Indexes view appears as shown in Figure 87 Thermo Scientific Proteome Discoverer User Guide 119 4 Searching for Data Using FASTA Databases Figure 87 FASTA Indexes view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help ea ag Glev anuanan 20aa RK SequestHT Mascot AAR HRMeE RBEBRRBPARE Administration x 4 ecoli fasta uniprot_sprot_2011_05 fasta bovine fasta Trypsin Full Tr
95. cellular component Protein complex Description Any protein group composed of two or more subunits which may or may not be identical Protein complexes might have other associated non protein prosthetic groups such as nucleic acids metal ions or carbohydrate groups Ribosome An intracellular organelle about 200 Angstroms in diameter consisting of RNA and protein It is the site of protein biosynthesis resulting from translation of messenger RNA mRNA Vacuole A closed structure found only in eukaryotic cells completely surrounded by unit membrane and containing liquid material Cells contain one or several vacuoles that might have different functions from each other Vacuoles have a diverse array of functions They can act as a storage organelle for nutrients or waste products as a degradative compartment as a cost effective way of increasing cell size and as a homeostatic regulator controlling both the turgor pressure and the pH of the cytosol Organelle lumen The volume enclosed by the membranes of a particular organelle for example endoplasmic reticulum lumen or the space between the two lipid bilayers of a double membrane surrounding an organelle for example nuclear membrane lumen GO Slim Categories for Biological Processes Table 19 describes the GO Slim categories for biological processes Table 19 GO Slim categories for biological processes Sheet 1 of 3 Go Slim biological process Cell
96. classifications 312 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Classifying Peptides Figure 209 Classifying peptides for protein quantification Depending on the settings in the Quantification Method Editor the Proteome Discover application excludes the following peptides No Quan Labels No reporter label Indistinguishable Channels Not all defined channels can be distinguished Inconsistently Labeled Labels are from different channels Excluded by Method Quan channels are missing single peak channels are missing ratios exceed limits and so forth The Proteome Discoverer application does not consider high scoring peptides if they have no protein links The Proteome Discoverer application marks peptides that are not unique as Redundant This check depends on the setting of the Consider Protein Groups for Peptide Uniqueness check box on the Protein Quantification page of the Quantification Method Editor dialog box It classifies the peptides as Unique if they match the proteins within the same protein group Thermo Scientific Quantification results For all quantification results Collect all peptides associated with the quantification result For all peptides Is the peptide filtered out Mark peptide as Filtered Out Mark peptide as No Quan Labels reporter ion Indistinguishable Channels precursor ion Inconsistently Labeled precurs
97. communication Description Any process that mediates interactions between a cell and its surroundings Cell communication encompasses interactions such as signaling or attachment between one cell and another cell between a cell and an extracellular matrix or between a cell and any other aspect of its environment Cell death Thermo Scientific The specific activation or halting of processes within a cell so that its vital functions markedly cease rather than simply deteriorating gradually over time which culminates in cell death Proteome Discoverer User Guide 237 6 Protein Annotation GO Slim Categories Table 19 GO Slim categories for biological processes Sheet 2 of 3 Go Slim biological process Cell differentiation Description The process in which relatively unspecialized cells for example embryonic or regenerative cells acquire specialized structural features functional features or both that characterize the cells tissues or organs of the mature organism or some other relatively stable phase of the organism s life history Differentiation includes the processes involved in commitment of a cell to a specific fate Cell division The processes resulting in the physical partitioning and separation of a cell into daughter cells Cell growth The process by which a cell irreversibly increases in size over time by accretion and biosynthetic production of matter similar to that already present
98. conditions MH Da Greater than or equal to gt 1100 7 MH Da lt Less than or equal to x 1300 x 4 Click Delete 5 Click OK or if you are deleting all filters click No Filters which appears instead of the OK button Displaying Filtered Out Rows If you choose Enable Row Filters in the shortcut menu the Proteome Discoverer application hides the filtered out rows on the Proteins Peptides or Search Input page so that you can easily view your results However you can still display these filtered out rows to perform a comparative analysis To display filtered out rows 1 Follow the procedure given in Filtering Results with Row Filters on page 167 to set any row filters Right click to display the shortcut menu as shown in Figure 126 and choose Show Filtered Out Rows The application now displays both the filtered out and unfiltered rows The peptides or proteins filtered out by filters set on the Result Filters page appear in light gray rows The peptides or proteins filtered out by row filters appear in darker gray rows Figure 126 shows both types of filtered out rows 170 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results Figure 126 Displaying filtered out rows File Search Report Quantification Processing Workflow Editor Administration Tools Window Help GOH BE BPSOTSHHORH LOM n i K seth Q Maot AKL AEBTEBARE Sequest iTRAQ 8 plex Benchmark
99. create and apply more than one filter to your search results In addition to the Result Filters page you can filter the data while opening your MSF file by setting filters on the Result Filters page that appears when you choose File gt Open Report refer to the Help These filters are identical to the filters on the Result Filters page for an already opened MSF file except that you can only set protein filters on the Result Filters page for an already opened MSF file Protein scores give some indication of the relevance of a protein They are calculated from a list of peptides identified for a particular protein and can be expected to change as soon as the peptides are removed by the application of result filters The Proteome Discoverer application recalculates the protein scores after you apply peptide filters or change the score thresholds on the Peptide Confidence page For information on how the application calculates protein scores refer to the Help Thermo Scientific Proteome Discoverer User Guide 153 5 Filtering Data Filtering the Search Results To filter the number of proteins and peptides visible on the Proteins and Peptides pages in an MSF file that is already open use the Result Filters page shown in Figure 111 For information about filtering MSF files while opening them refer to the Help To display the Result Filters page in an open MSF file e In an open report click the Result Filters tab The Result Filters pag
100. dynamic chemical modifications Name and describe the search Start the search Analyze the search results Starting a New Search The following procedure describes how to search your data by using a search wizard using Sequest HT as an example The procedure is very similar for Mascot searches differences between the two procedures are noted where appropriate Note Although the basic procedure for using the Mascot wizard and the Sequest HT wizard is the same see Configuring the Mascot Search Engine on page 25 for information about the unique aspects of conducting Mascot searches If you have not selected a FASTA database to search you must add one before you start a search wizard For instructions on adding a FASTA file see Adding FASTA Files on page 104 Note The available FASTA files are registered and available through the Proteome Discoverer application See Using FASTA Databases on page 101 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards 1 To start a new search using a search wizard Optional Open the job queue by choosing Administration gt Show Job Queue or clicking the Show Job Queue icon i You can find more information about the job queue in the Help 2 Choose Processing gt Start Wizard_name Search Wizard as shown in Figure 14 or click the appropriate wizard icon in the toolbar g Sequest
101. e N Terminal Modification Indicates that the label occurs on the N terminus e C Terminal Modification Indicates that the label occurs on the C terminus 8 From the Modification list select the modification to label the amino acid with This example shows Label 13C 6 6 020 Da 9 From the list adjacent to the Modification list select the abbreviation of the amino acid selected in the Quantification Labels box on which the modification should occur In this example K is selected The completed Quan Channels page will resemble Figure 180 Figure 180 Completed Quan Channels page Quantification Method Editor SILAC 2plex Arg10 xs Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias ae Channel Name Medium Quantification Labels Arg6 Lys6 tae tyes Modification Target N Teminal Modification C Teminal Modification Modification Label 13C 6 6 020 Da za z m a Canos Hep 10 Continue setting up the quantification method by following the instructions in Setting Up Quantification Channels for Ratio Reporting on page 273 270 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Setting Up the Quantification Method To specify label modifications for reporter ion quantification already selected see Figure 181 can select the following defa
102. e Updating Chemical Modifications e Using the Qual Browser Application e Customizing Cleavage Reagents Using FASTA Databases You can use the FASTA database utilities to add delete and find protein references and sequences You can also extract information from an existing FASTA file place it into a new FASTA file and compile it for availability in the Proteome Discoverer application For more information about FASTA databases see FASTA Reference on page 339 Displaying FASTA Files You can list all the FASTA files that you have downloaded from other sources onto your hard drive and registered To list the available FASTA files e Choose Administration gt Maintain FASTA Files or click the Maintain FASTA Files icon either in the toolbar or on the Administration page The FASTA files view shown in Figure 74 appears It lists all the FASTA files that you have downloaded from other sources and registered It displays the processed FASTA file properties such as the file name file size and the number of proteins stored The Proteome Discoverer application analyzes each protein entry to determine if the FASTA file meets the application requirements for use in a spectra search It processes the FASTA file and makes it available for use Thermo Scientific Proteome Discoverer User Guide 101 4 Searching for Data Using FASTA Databases Figure 74 FASTA files view Add icon Remove icon Cancel icon Refresh icon
103. for charge state 7 3 72 Minimal Score for charge state gt 7 3 725 Minimal Score for charge state gt 7 3 725 Ready 6021606 Protein Group s 6624 6624 Protein s 8628 8628 Peptide s 9571 9571 PSM s 4766 4766 Search Input s q Go to the following sections e To change the target rate e To change the filter settings e To save the peptide confidence and FDR settings on the Result Filters page To change the target rate 1 Change the value in the Target box of the Actual Relaxed False Discovery Rate area for medium confidence the Actual Strict False Discovery Rate area for high confidence or both 2 Click Apply FDRs 198 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates To change the filter settings 1 Select the filter settings that you want to change in the Filter Settings area in the upper left corner of the Peptide Confidence page and enter the new values in the FDR Settings area The Minimal Score for Charge State values in the FDR Settings area specify the charge state above which peptides are filtered out Charge state values can range from 0 to 20 2 Click Apply Filters If you set any filters except the Peptide Confidence filter on the Result Filters page when you loaded the report the warning shown in Figure 145 appears Figure 145 FDR recalculation message box for all filters except Peptide Confidence Warning 3
104. for processing the data It is only used to verify the peptides when the Proteome Discoverer application loads the reports When you specify at least one of the label modifications in the quantification method the Proteome Discoverer application verifies that each identified peptide has at least one of the specified modifications It does not matter if only one terminal or only one residue is modified with the specified label modification e When the application identifies a peptide with none of the specified label modifications this peptide cannot be the source of reporter peaks in the MS MS spectra As a result the application marks the peptide No Quan Labels in the MSF report It does not use these peptides when it calculates the protein quantification values from the peptides e When the application finds a peptide that does not have an iTRAQ or TMT label as a modification even though reporter ions were present it leaves the Ratio columns blank When you install the Proteome Discoverer application the default methods for TMT and iTRAQ include the correct label modification The application does not automatically update already existing reporter methods you must manually specify the label modifications When you open old MSF files that contain reporter quantification data the label modifications of the quantification method of the MSF file appear as None on the Quan Channels page of the Quantification Method Editor dialog box You
105. gas molecules such as helium nitrogen or argon The collision breaks the bonds and fragments the molecular ions into smaller pieces e ECD Uses the electron capture dissociation ECD method of fragmentation where 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 HCD Uses the high energy collision induced dissociation HCD method of fragmentation where the projectile ion has laboratory frame translation energy higher than 1 keV HCD produces a highly abundant series of reporter ions for TMT and iTRAQ quantification e ETD Uses the electron transfer dissociation ETD method of fragmentation where singly charged reagent anions transfer an electron to multiply protonated peptides within an ion trap mass analyzer to induce fragmentation ETD cleaves along the peptide backbone while side chains and modifications such as phosphorylation are left intact This method is used to fragment peptides and proteins 8 Proteome Discoverer User Guide Thermo Scientific 1 Introduction Workflow e IRMPD With the infrared multi photon dissociation IRMPD method of fragmentation an infrared 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 b
106. in the new GO Terms column in the output MSF file For more information on this feature see Displaying GO Accessions on page 212 16 Proteome Discoverer User Guide Thermo Scientific 1 Introduction New Features in This Release Mascot Quantification Mode Thermo Scientific When you use the Mascot node on the Mascot server as the search engine in a quantification workflow you can set up to nine dynamic and static modifications as parameters However if you want to set more modifications as parameters you can use the Mascot node to configure quantification methods on the Mascot server Modifications in a quantification method are organized into groups classified as fixed variable or exclusive You can use the node s From Quan Method parameter to select the dynamic modifications to search for rather than manually specifying each modification with a Dynamic Modifications parameter For detailed information on this capability see Searching for Quantification Modifications with Mascot on page 261 Proteome Discoverer User Guide 17 ee i Getting Started This chapter describes how to use Proteome Discoverer search wizards and the Workflow Editor to define your search parameters The search wizards are the quickest way to start using the Proteome Discoverer application Contents e Starting the Proteome Discoverer Application e Closing the Proteome Discoverer Application e Configuring Search Engine Parameters e S
107. indicate where a quantification pattern peak was expected but is absent Figure 206 shows these regions This ion pattern peak is not used in calculating the quantification values for the different quantification channels 306 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart Figure 206 Expected but absent peak in the Quantification Spectrum chart File Search Report Quantification Processing Workflow Editor Administration Tools Window Help Oa Se B eTl SanTDef zon PR SequestHT G Mascot AA LHe AEBRABARE Sequest SILAC Dimethylation Search 1 3 0 297 msf x C Documents and Settings ussjo prodeval2 My Documents Smoke_Test_Files SILAC Smoke Tests 081023_RR_BSA_1_1_1_2plus RAW RT 25 45 26 39 min Event Spectrum FTMS Quantified lon 2 2 Mono m z 564 79120 Da MH 1128 57512 Da Area counts sec 103 8 8 6 8 8 8 Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s Regions in blue shown in Figure 207 indicate where a quantification pattern peak was expected but is unsuitable Pattern peaks might be unsuitable because of the wrong centroid retention time a range out of the delta mass the wrong intensity or a peak that has been used by another isotopic pattern This ion pattern peak is not used in calculating the quantification values for the different quantifi
108. linear equations intensity_of_loss_because_of_116_impurity and intensity_of _gain_because_of_other_label_impurities For this correction you must enter the isotopic distribution of each of the labels used in the quantification method as shown in Figure 208 The values are part of each of the iTRAQ or TMT label kits used Figure 208 Entering values for the isotopic distribution of a specific reporter tag Quantification Method Editor TRAQ 4plex Ea Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Residue Modification _ iTRAQ4plex 144 102 Da x KY N Terminal Modification TRAQ4plex 144 102 Da EJ E 115 Tag Name 114 116 117 Monoisotopic m z 114 1106798 Average miz 114 17347 Reporter lon Isotopic Distribution i Name 2 1 7 r 42 Isotope Shift 26 Isotope Intensity 0 0 You can also deconvolute the overlapping labels using other methods Compatible with the Mascot search engine the Proteome Discoverer application uses a first order approximation to the solution The error made is small when the intensities of all possible contributing labels are of similar height and it becomes larger if the intensity differences become larger Excluding Peptides from the Protein Quantification Results You can manually exclude and include certain peptides from the protein quantification results You can also retu
109. mass RDB equivalent or mass delta click the Composition tab and select the labels for display Customizing Cleavage Reagents In the Cleavage Reagents view you can explore the default types of reagents and their corresponding settings You can also add remove and modify the reagents and their corresponding settings The Cleavage Reagents view contains the cleavage sites cleavage inhibitors abbreviations and cleavage specificities 150 Proteome Discoverer User Guide Thermo Scientific To display the Cleavage Reagents view 4 Searching for Data Customizing Cleavage Reagents e Choose Administration gt Maintain Cleavage Reagents or click the Maintain Cleavage Reagents icon on the toolbar or on the Administration page The Cleavage Reagents view appears as shown in Figure 109 Figure 109 Cleavage Reagents view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 8a BH 8 eT SB20008 Z omm 0 SequestHT Macot A R hE aA BB RIAR E Administration x 4 3 Remove Apply oe Name VY CleavageSites Y Cleavage Inhibitors Y Offset Y Abbreviation Y CleavageSpecifictes V re 4 Job Queue Click here to add a new record y i m Trypsin R R 1 Full Content Management Trypsin K K 1 Full m Trypsin KRLNH KRLNH 1 Try_a Full fe FASTA Files gt Trypsin KR 1 Try Full FASTA Indexes Staph_Protease E 1 Full i Proline_Endopept P 1 Full m Spectral Li
110. modification or indirectly specified a modification from the chosen quantification method As an exception when you select an exclusive modification group the Mascot search engine modifies all or none of the affected residues of a peptide sequence Peptide matches with inconsistent labeling therefore no longer occur Proteome Discoverer User Guide 263 7 Quantification Setting Up the Quantification Method Note Using a Mascot quantification method to retrieve the modification groups to use does not affect how the Proteome Discoverer application performs the quantification The application itself exclusively performs the quantification You must specify in the application s methods any quantification labels used for the quantification Setting Up the Quantification Method Setting up the quantification method is similar for both precursor ion quantification and reporter ion quantification Both methods use values called quantification quan channels as the basis for the ratio reporting You do not need to set up a quantification method for peak area calculation quantification For reporter ion quantification a quantification channel is one of several masses states or tags depending on which quantification method you use for which you measure a quantification value The Proteome Discoverer application calculates the reported quantification ratios from the quantification values of the different quantification channels For ex
111. of your spectra The search wizards are predefined to enable you to quickly set your search parameters and obtain results The Proteome Discoverer application includes a wizard for the Sequest HT and Mascot search engines For information about how to use the wizards see Starting a New Search by Using the Search Wizards on page 29 The Workflow Editor provides greater flexibility in creating custom search results Use its three pane display to create a custom workflow The Workflow Nodes pane of the application s interface contains seven categories of workflow choices A typical workflow uses three or more options from these categories as shown in Figure 1 To start a new workflow begin with a node from the Data Input category For more information see Starting a New Search by Using the Workflow Editor on page 42 When you activate any node from the Workflow Nodes pane the parameters appear in the Parameters pane Proteome Discoverer User Guide 5 1 Introduction Features Figure 1 Workflow Editor workspace File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BRO RH Zomm RM SequethT g Mascot i A R AAE AZESBRRBSARE ifjau saalslor A Event Detector Spectrum Selector Spectrum Processing lg Noise Peak Filter ug Spectrum Grouper ug Spectrum Normalizer ig Top N Peaks Filter i Spectrum Confidence Filter Ti Spectrum Properties Filter lih F
112. opens 6 Save the sequence as follows a In the File Summary Information box click OK b In the File Name box type a unique name for the sequence c Inthe Save In list select the appropriate folder location for the sequence d Click Save The Xcalibur data system adds the sequence to the acquisition queue For each sequence row after the data system acquires a raw file it sends the processing method and the raw data file to the Proteome Discoverer application which stores the raw file and the MSF file in the server output directory specified in the Server Output Directory box of the Export Parameter File page of the Start Jobs page All the search results of the batch processing are stored in the same directory If the same directory name is used for the results of another batch process the date and an index number that increments are appended to the folder name Batch Processing with Multiple Processing Methods In some cases you might need to use more than one processing method in the sequence For example the sequest pmd method runs the Proteome Discoverer Daemon application with a parameter file containing a simple Sequest workflow and the export pmd method runs the Proteome Discoverer Daemon application with an export workflow To use more than one processing method in a sequence 1 In the Sequence Setup view choose File gt New The New Sequence Template dialog box opens 2 Enter the appropriate values in each of
113. precursor 7 definition 7 348 generated by ETD and ECD 3 C terminus 40 Cancel icon 103 catalytic activity GO Slim category 233 cell communication GO Slim category 237 cell death GO Slim category 237 cell differentiation GO Slim category 238 cell division GO Slim category 238 cell growth GO Slim category 238 cell homeostasis GO Slim category 238 cell motility GO Slim category 238 cell organization and biogenesis GO Slim category 238 cell proliferation GO Slim category 238 cell surface GO Slim category 234 cellular component codes 229 Cellular Component column 206 Proteome Discoverer User Guide 349 Index D cellular components 201 Cellular Components page 202 221 228 Centroid Sum integration method 256 Centroid With Smallest Delta Mass integration method 256 Change Instruments In Use dialog box 86 chemical modifications adding 144 adding amino acids 145 deleting 146 deleting amino acids 148 displaying 142 dynamic 141 importing 146 importing from local file 147 importing from UNIMOD 146 static 141 updating existing 145 Chemical Modifications view adding modifications 144 description 141 142 opening 142 Position column 145 chromosome GO Slim category 234 CID activation type analyzed by Sequest 3 description 8 fragmenting ions 7 347 ion factors 37 selecting in search wizards 33 specifying in Reporter Ions Quantifier node 256 CID libraries 129 cleavage properties 346 cleavage reagents adding 151
114. protein ratios in a multiconsensus report when the quantification results of the single result files are treated as replicates of the same sample 330 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Calculating and Displaying Protein Ratios for Multiconsensus Reports Figure 224 Protein ratios when single quantification result files are treated as replicates of the same sample File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH aoao SABO SH gonm K sme Kut AR AME AEBSBTABIARE Multi Report from 3 Reports ac v4 Y IgG1 Fab Fragment Anti 28 68 989 serum albumin Bos taur 19 28 BUMIN PLAKALBUMIN ALL 18 44 casein variant CnH bovine 17 22 ERALDEHYDE 3 PHOSPHATE 14 11 nic Trypsin Wild Type 11 21 BONICANHYDRASE II CARB 10 04 onate dehydratase EC4 2 1 1 5 02 P DEPENDENTPROTEINKINA 2 83 icidin Ssynthase 2 Bacillus 135 2 160 7 7 7 0 0 2 345 7 7 7 0 0 12 170 7 7 7 2 253 3 3 3 0 0 2 069 3 3 3 0 0 12 289 3 3 3 2 266 Bick 0 0 2 517 HDI 0 0 13 890 HDI 2 204 4 4 4 0 0 2 297 4 4 4 0 0 12 585 45454 1 797 2 2 2 0 0 2 282 2 2 2 00 10 762 2 2 2 2 293 2 2 2 0 0 2 087 2 2 2 0 0 11 634 2 2 2 4 137 1 1 1 0 0 4 040 l l 1 0 0 22 176 1 1 1 Ready 12 12 Protein Group s 371 371 Merged Protein s 536 536 Peptide s 3267 3267 PSM s 17571 17571 Search Input s The Proteome Discoverer application combines on
115. protein references e Sequences Searches for the specified amino acid sequence within the protein sequences You can further refine the results by using filters either before or after you run the search For instructions on filtering see To filter a protein reference search on page 109 In the Maximum Number of Matches Reported box select the maximum number of references or sequences to report Click Start Search Results appear if the search parameters match the data as shown in Figure 78 Click a protein row to see the amino acid sequences that constitute that protein 108 Proteome Discoverer User Guide Thermo Scientific 9 To suspend the search click Stop Search Figure 78 Find Protein References page in the FASTA Database Utilities dialog box Add Protein References Compile FASTA Database Find Protein References FASTA Database C Program Files Proteome Discoverer source files FASTA_Files bovine fasta uaa Search for ASE Searchin References Sequences Madmum number of matches reported 10 Reference Starts with a gt gil3212389 pdb 1A5P C 40 95 aVariant Of Bovine PancreaticRibonucleaseA 1 2 gt gi 3212390 pdb 1A5Q P93aVariant Of Bovine PancreaticRibonucleaseA 3 5 gt gi 1827672 pdb 1FON B Chain B Crystal Structure Of Bovine Procarboxypeptidase A S6 Subunit Iii AHighly Structured Truncated ZymogenE gil18276 JE gt gi 3660252 pdb 1NBMIE Chai
116. quantification you might select the following two dynamic modifications e 13C 6 6 020 Da K e 13C 6 15N 4 10 008 Da R If you do not find these labels you can enable them by following the instructions in Updating Chemical Modifications on page 141 In the Static Modifications area select the static modifications For example for SILAC 2plex Arg10 Lys6 quantification select Carbamidomethyl 57 021 Da C in the Static Modification box Set any other parameters that you prefer Set the parameters for all other nodes in the Parameters pane For information about all the parameters that you can set for each node refer to the Help For information on the parameters that you can set for the Precursor Ions Quantifier node see step 14 of this procedure Thermo Scientific 7 Quantification Performing Reporter lon Quantification 14 Click the Precursor Ions Quantifier node and set the parameters for it in the Parameters pane a Set up the quantification method Click the Quantification Method parameter and follow the procedure in Setting Up the Quantification Method on page 264 to specify the quantification method b Set the parameters that identify the isotope patterns i Inthe RT Tolerance of Isotope Pattern Multiplet min box specify the maximum retention time tolerance of the AO peak in the isotope pattern of a quantification multiplet in minutes The default is 0 2 minutes ii In the Single
117. remove the completed job from the job queue by clicking Remove and then clicking OK in the Delete Jobs dialog box Compressing a Protein Database A protein database contains the proteins of imported FASTA files It also contains proteins found during a Mascot search that are inserted into the database When you remove a FASTA file from the database by using the FASTA file manager it automatically deletes protein entries but does not make the storage space available Although following this next procedure can explicitly make the storage space available it can be time consuming for large databases To compress a protein database 1 Choose Administration gt Maintain FASTA file The Administration page appears with the FASTA files view 2 Click Compact A message informs you that compressing the protein database can take a long time 3 To continue with the database compression click OK in the message box A job starts and appears in the job queue Before you start the job you can remove it if necessary However you cannot cancel the job and it will restart automatically if you shut down the Proteome Discoverer application during job execution Displaying Temporary FASTA Files The Proteome Discoverer application temporarily imports FASTA files that contain the proteins found by a Mascot search but these files are not available for Sequest searches You can optionally display these files in the FASTA files view To displa
118. reports all calculated protein ratios of the single result files side by side for the multiconsensus report It prefixes each file by a single letter identifier of the particular report and the number of the quantification node in the processing workflow In the example given in Figure 222 the protein and corresponding variability and ratio count columns are A4 115 114 B4 115 114 C4 115 114 A4 116 114 B4 116 114 C4 116 114 A4 117 114 B4 117 114 and C4 117 114 At the peptide level there is no difference between treatments or replicates in multiconsensus reports and single reports Figure 222 shows the protein ratios in a multiconsensus report when the quantification results of the single result files are treated as different treatments of the sample Figure 222 Protein ratios when single quantification result files are treated as different treatments of the sample File Search Report Quantification Processing Workflow Editor Administration Tools Window Help H ag eov onono Multi Report from 3 Reports x Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary gz Bam W SequestHT W Mascot amp MH A 3S i amp SERRPARE Ea Description ZCoverag E Proteins Peptides 5 PSMs A4 115 114 A4 115 114 Count A4 115 114Variability B4 115 114 B4 115 114 Count B4 115 114Variability C4 115 114 C 2 7
119. state and the quality of the fragmentation spectrum The Sequest search algorithm correlates experimental MS MS spectra through comparisons to theoretical in silico peptide candidates derived from protein databases The proprietary cross correlation identification algorithm at the core of Sequest uses a sophisticated scoring system to help assess results Sequest looks for characteristic spectral patterns and then critically evaluates the equivalence of experimental and theoretical MS MS spectra The identification algorithm extracts information and correctly identifies proteins even when protein sample sizes are limited and the signal to noise ratio of spectra is low You can extract specific information from your results through the interactive data summary screens With a click you can examine a fully annotated MS MS spectrum or view the percent peptide coverage of an identified protein Sequest provides excellent search results on data acquired with Thermo Scientific ion trap mass spectrometers Using accurate mass windows decreases the search time increases the accuracy of the result and decreases the false positive rate The Proteome Discoverer probability based scoring system rates the relevance of the best matches found by the Sequest algorithm With this probability based scoring the application can independently rank the peptides and proteins and increase the confidence in protein identification Additionally this scoring system minim
120. than or equal to a specified value for inclusion in the protein grouping process specify a value in the Consider Only PSMs with Delta Cn Better Than box 176 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 5 Filtering Data Grouping Proteins The default A Cn value is 0 15 To have the Proteome Discoverer application consider all PSMs set the value to 1 0 If you want to remove all protein groups that are not necessary to explain the found peptides select the Apply Strict Maximum Parsimony Principle check box The Apply Strict Maximum Parsimony Principle option ensures that only one PSM per spectrum is used for protein grouping If the A Cn range of the spectrum includes more than one PSM the Proteome Discoverer application selects the best PSM and rejects the others for grouping and quantification To display other proteins belonging to the same protein group Open the MSF file On the Proteins page click anywhere in a protein row Choose Search Report gt Show Protein Group Members or click the Show Protein Group Members View icon f The Protein Group Members view appears below the Proteins page as shown in Figure 132 Proteome Discoverer User Guide 177 5 Filtering Data Grouping Proteins Figure 132 Proteins in the same group a a00 ajeta nn0a m i Z DTT i K suth g Mso ARARS RCESBTABARE Sequest TRAQ 8 plex Benchmark 1 4 0
121. the Proteome Discoverer application after the first sample is finished Real Time Plot Home Page le Actions View GoTo Help wwen pmm 1 1 S melo Aa Status Acquisition Queue All Sequences HOMEPAGE C calibur Data test_09071 Sequence Row 1 Sequence Row 2 Sequence Row 3 O O O The Proteome Discoverer application synchronously processes the raw files on the remote host as shown in Figure 66 Thermo Scientific Proteome Discoverer User Guide 91 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 66 Processing the raw files synchronously on the remote host H8 Real Time Plot File Actions View GoTo Help Eafe plm m S S ee ea BatchSample3 2 RT 0 16 AV 1 NL 8 27E2 Status Acquisition Queue T ITMS p MALDI d w Full ms2 1159 61 cid40 00 305 00 1170 00 ps 1013 50 All Sequences HOMEPAGE C Xcalibur D ata test_09071 Sequence Row 1 Sequence Row 2 COMP Sequence Row 3 Relative Abundance a i tit 773 50 494 17 528 00 657 17 578 50 i Thermo Proteome Discoverer Eile Search Report Quantitation Processing Workflow Editor Administration Tools Window Help F OU SSBSOTSAGOSH Zoom Keer Kemet Kao AM A Administration x re 4 Job Queue al FASTA Files cma E r
122. the Refresh icon Refresh Thermo Scientific Proteome Discoverer User Guide 125 4 Searching for Data Using FASTA Databases The new FASTA index appears in the FASTA Indexes view on the Administration page The Proteome Discoverer application creates an index for the specified FASTA file and the decoy version of the FASTA file Controlling Automatic FASTA Index Removal After the number of FASTA indexes reaches the specified maximum the Proteome Discoverer application automatically removes from memory the number of FASTA indexes over the maximum It first removes the oldest indexes that is the ones with the earliest access time However you can mark specific FASTA indexes so that they will not be removed from memory even after the maximum is reached e To deactivate automatic FASTA index removal Deleting a FASTA Index To activate automatic FASTA index removal To deactivate automatic FASTA index removal In the FASTA Indexes view on the Administration page clear the Auto Remove check box The Apply icon now becomes available Click the Apply icon Apply In the Remove FASTA indexes confirmation box click OK To activate automatic FASTA index removal Select the Auto Remove check box Click the Apply icon Apply In the Remove FASTA indexes confirmation box click OK You can only delete FASTA indexes that have an Auto Remove check box selected To delete a FASTA index To restore a
123. the URL of the ProteinCenter server to use to upload your search results User Name Specifies the user name of your ProteinCenter user account Password Specifies the password of your ProteinCenter user account Test Verifies that the URL that you specified in the URL box is valid However it does not verify that the user name and password are valid Accessing ProteinCards Thermo Scientific You can access the data in ProteinCenter through the ProteinCard for each protein In ProteinCard a protein is considered a specific amino acid sequence in a given species To access the data in ProteinCenter 1 Double click a grid cell on the Proteins page of the MSF file or select a cell and choose Search Report gt Show Protein ID Details or click the Show Protein Peptide ID Details icon i You might experience a short delay as the Proteome Discoverer application accesses the URL 2 In the Protein Identification Details dialog box click the ProteinCard tab After loading data from the ProteinCenter server the Proteome Discoverer application displays the data in the ProteinCard tab By default it shows the General tab shown in Figure 163 on page 223 3 Click the tab of the page containing the information that you are seeking General Page Keys Page Features Page Molecular Functions Page Cellular Components Page Proteome Discoverer User Guide 221 6 Protein Annotation ProteinCard Parameters e Biological
124. the boxes 3 In the Bracket Type area select the None option as shown in Figure 60 With this bracket type you can change the processing methods individually for each sample Thermo Scientific Proteome Discoverer User Guide 87 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 60 New Sequence Template with the selection of None for the bracket type General Base File Name DiscovererD aemonBracket Starting Number 1 Path C Projects Daemon Instrument Method C Xcaliburkmethods Daemon i Browse Processing Method C Xcalibur methods batch Calibration File Samples Number of Samples 3 Tray Type 6x8 vials v Injections per Sample 1 j Initial Vial Position Al V Re Use Vial Positions Base Sample ID Bracket Type None Open Non Overlapped Overlapped Calibration ac Add Standards Add OCs Number of Calibration Sets 1 After First Calibration Only Penns perle After Every Calibration Add Blanks Add Blanks Fill in Sample ID for Standards Fill in Sample ID for OCs Figure 61 shows a sequence using two different processing methods Figure 61 Sequence with two different processing methods Sample Type File Name Sample ID Path Inst Meth Proc Meth Position Inj Vol Unknown Unknown Unknown BSA1 1 C Projects Daemon C calibur methods D aemon C calibur
125. the matches that pass a given set of filter thresholds from the decoy database and from the non decoy database It counts only the top match per spectrum assuming that for any given spectrum only one peptide can be the correct match Target FDRs If you set an FDR target value for a decoy database search the application determines and applies filter thresholds to identified matches so that the resulting FDR is not higher than the set target value The confidence indicators applied to each peptide match are distributed according to these calculated filter thresholds see Figure 142 on page 195 You must specify two target values for a decoy database search a strict target FDR and a more relaxed FDR Figure 139 on page 191 shows the decoy search setting with target FDRs of one percent and five percent respectively After completing the search the system automatically determines two sets of filter settings so that the resulting separate FDRs do not exceed their corresponding target value Thermo Scientific Proteome Discoverer User Guide 187 5 Filtering Data Calculating False Discovery Rates Peptide Confidence Indicators The filter settings that determine FDRs are used to distribute the confidence indicators for the peptide matches these are the green yellow and red circles attached to each peptide match Whenever you perform a decoy database search during the database search and apply filter settings to achieve the specified target FD
126. to resources containing information about the protein Thermo Scientific Proteome Discoverer User Guide 231 6 Protein Annotation ProteinCard Parameters Figure 170 External Links page of the ProteinCard page Coverage ProteinCard a General Keys Features Molecular Functions Cellular Components Biological Processes Diseases External Links HPRD MM HPA Click the appropriate link to open the browser for the database The external links contains links to resources containing information about the respective protein 232 Proteome Discoverer User Guide Thermo Scientific GO Slim Categories 6 Protein Annotation GO Slim Categories This section defines the GO Slim terms for molecular functions cellular components and biological processes GO Slim Categories for Molecular Functions Thermo Scientific Table 17 describes the GO Slim categories for molecular functions Table 17 GO Slim categories for molecular functions Sheet 1 of 2 GO Slim molecular function Antioxidant activity Description Inhibition of the reactions brought about by dioxygen O2 or peroxides Usually the antioxidant is effective because it can be more easily oxidized than the substance protected The term is often applied to components that can trap free radicals breaking the chain reaction that normally leads to extensive biological damage Catalytic activity Catalysis of a biochemical reactio
127. to 55 peak area calculation quantification 259 260 precursor ion quantification 247 248 evidence codes 227 229 230 Experimental Bias page 280 Export Parameter File page 81 94 Export to ProteinCenter dialog box 219 Extensible Markup Language files See XML files External Links page 202 222 231 extracellular GO Slim category 235 Thermo Scientific Index E F value 137 138 false discovery rate definition 186 recalculating 197 relaxed peptide confidence indicators 188 Peptide Confidence page 195 specifying for decoy database search 187 setting up in search wizards 189 setting up in Workflow Editor Percolator node 190 Target Decoy PSM Validator node 190 strict peptide confidence indicators 188 Peptide Confidence page 195 specifying for decoy database search 187 target 187 188 FASTA Database Utilities dialog box Add Protein References page 106 Compile FASTA Database page 112 116 Find Protein References page 107 opening 106 FASTA files adding 103 104 adding before using search wizards 30 adding protein reference 106 adding protein sequence 106 cancelling addition or removal 103 compiling 112 deleting 104 displaying 101 current status 103 date last modified 103 name 103 number of amino acids found 103 number of sequences found 103 size 103 temporary 105 excluding protein sequences or references 116 filtering protein reference searches 109 finding protein sequences or references 107 input to search wizards 29 m
128. 0 and there is no maximum value The default is 20 4 In the Time Interval between Attempts to Submit a Search sec box specify the interval of time in seconds that elapses between attempts to submit a search when the Mascot server is busy The minimum value is 20 and there is no maximum value The default is 90 seconds 5 If you are accessing a Mascot server through your own network and security for that server is enforced enter your user name and password in the boxes beneath the Mascot Server Authentication parameter 6 If you are accessing a Mascot server through the Web and security for that server is enforced enter your user name and password in the boxes beneath the Web Server Authentication parameter 7 Set the Default Confidence Thresholds parameters e Significance High Calculates the thresholds for high confidence peptides The Proteome Discoverer application automatically sets this value to the calculated relaxed significance when it performs a decoy search The minimum value is 0 0 and the maximum value is 1 0 The default is 0 01 e Significance Middle Calculates the thresholds for medium confidence peptides The Proteome Discoverer application automatically sets this value to the calculated relaxed significance when it performs a decoy search The minimum value is 0 0 and the maximum value is 1 0 The default is 0 05 8 If you changed any settings click Apply The message box shown in Figure 12 appear
129. 0 0000 is W 10 I eVVGSAEAGVDAASVSEER Unambiguous 1 1 129293 N Term TMT6plex S5 Ph CID 0 3633 0 0000 1 1g 11 eLINSWVESQTNGIR Unambiguous 1 1 129293 N Term TMT plex cID 0 5517 0 0000 i 1 12 QGLEPINFQTAADQAR Unambiguous 2 1 129293 N Term TMT plex CID 0 5599 0 0000 1 1 a 13 M GILNQITKPNDVYSFSLAR Unambiguous 2 1 129293 N Term TMT6plex K8 TM CID 0 4954 0 0000 1 1 14 7 ITEWTSSNVMEER Unambiguous 1 1 129293 N Term TMT6plex CID 0 4780 0 0000 1 1 E 15 M ITEWTSSNVMEER Unambiguous 1 1 129293 N Term TMT6plex cID 0 4617 0 0000 1 1 mH 16 M dILNQITKPNDVYSFSLAR Unambiguous 2 1 129293 N Term TMT 6plex K8 TM CID 0 4031 0 0000 1 f 17 M VASMASEk Unambiguous 1 1 129293 N Term TMT plex K8 TM CID 0 4766 0 0000 1 1 a i Tf Unambiguous 1 1 129293 N Term TMT plex K3 T M cID 0 3810 0 0000 1 1 E J 19 eVVGSAEAGVDAASVSER Unconsidered 1 1 129293 N Term TMT 6plex 14 P CID 0 5966 3 3 S f 20 m IYAEER Unambiguous 2 1 129293 N Term TMT6plex cID 0 2233 0 0000 1 1 m 21 M glWek Selected 1 1 129293 N Term TMT6plex K5 TM cD 0 0558 2 2 m 2 M wR Unambiguous 2 2 129296 129293 N Term TMT6plex cD 0 4278 0 0000 1 1 ra A T Quan Channel Values 4 X Quantification Spectrum vax C Smoke_Test_Files 10mcTMT_45CE_1us RAW 6104 RT 84 05 mn C ASmoke_Test_Files 10mixTMT_45CE_1us RAW 6104 RT 84 05 min FTMS HCD Precursor z 3 Mono m z 672 02185 Da MH 2014 05 100 Da Integration Most Confid
130. 002 0 300 0 010 100 Yes 2000 000 3 000 100 000 3 000 100 Yes 0 300 0 002 0 300 0 010 100 No 2000 000 0 002 100 000 0 010 100 Yes 2000 00 0 002 100 000 0 010 Calculating Protein Ratios from Peptide Ratios This section describes seven different scenarios that can occur when you derive protein quantification ratios from peptide quantification ratios These cases show how the validity of using a given quantification result for the quantification of a certain protein depends on whether this particular quantification result is unique or shared among other peptides The peptide quantification ratios are taken from the associated quantification results The term quantification result in this section refers to MS MS reporter intensities taken from the same scan as the identification for example ID CID or from a separate quantification scan for example Quan HCD The term also refers to intensities derived from the precursor scans in precursor ion quantification A quantification result here is a general quantity associated with one or more peptides that are in turn associated with one or more proteins Case 1 Quantification Result Associated with One Spectrum One Peptide and One Protein Case 1 shown in Figure 214 is the simplest case The quantification result is associated with one identification spectrum whether the quantification results come from the same identification spectrum from a different quantification spectrum or from the pr
131. 030016 GO 0016459 GO 0043292 GO 0044422 GO 0044444 GO 0015629 GO 0043234 GO 0044430 GO 0043232 GO 0005575 GO 0043226 GO 0005737 G0 0044424 GO 0005856 GO 0032991 GO 0044446 GO 0043228 GO 0043229 GO 0005622 GO 0044464 GO 0005623 j G0 0005874 GO 0015630 GO 0043234 GO 0044430 GO 0005856 GO 0032991 GO 0044446 GO 0043232 GO 0005575 GO 0043229 GO 0044422 GO 0044424 GO 0043228 GO 0043226 GO 0005622 GO 0044464 GO 0005623 G0 0007130 GO 0007129 GO 0070192 GO 0070193 GO 0071844 GO 0007127 GO 0007126 GO 0022402 GO 0051276 GO 0022607 GO 0071842 GO 0022403 GO 0051327 GO 0007049 GO 0009987 GO 0006996 GO 0016043 GO 0044085 GO 0071841 GO 0000279 GO 0051321 GO 0008150 GO 0071840 GO 0048610 GO 0022414 GO 0000003 107626 GO 0007610 GO 0050896 GO 0008150 18017 GO 0015631 GO 0008092 GO 0005515 GO 0005488 GO 0003674 8092 GO 0005515 GO 0005488 G 103674 109792 GO 0009790 GO 0007275 GO 0048856 GO 0032501 GO 0032502 GO 0008150 10171 GO 0009653 GO 0032502 GO 0048856 GO 0008150 G0 0015078 GO 0015077 GO 0022890 GO 0008324 GO 0015075 GO 0022891 GO 0022857 GO 0022892 GO 0005215 GO 0003674 G0 0015991 GO 0015988 GO 0015992 GO 0034220 GO 0006818 GO 0015672 GO 0006811 GO 0055085 GO 0006810 GO 0006812 GO 0009987 GO 0051234 GO 0008150 GO 0051179 cy 2 a aaa C aa C a aaa C a a A A G G G G Gi G0 0016021 GO 0031224 GO 0044425 GO 0005
132. 0mixTMT_45CE_ius RAW 0 878 0 918 0 993 HCD MS2 47 78 421 28717 841 56706 40 45 2515 10mixTMT_45CE_ius RAW 1 012 1 116 0 964 0 978 1 135 HCD MS2 52 129 392 55481 1175 64988 40 47 2516 10mixTMT_45CE_1us RAW HCD MS2 71 150 335 90640 1005 70465 40 49 2517 10mixTMT_45CE_1us RAW HCD MS2 0 300 346 42017 1382 65883 40 54 2522 10mixTMT_45CE_1us RAW HCD MS2 9 150 588 32861 1175 64995 40 56 2523 10mixTMT_45CE_ius RAW HCD MS2 0 300 829 89471 1658 78215 40 60 2527 10mixTMT_45CE_1us RAW HCD MS2 58 132 359 91354 1077 72608 40 64 2530 10mixTMT_45CE_ius RAW 0 923 2 079 1 267 1 180 2 055 HCD MS2 16 150 437 30069 873 59410 40 66 2531 10mixTMT_45CE_lus RAW 0 801 1 016 1 299 0 807 1 247 HCD MS2 66 150 432 27805 863 54881 40 68 2532 10mixTMT_45CE_ius RAW 1 490 1 144 1 629 0 998 HCD MS2 a 300 424 93628 1696 72329 40 76 2539 10mixTMT_45CE_1us RAW HCD MS2 49 150 321 57114 962 69886 40 78 2540 10mixTMT_45CE_ius RAW 0 636 1 221 0 604 1 342 1 197 HCD MS2 34 150 560 92474 1680 75968 40 80 2541 10mixTMT_45CE_1us RAW HCD MS2 43 85 340 71921 680 43114 40 85 2546 10mixTMT_45CE_lus RAW 0 783 1 085 0 876 0 890 1 100 HCD MS2 18 72 533 63068 1598 87748 40 87 2547 10mixTMT_45CE_ius RAW 0 576 0 643 0 551 0 635 HCD MS2 29 300 400 47482 1598 87746 40 88 2548 10mixTMT_45CE_1us RAW HCD MS2 z 40 432 58578 1295 74280 40 93 2553 10mixTMT_45CE_1us RAW 1 713 1 283 1 132 1 824 HCD MS2 38 300 389 47742 1554 88784 40 95 2554 10mixTMT_45CE_ius RAW 1 189 HCD MS2 80 150 648 37445 1295 74162 40 97 2555 10mixTMT_
133. 11_05 fasta Target database options Create Replace Append Search in References Sequences Ignore case of reference strings Step 1 String s to Include Gp BM Step2 String s to Exclude From the Results of Step 1 Select Operator 4 j Select Operator Condition 1 piv Contains y influenza 11 Click Compile Database Click Stop if you want to halt the compilation 12 After the compilation click Start Search on the Find Protein References page to view the results of the extraction as shown in the example in Figure 84 You do not have to enter information into the Search For box 114 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases Figure 84 Results of search Add Protein References Compile FASTA Database Find Protein References FASTA Database C Program Files Proteome Discoverer source files FASTA_Files uniprot_sprot_2011_05 fasta ua Search for Searchin References Sequences Maximum number of matches reported 1000 j gt sp 005011 NQRB_HAEIN Na translocating NADH quinone reductase subunit B OS Haemophilus influenzae GN nqrB PE 3 SV 1 gt sp 005012 NQRF_HAEIN Na translocating NADH quinone reductase subunit F OS Haemophilus influenzae GN ngrF PE 3 SV 1 gt sp 005029 ISPD_HAEIN 2 C methyl D erythritol 4 phosphate cytidylyltransferase OS Haemophilus influenzae GN ispD PE 3 SV 1 gt sp 005052 TIRH_HAEIN Putativetyp
134. 175 msf X 1000ngYeast_Top10_DE30 01 msf X 4b Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary n E Accession Description Score Coverage Proteins UniquePeptides Peptides PSMs 114 113 114 113 Count 114 113 Varia lt m a I 1942750 Myoglobin Horse Heart Mutant With Ser 92Replaced By 63105 545 8 1 7 173 0 980 10 E 2 gt C 494711 Myoglobin Horse Heart Mutant With His 64Replaced By 567 37 54 25 5 1 7 158 1 102 3 3 I 1171661 NITRITE REDUCTASE NAD P H 72 60 124 1 1 1 32 _ g A2 Sequence PsMs Proteins ProteinGroups Protein Group Accessions Modifications ACn 114 113 114 113 Count 114 113 Variability 115 113 1 DTDTmLAASK 2 1 1 1171661 M5 Oxidation 0 0000 2 DTDTMLAAK 30 1 1 1171661 0 0000 4 Accession Description Score Coverage Proteins UniquePeptides Peptides PSMs 114 113 114 113 Count 114 113 Vari m 4 4127521 AJ011099 viral non structural polyprotein Bovine cdv 33 18 202 1 1 2 4 e s F mm0 CALPAIN INHIBITOR CONTAINS ERYTHROCYTE CALPAS 23 80 153 1 1 1 u m 6 T 4699596 Crystal Structure Of Calsequestrin From Rabbit Skeletal M 8 88 3 00 2 1 1 3 1 028 2 a E m 118838 DNA POLYMERASE DELTA CATALYTIC CHAIN 8 76 3 16 3 2 3 4 0 993 2 e s 7429315 probable ferrichrome ABC transporter yclQ Bacillus subtilis 755 442 1 1 1 4 0 718 1 a 9 M 1705470 6 CARBOXYHEXANOATE COA LIGASE PIMELOYL COAS 730 734 1 1 1 3
135. 2 126 0 00 0 00 89 80 9 80 0 40 127 0 00 0 70 90 10 8 80 0 40 128 0 00 1 20 91 60 7 00 0 20 129 0 00 1 70 92 00 6 10 0 20 130 0 10 1 90 92 70 5 20 0 10 131 0 10 3 60 91 60 4 60 0 10 Ratio Calculation Ready 10 10 Protein Group s 10 10 Protein s 525 525 Peptide s 6470 6470 PSM s 14702 14702 Search Input s Displaying Quantification Spectra After you perform reporter ion quantification you can display the Quan Spectra page This page displays the TMT intensities and ratios for all spectra in reporter ion quantification regardless of whether they have been identified To display the Quan Spectra page 1 Perform reporter ion quantification 2 Choose File gt Open Report to open the resulting MSF file 3 On the Input files page click Add 4 In the Add Analysis File s dialog box select the file to open and click Open 5 Select the Show Quan Spectra on Separate Tab check box Thermo Scientific Proteome Discoverer User Guide 293 7 Quantification Displaying Quantification Spectra This option generates the Quan Spectra page in the MSF report only if you included a Reporter Ion Quantification node in your workflow 6 Click Open Figure 197 gives an example of the Quan Spectra page Figure 197 Quan Spectra page File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE SEB SOT SODAS m i Z Dmm K smetit G Mas
136. 2 Tubulin Beta family member tbb 1 Caenorhabditis deg 6 3 M E mM Pf00091 Pf03953 176501 G0 0000166 G 44 23 T22F3 3b Caenorhabditis elegans B 1s ff I PF00343 178777 G0 0004645 G 84 a 24 7 R11A5 4d Caenorhabditis elegans 8 14 i Pf00821 172556 G0 0004611 G 61 Displaying UniProt Annotation Data For information on displaying UniProt PTM annotation data refer to the Help Thermo Scientific Proteome Discoverer User Guide 215 6 Protein Annotation Reannotating MSF Files Reannotating MSF Files You can use the Re Annotation node in the Workflow Editor or the batch processing function in Discoverer Daemon to update existing annotations or annotate existing MSF files that do not yet include annotations Use the Re Annotation node in the Workflow Editor to reannotate a single file The Re Annotation node must be the only node in a workflow It takes an existing MSF file as input retrieves up to date annotations for the proteins contained in the MSF file and stores them in the same MSF file Note If you used a previous version of the Proteome Discoverer application to create the MSF file to reannotate the application first updates the file to comply with the current results file schema Use the batch processing function in Discoverer Daemon to reannotate multiple files e T
137. 2 06 50 AM 09 25 2012 04 54 AM Remove Thermo Scientific Description c_elegans_sequest flow c_elegans_100_021411_FWD_combined fasta basic SEQUEST workflow WF_Spectrum_Export_MZML WF_Re Annotation WF_Q_Exactive_SEQUEST_vs_Mascot_Search_Percolator Workflow for processing Q Exactive raw files with Sequest and Mascot WF_LTQ_Orbitrap_Sequest_SILAC_2plex_ Arg10 Lys6 WF_LTQ_Orbitrap_Sequest_Precursor_Ions_Area Detector WF_LTQ_Orbitrap_Sequest_HCD_ReporterQuantitation WF_LTQ_Orbitrap_Sequest StaticMods _Dimethyl_SILAC WF_LTQ_Orbitrap_SEQUEST_vs_Mascot_Search_Phospho WF_LTQ_Orbitrap_SEQUEST_vs_Mascot_Search_Annotaii WF_LTQ_Orbitrap_ETD_Sequest_Mascot_new Workflow for quantifying SILAC Arg10 Lys 6 duplex labeled samples using Se Workflow for reporting the peptide and protein areas Workflow for processing raw files with HCD spectra for quantitation and all spec Workflow for quantifying Dimethyl SILAC Triplex labelled samples using Sequest Workflow for processing LTQ Orbitrap raw files with Sequest and Mascot Workflow for processing LTQ Orbitrap raw files with Sequest and Mascot Workflow forprocessing LTQ Orbitrap rawfiles withETD and CID spectra with Mascot Quan 2 Select a workflow from the list 3 Click Open The Workflow Editor window opens displaying the selected workflow The Based on Template area now displays the name of the template that you chose When you open an existing workflow template
138. 2 11 50 AM E M bovine fasta Trypsin Ful 1278 10 04 2012 11 51 AM ae eee A la Ww yeast5protmix fasta Trypsin Full 56 10 04 2012 01 37 PM a FASTA Fil ASTA Files B FASTA Indexes T Spectral Libraries a Chemical Modifications 3 Cleavage Reagents A Quantification Methods FASTA Indexes Thermo Scientific m Ed Indexed FASTA File Hb ecoli fasta Last Access Time 9996 10 04 2012 11 31 AM Trypsin Full To restore a deleted FASTA index In the Deleted FASTA Indexes table select the deleted index by clicking the Right Arrow icon Click the Restore icon WA Restore In the Restore FASTA indexes confirmation box click OK The restored index appears in the FASTA Indexes table and disappears from the Deleted FASTA Indexes table Proteome Discoverer User Guide 127 4 Searching for Data Using FASTA Databases Changing Number and Location of Stored FASTA Indexes You can specify a new directory for storing the FASTA indexes and change the maximum number of FASTA indexes stored The Proteome Discoverer application counts all FASTA indexes even the indexes that cannot be automatically removed with the Auto Remove option e To change the number and location of stored FASTA indexes e To reset the changes made in a previous FASTA index session To change the number and location of stored FASTA indexes 1 Click the Options icon gy Options
139. 236 metabolic process GO Slim category 239 metal ion binding GO Slim category 233 MGF files contents of 26 importing into Workflow Editor 65 input file type 12 29 44 output file type 13 66 splitting 26 mirror plots 15 140 missing reporter ions 300 mitochondrion GO Slim category 236 molecular function codes 227 Molecular Function column 206 molecular functions 201 Molecular Functions page 202 221 227 Most Confident Centroid integration method 256 Most Intense Centroid integration method 256 motor activity GO Slim category 233 MPD activation type selecting in search wizards 33 specifying in Reporter Ions Quantifier node 256 MS MS spectra fragmenting reporter ions in 295 generating CID 7 347 minimum ion count 34 processed by Mascot 157 processed by SEQUEST 157 reporter ion quantification 252 SEQUEST processing 4 types of fragment ions observed in 7 validating searches with FDRs 186 MSDB database 342 MSF files activating Quantification menu 242 filtering data 154 input to Proteome Discoverer application 64 354 Proteome Discoverer User Guide multiple files in quantification 282 output by Proteome Discoverer application 13 reannotating 216 ungrouping proteins 178 MSIPI database 340 MSP files 131 132 MSPepSearch node description 14 129 139 dot score 139 MSPepSearch score 139 reverse dot score 139 scores reported 139 MSPepSearch score 139 MudPIT creating a search workflow 53 creating parameter file to call Discoverer
140. 27 995 Da CO 32 026 Da CH30H 34 053 Da N2H6 2xNH3 35 037 Da H4NO 36 021 Da H402 2xH20 44 037 Da CH4N2 45 021 Da CH3NO 46 006 Da CH202 46 042 Da C2H60 59 037 Da C2HS5NO 59 048 Da CH5N3 73 089 Da C4H11N 74 019 Da C3H6S 82 053 Da C4H6N2 86 072 Da C3H8N3 99 068 Da C4H9IN3 101 095 Da C4H11N3 108 58 Da C7H80 131 074 Da CIHIN Opening an Existing Workflow You can open an existing workflow from a template that you saved or you can open it from an MSF or XML file See the following e Opening an Existing Workflow from a Template e Opening an Existing Workflow from an XML or MSF File 60 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Opening an Existing Workflow from a Template You can open an existing workflow that you previously saved when you chose Workflow Editor gt Save As Template To open an existing workflow from a template 1 Choose Workflow Editor gt Open From Template or click the Open From Template icon amp The Open Processing Workflow Templates dialog box appears as shown in the example in Figure 37 listing the available workflow templates Figure 37 Open Processing Workflow Templates dialog box 10 02 2012 08 46 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 2012 06 50 AM 10 02 201
141. 290 restoring quantification method template defaults 282 setting up quantification method 265 Quantification Spectrum chart 297 298 304 Quantification Summary page description 276 292 displaying 292 parameters for reporter ion quantification 294 quantification workflow creating 55 See also precursor ion quantification See also reporter ion quantification QuickGO browser 227 Ratio Calculation page 275 300 Ratio columns 273 Ratio Count columns calculating ratio count 323 324 Protein Quantification page 279 Proteins page 323 ratio counts 279 324 Ratio Reporting page 273 287 Ratio Variability columns calculating ratio count 323 Proteins page 323 Quantification Method Editor 279 Ratio Variability columns description 324 raw files base peak chromatogram of search wizards 32 contents of Xcalibur data system 12 determining charge state 34 in parallel workflows 57 58 input file type 29 44 passing for Qual Browser operations 149 performing multiple searches on multiple 69 processing multiple from multiple samples 44 69 processing multiple from one sample 44 53 69 processing one from one sample 44 processing synchronously in Xcalibur data system 91 search wizards excluding first and last minutes of data in 33 Thermo Scientific Index R grouping spectra 34 specifying multiple files in 32 specifying name in 32 selecting in Workflow Editor 44 specifying instrument that produced 37 submitting multiple files to Workf
142. 4 42 244 117 time 16ims TTL 52 Reply from 194 42 244 117 time 159ms TTL 52 Reply from 194 42 244 117 time 159ms TTL 52 Reply from 194 42 244 117 bytes 32 time 162ms TTL 52 ics for 194 42 244 117 Sent 4 Received 4 Lost 6 z los Approximate round trip times in milli seconds Minimum 159ms Maximum 162ms Average 166ms C WINDOWS Ns ys tem32 gt If the pin command is unsuccessful a firewall on your computer or on the Mascot server computer or a bad network connection might be blocking the ping command Contact your system administrator to assist you in resolving this problem If you can obtain Web access to the Mascot server and the ping test is successful but the same URL is not accepted in the Proteome Discoverer application a type of user authentication restriction might be active In this case the error message issued by the Proteome Discoverer application should provide information about missing authentication If it does not send an error report Configuring Mascot Parameters Before using the Mascot search engine set the parameters that govern access to the Mascot server To configure the Mascot search engine 1 On the Administration page click Mascot under Workflow Nodes in the Configuration section The Proteome Discoverer application generates an MGF file that contains the search settings and all mass spectral information It submits this file to the Mascot server through a Web server which
143. 433 7 ELVCTLSALCRGPLPGR Unambiguous 1 1 Q8wT44 ETD 0 2870 0 0000 1 1 H 1434 GVRPAINVGLSVSR Unambiguous 2 1 Q9XXxK1 CID 0 4709 0 0000 1 1 1435 GQPVADTGDPIKIPVGPEIL Unambiguous 1 1 P46561 CID 0 5112 0 0000 1 1 1436 LTTDPHFSESGTAVNLFVDYK Unambiguous 4 1 Q7IL40 0 4215 1 1 7 E S warzone mine ame a a 1438 7 ELVEVINQAEGIIHDTEAK Unambiguous 1 1 P11141 0 6188 1 1 1439 NLLASEEDNLPVLAV Unambiguous 1 1 P34462 CID 0 6816 0 0000 1 1 H 1440 GIMDVLR Unambiguous 4 1 045865 CID 0 2870 0 0000 1 7 H 1441 M AILTDYFASK Unambiguous 1 1 P49196 CID 0 6996 0 0000 1 1 1442 GKDSRYCFLDTPLLAPFPDFF Unconsidered 1 0 ETD 0 0314 0 0000 1 1 1443 ISNVSDCWNYMQTGSDAK Unconsidered 2 0 ETD 0 2960 0 0000 1 1 1444 LDLGSVVPSVSGPK Unambiguous 1 1 Q23500 CID 0 4369 0 0000 i 1 1445 GVLEMSVFLRIFHDCDDK Unconsidered 1 0 ETD 0 1802 0 0000 1 1 1446 RTTIESTETFDVIEPR Unconsidered 1 1 Q7Z193 ED 0 1532 0 0000 1 1 1447 EFVNKAQNQEQAEFAVEAIA Unconsidered 1 0 ETD 0 0811 0 0000 1 1 4448 7 KQNPGLISQISCCNETRMLG Unconsidered 1 0 ED 0 1802 0 0000 1 1 1449 7 TPEIAPIFVEIFTK Unambiguous 1 1 B3034 cID 0 5721 0 0000 1 a 1450 ALIAITTNPVNSTVPIASEVLK Unambiguous 1 1 002640 CID 0 8378 0 0000 1 a 1451 VTLIPGDGIGPEISASVQK Unambiguous 1 1 Q93714 CID 0 5158 0 0000 1 1 cy 1452 M DFGVVYQAK Unambiguous A 1 Q9xTI0 cID 0 4932 0 0000 1 1 1453 DLDLTFLAK Unambiguous 1 1 P54811 CID 0 5430 0 0000 1 1 1454 AIEI
144. 45CE_ius RAW 0 959 1 081 1 160 1010 41 647 HCD MS2 10 90 440 61414 1319 82785 41 02 2560 10mixTMT_45CE_ius RAW 0 970 0 995 0 845 0 769 0 775 HCD MS2 32 150 522 23822 1043 46916 41 04 2561 10mixTMT_45CE_1us RAW HCD MS2 39 300 362 97491 1448 87783 41 06 2562 10mixTMT_45CE_1us RAW 1 457 0 971 HCD MS2 20 63 359 24524 717 48320 41 10 2567 10mixTMT_45CE_ius RAW 1 076 1 142 1 018 0 954 1 427 HCD MS2 28 134 598 90906 1196 81084 41 13 2568 10mixTMT_45CE_lus RAW 1 432 1 301 1 233 1300 1451 gt Ready 1021 3819 Protein Group s 9007 9007 Protein s 16804 16804 Peptide s 22705 22705 PSM s 2504 2504 Search Input s Quan Spectra Page Parameters The parameters on the Quan Spectra page are basically the same as those on the Search Input page refer to the Help However they also include reporter ion quantification ratio columns that display the corrected ratio of the intensity of the fragmented tag in a sample to the intensity of the fragmented tag in the control sample for all spectra regardless of whether they have been identified 294 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Channel Values Chart The Proteome Discoverer application generates the Quan Spectra page only if you included a Reporter Ion Quantification node in your workflow and select the Show Quan Spectra on Separate Tab check box on the Result Filters page when you open an MSF file For more information on generating this page
145. 47 member 1 collagen binding protein 1 isoform CRA_a 119595383 GI EAW74977 1 GB serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 isoform CRA a 119595381 GI EAW74975 1 GB serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 isoform CRA a 119595380 GI EAW74974 1 GB serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 isoform CRA a 53831040 GI AAU95378 1 GB arsenic transactivated protein 3 37589973 GI AAH36298 2 GB Serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 33090237 GI AAP93914 1 GB proliferation inducing gene 14 E 32454741 GI NP 001226 2 REF serpin H1 precursor 30583027 GI AAP35758 1 GB serine or cysteine proteinase inhibitor clade H heat shock protein 47 member 2 20141241 GI P50454 2 SP Serpin H1 15779117 GI AAH14623 1 GB Serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 8574447 GI BAA96789 1 DBJ rheumatoid arthritis related antigen RA A47 8574445 GI BAA96788 1 DBJ rheumatoid arthritis related antigen RA A47 1P100032140 4 IPI Serpin H1 P50454 SP STY96 UNI Serpin H1 SERPH HUMAN UNI SPH2 HUMAN UNI ad He 224 Proteome Discoverer User Guide Thermo Scientific Features Page Thermo Scientific 6 Protein Annotation ProteinCard Parameters Table 12 lists the parameters on t
146. 5 47 z 2068 14897 4 44 65 88 I7 GLQAQIASSGLTVEVDAPK 1 1 1 4557888 0 0000 0 0 00049 5 40 2 1884 01665 4 92 58 75 I WTAISALEYGVPVTLIGEAVF 2 1 1 40068518 0 0000 0 3 31e 05 5 36 3 2463 34293 5 92 83 13 I DYEEVGVDSVEGEGEEEGEEY 1 2 1 17986283 0 0000 0 1 54e 05 5 33 2 2348 91753 5 35 56 59 I IQENEQLDVTTSEYEK 1 1 1 31542947 0 0000 0 2 78e 05 5 28 2 2038 03362 5 34 63 17 J7 DYEEVGADSADGEDEGE Y 1 1 14389309 0 0000 0 0 00142 5 25 2 2078 75640 4 53 50 63 I7 LAPITSDPTEATAVGAVEASK 2 1 1 33286418 0 0000 0 0 000248 5 25 3 2175 12998 5 47 65 33 I DATNVGDEGGFAPNILENK 1 1 1 4503571 0 0000 0 2 45e 05 5 19 2 1960 93547 5 45 58 27 7 GINSSNVENQLQATQAAR 1 1 1 4504897 0 0000 0 0 000656 5 17 2 1900 95378 3 45 50 23 I VDATEESDLAQQYGWR 1 1 1 20070125 0 0000 0 8 94e 05 5 17 2 1780 84551 5 97 53 13 J7 STGEAFVQFASKEIAENALGK 2 2 1 14141159 0 0000 0 0 000204 5 16 3 2197 12607 5 65 73 17 I LFVGGLDWSTTQETLR i 2 1 25470890 0 0000 0 4 87e 05 5 15 2 1822 94316 5 32 65 32 I7 ALMLQGVDLLADAVAVTMGPK 2 1 1 31542947 0 0000 0 0 000724 5 13 3 2113 15079 5 25 83 91 I FTASAGIQVVGDDLTVINPK 3 1 1 4503571 0 0000 0 8 32e 05 5 13 2 2033 06462 4 70 60 33 I AVFVDLEPTVIDRR 2 3 2 17986283 14389309 0 0000 0 6 55e 05 5 12 3 1701 91422 4 90 65 79 I EVAAFAQFGSDLDAATQQLL 2 1 1 4757810 0 0000 0 0 000125 5 11 2 2338 18462 7 33 77 50 I VPSTEAEALASSLMGLAK 2 1 6598323 0 0000 0 0 000229 5 06 2 1980 00871 4 64 80 78 I ALDLFSDNAPPPELLEINED 2 ul 1 5031973 0 0000 0 4 25e 05 5 06 3 2
147. 575 GO 0016020 El rE G0 0016459 GO 0015629 GO 0043234 GO 0044430 GO 0005856 GO 0032991 GO 0044446 G0 0043232 GO 0005575 GO 0043229 GO 0044422 G0 0044424 GO 0043228 G0 0043226 GO 0005622 GO 0044464 G0 0005623 fH 4 fe 0019898 GO 0044425 GO 0005575 GO 0016020 a 46 30016 GO 0043292 GO 0043232 GO 0044444 GO 0043228 GO 0043229 GO 0005737 GO 0044424 GO 0043226 GO 0005622 GO 0044464 GO 0005575 GO 0005623 4 G 31672 GO 0030017 GO 0044449 GO 0030016 GO 0043292 GO 0044422 GO 0044444 GO 0043232 GO 0005575 GO 0043226 GO 0005737 GO 0044424 GO 0043228 GO 0043229 GO 0005622 GO 0044464 GO 0005623 H 4 G 32982 GO 0016459 GO 0015629 GO 0043234 GO 0044430 GO 0005856 GO 0032991 GO 0044446 G0 0043232 GO 0005575 G0 0043229 G0 0044422 G0 0044424 GO 0043228 G0 0043226 GO 0005622 G0 0044464 GO 0005623 a d G 33177 GO 0016469 GO 0043234 GO 0044425 G0 0032991 GO 0005575 GO 0016020 j G0 0040007 GO 0008150 E3 G0 0040011 GO 0008150 a 4 60 0040017 GO 0040011 GO 0040012 GO 0048518 GO 0008150 GO 0050789 GO 0065007 T 27 PA TPA_exp ANC 1 Caenorhabditis elegans 34 39 I I Pf00307 PF10541 G0 0003779 G 854 28 FO1F1 12a Caenorhabditis elegans 6 12 it PF00274 175827 G0 0000003 G 36 29 Ribosomal Protein Largesubunit family member rp 4
148. 637 38193 6 07 79 43 M FDQLFDDESDPFEVLK 4 1 66346685 0 0000 0 6 46e 05 5 03 2 1943 90129 5 29 69 52 M NQILNLTTDNANILLQIDNAR 1 1 1 40354192 0 0000 0 8 44e 05 5 03 3 2367 27298 4 32 69 44 I ITGEAFVQFASQELAK 1 1 4826760 0 0000 0 1 09e 05 5 01 2 1867 95244 4 67 69 09 TE Peptide Group Members ax Ea Sequence PSM Ambiguity Proteins ProteinGroups Protein Group Accessions Activation Type AScore ACn Rank Search Engine Rank q Value PEP XCorr 117 SLQDIIAILGMDELSEDK Unambiguous 1 1 32189394 CID 0 7304 0 0000 1 a 0 0 000124 2 7 SLQDIIAILGMDELSEDK Unambiguous 1 1 32189394 CID 0 7240 0 0000 1 1 0 0 000325 2 Ready 378 398 Protein Group s 16500 16500 Protein s 26480 26480 Peptide s 28142 28142 PSM s 13472 13472 Search Input s Viewing the Results on the Peptide Confidence Page After the Proteome Discoverer application completes the search open the results MSF file and view the decoy database search results on the Peptide Confidence page This page shows the relaxed and strict FDRs with their corresponding filter settings listed above them To display the Peptide Confidence page In an open report click the Peptide Confidence tab The Peptide Confidence page of your search report appears as shown in Figure 142 It filters out peptides to two predefined FDRs and sets the confidence levels for database searches Use the splitter bar to separate the two columns in the FDR Settings panes
149. 645 3 51 0 6 1 221 3 860e5 Medium Unique 0 884 1 112 37 3 50 0 1 48 7 866e5 Light Redundant 0 786 0 985 5 3 49 wy Xx C Documents and Settings ussjo prodeval2 My Documents Smoke_Test_Files SILAC Smoke Tests 081023_RR_BSA_1_1_1_2plus RAW RT 30 67 31 62 min Event Spectrum FTMS Quantified lon z 2 Mono miz 758 40454 Da MH 1515 80181 Da 750 36074 751 86204 120 z 2 z 2 QuaniD 224 janID 222 759 90722 Light Light 755 88721 100 eRT 31 17 cRT 30 96 z 2 janID 222 g i JQuaniD 222 Heavy Medium cRT 30 92 7 so RT 30 94 a g H 60 2 752 36198 Fa z 2 40 747 34440 Light oRT 31 14 cRT 30 96 746 39147 i 757 90231 747 84181 5 20 4 cRT 31 57 ptet sein sens cRT 31 13 iB Lede THNH A i 750 755 760 a mz lt am r Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s Table 22 shows what the various colors mean on the Quantification Spectrum charts in Figure 206 on page 306 and Figure 207 Table 22 The meaning of colors in the Quantification Spectrum chart Sheet 1 of 2 Color Meaning Filled blue circle Indicates the isotope pattern peaks that are used in calculating the quantification values for the different quantification channels Unfilled blue circle Indicates the isotope pattern peaks that are not used in calculating the quantification values for the different quantification channels 308 Proteome Discoverer User Guide Ther
150. 7 FHGATSIQLVGDDLTVINPK Unambiguous 1 Q27527 3 9 7 VDLGTHIDGLIATAAHTWV Unambiguous 1 1 Q9NS5B3 168 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results Figure 123 Logic operator menu File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH SSS OTISBD OOS amp B BD BY RW SequestHT 9 Mas Celegans_FT_6ITDDDT_01 01 msf x WorkflowEditor x Search Input Result Filters a PSM Ambiguity al Proteins Protein Groups Protein Group Accessions A m J we 1 GHYTEGAELVDNVLDVR Unarpeessucils 1 017921 2 M GHYTEGAELVDNVLDVR Unal than 1 017921 a 3 7 KADADLTAISNDSSLSVQAK Unan lt Less than or equal to 1 Q17698 E 4 I NLITSVSSGAGSGPAPAAAA Unar gt Greater than 1 P91913 a 5 NKLETELSTAQADLDEVIK Una 2 Greater than or equal to 1 P10567 re 6 I HTDAVAELTDQLDQLNK Unambiguous 1 1 P02566 7 7 LVAQKPINDAPAIDLHVGSK Unambiguous 1 1 017759 Figure 124 gives an example of simple filter criteria being entered in the row filter line In this example Score is set to be greater than 100 and PSMs is set to be greater than 20 Figure 124 Setting row filter criteria File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH BEBOTSDOOAH LOAD Ksmeth g Mascot AMAR ACEBRABARB Celegans_FT_6ITDDDT_01 01 msf x Loading x
151. 7 1P100691212 1 Alpha 1 acid glycoprotein 14 85 2 2 3 5 1 674 0 861 2 fe A2 A4 AS Sequence PSM Ambiguity Proteins Protein Groups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Qu 1 eYQTIEDk Unambiguous t 1 1P100691212 1 N Term Dimethyl 2H 6 13 CID 0 4829 0 0000 1 1 2 gt Unambiguous 1 1 1P100691212 1 N Term Dimethyl K8 Dim cD 0 3093 0 0000 1 1 3 e eYQTIEDk Unconsidered 1 1 1P100691212 1 N Term Dimethyl 2H 4 j CID 0 3694 0 0000 1 aH 4 eFLDVIk Unambiguous 2 1 1P100691212 1 N Term Dimethy 2H 6 13 CID 0 1858 0 0000 1 1 5 HFRTFMLAASWNGTIK Unconsidered 2 1 1P100691212 1 CID 0 6111 9 4 fe Accession Description ECoverage V Z Proteins UniquePeptides Z Peptides E PSMs Area A7 Heavy Light A7 Heavy Light Count A7 Heavy L lis 4 7 1P100823795 1 Vitamin D binding protein 8 65 1 1 5 8 6 412 0 779 1 5 5 p 1P100867199 1 KTN1 protein 6 61 1 1 9 15 2 262 gt l Quan Channel Values 7 vax C Documents and Settings ussjo prodeval2 My Documents Smoke_Test_Files SILAC Smoke Tests 081023_RR_BSA_1_1_1_2pusRAW 1 RT 28 44 2938 mn Event Spectrum FTMS Quantified lon z 2 Monom z 541 27679 Da MH 1081 54631 Da 600 500 428938 445274 x 365166 2 300 5 3 s 2 200 100 of T T 1 Light Medium Heavy Quan Channels Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Inpu
152. 70 Proteome Discoverer Daemon application batch processing with a single processing method 85 batch processing with multiple processing methods 87 connecting to remote server 76 77 connecting to server 70 creating parameter file for calling from Xcalibur data system 81 creating processing method 82 description 69 Job Queue page 75 Load Files page 217 monitoring job execution 75 output files preparing to run Proteome Discoverer Daemon 79 starting a workflow 73 217 reannotating MSF files 217 Refresh icon 73 running on local server 98 100 running on remote server 98 100 specifying sample types to be sent to 84 Start Jobs page 71 73 217 starting from Xcalibur data system 78 starting in window 70 starting on command line 97 starting workflow for batch and MudPIT processing 71 using as post acquisition method 89 See also batch processing See also MudPIT Proteome Discoverer icon 19 ProtXML files 13 PSM Ambiguity column 182 PTM analysis workflow 55 PubMed database 227 purity correction factors applying in Ratio Calculation page 277 318 358 Proteome Discoverer User Guide iTRAQ methods including 266 selecting in Quan Channels page 272 using in reporter ion quantification 308 Q QTOF libraries 129 Qual Browser 7 149 Quan Channel Values chart 295 296 Quan Channels page creating new quantification method 287 specifying label modifications for reporter ion quantification 271 specifying quantification channels for precusor ion
153. 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s Handling Missing and Extreme Values in Calculating Peptide Ratios Table 23 and Table 24 on page 319 list some of the different circumstances that can arise in calculating quantification ratios for peptides from the selected quantification values A quantification value is the intensity or area detected for a given quantification channel For reporter ion quantification a quantification channel is one of the mass or reporter tags and for precursor ion quantification it is one of the different possible labeling states of a peptide corresponding to the different heavy amino acids used in the cell cultures Intensity refers to both the intensity of the reporter peaks in reporter ion quantification and to the areas detected in precursor ion quantification When the Proteome Discoverer application detects the quantification values for the different quantification channels some of the quantification values might be missing probably because they fell below the detection limit In addition some channels might show very low or very high intensities leading to the calculation of very high or very low ratios Major changes might indicate exceptional cases which you can exclude from the calculation of the protein ratios by using the settings on the Ratio Calculation page of the Quantification Method Editor dialog box 318 Proteome Discoverer User Guide Thermo Scientific 7 Quant
154. 802 180 labeling method for fully labeled samples Vv v Incomplete 180 Labeling 02 0180 1802 180 labeling method for incompletely labeled samples Vv vY iTRAQ 4plex Method for iTRAQ 4 plex mass tags by Applied Biosystems Vv v iTRAQ 4plex Thermo Scientific Instruments Method For ITRAQ 4 plex mass tags by Applied Biosystems o Vv v iTRAQ 8plex Method for iTRAQ 8 plex mass tags by Applied Biosystems Vv v iTRAQ amp plex Thermo Scientific Instruments Method for iITRAQ 8 plex mass tags by Applied Biosystems o Vv A SILAC 2plex Arg10 Lys6 SILAC 2plex 4rg10 Lys6 Method Vv v SILAC 2plex Arg10 Lys8 SILAC 2plex Arg10 Lys8 Method Vv SILAC 2plex Ile6 SILAC 2plex Ile6 Method Vv SILAC 3plex Arg6 Lys4 Arg10 Lys8 SILAC 3plex Arg6 Lys4 Arg10 Lys8 Method Vv A SILAC 3plex Arg6 Lys6 Arg10 Lys8 SILAC 3plex Arg6 Lys6 Argi0 Lys8 Method Vv v TMT 2plex Method for 2 plex Tandem Mass Tag of Proteome Sciences Vv vY TMT 6plex Method for 6 plex Tandem Mass Tag of Proteome Sciences Vv Wadd IIIA IIIR Thermo Scientific 7 Quantification Adding a Quantification Method 2 Click Add The Create Quantification Method dialog box now appears as shown in Figure 192 Figure 192 Create Quantification Method dialog box Create Quantification Method New Method Name newMethod1 D Clone From Existing Method iTRAQ 4plex New Empty Quan Method Reporter lon Quan Method Create From Fa
155. 82 gives an example of a search for protein references that meet both of the conditions Figure 82 Specifying two conditions Custom Filter Fiterbasedon All of the following conditions Delete Reference m Contains asel 6 Click OK bd To delete conditions in filtered protein reference searches e To delete a condition in the Custom Filter dialog box select the check box to the left of the appropriate condition in the Operator column and click Delete e To delete the condition in the Reference area on the Find Proteins References page click the Clear Reference Filter Criteria icon Eal in the line below the operator Proteome Discoverer User Guide 111 4 Searching for Data Using FASTA Databases e To delete all conditions in both the Custom Filter dialog box and the Reference area on the Find Proteins References page click the Clear All Filter Criteria icon Eal in the box to the left of the filters Compiling a FASTA Database You can extract information from an existing FASTA file and place it into a new FASTA file replace an existing FASTA file or append it to an existing FASTA file Then you must compile the new or changed FASTA file to make it available in the Proteome Discoverer application To compile a FASTA database Choose Tools gt FASTA Database Utilities In the FASTA Database Utilities dialog box click the Compile FASTA Database tab The Comp
156. 9 5 539 87262 1078 73796 2 8 7 Unambiguous T s PQD Ms2 8 6 188 586 59283 2343 34951 4 9 Selected TMS PQD MS2 10 10 180 642 35065 1925 03739 3 10 J7 Unambiguous FIMS HCD MS2 15 300 559 84418 1118 68108 2 u J Seleted FIMS HCD MS2 47 300 573 86017 1146 71306 2 12 Selected IMS PQD MS2 10 15 300 559 84418 1118 68108 2 13 J Selected TT S PQD MS2 10 7 300 573 86017 1146 71306 2 14 I Selected FIMS HCD MS2 3 62 300 467 30148 933 59569 2 15 J Selected TT S PQD MS2 8 62 300 467 30148 933 59569 2 16 Unconsidered FIMS HCD MS2 5 65 300 459 79709 918 58690 2 17 M Seleted TT S PQD MS2 10 65 300 459 79709 918 58690 2 18 J7 Unambiguous FIMS HCD MS2 3 23 300 477 76236 954 51744 2 Selected FIMS HCD MS2 6 25 300 451 79852 902 58977 2 Unconsidered FIMS HCD MS2 5 54 300 509 31293 1017 61858 2 Selected TMS PQD MS2 4 23 300 477 76236 954 51744 2 I Selected I S PQD MS2 7 25 300 451 79852 902 58977 2 J7 Unambiguous FIMS HCD MS2 3 51 300 666 86005 1332 71282 rd Unconsidered FIMS HCD MS2 2 69 300 477 76410 954 52092 2 Unambiguous I S PQD MS2 8 51 300 666 86005 1332 71282 2 zarn 2 za nan ar aen ara naan l 292 1120 Protein Group s 2266 2266 Protein s 3286 3286 Peptide s 7721 7721 PSM s 1541 1541 Search Input s Proteins Containing Peptides with Sequences Not Belonging to a Master Protein Thermo Scientific Because the Proteome Discoverer application considers for inclusion in the protein grouping process
157. AM WF_LTQ_Orbitrap_SEQUEST_vs_Mascot_Search Pl Workflow for Processing LTQ Orbitrap raw files with Sequest and Mascot 10 02 2012 06 50 AM WF_LTQ_Orbitrap_Sequest_SILAC_2plex_ Argi0 Lys6 Workflow for quantifying SILAC Arg10 Lys 6 duplex labeled samples using Se 10 02 2012 06 50 AM WF_LTQ_Orbitrap_Sequest_Precursor_Ions_Area Detector Workflow for reporting the peptide and protein areas 10 02 2012 06 50 AM WF_LTQ_Orbitrap_Sequest_HCD_ReporterQuantitation Workflow for processing raw files with HCD spectra for quantitation and all spec 10 02 2012 06 50 AM WF_LTQ_Orbitrap_Sequest StaticMods _ Dimethyl SLAC Workflow for quantifying Dimethyl SILAC Triplex labelled samples using Sequest 10 02 2012 06 50 AM WF_LTQ_Orbitrap_SEQUEST_ws_Mascot_Search Phospho Workflow for processing LTQ Orbitrap raw files with Sequest and Mascot 10 02 2012 06 50 AM WF_LTQ_Orbitrap_SEQUEST_vs_Mascot_Search_Annotati Workflow for processing LTQ Orbitrap raw files with Sequest and Mascot 10 02 2012 06 50 AM WF_LTQ_Orbitrap_ETD_Sequest_Mascot_new Workflow forprocessing LTQ Orbitrap rawfiles withETD and CID spectra with 10 02 2012 06 50 AM WF_LTQ_Orbitrap_Annotation Workflow for processing LTQ Orbitrap raw files with Sequest and Mascot 09 25 2012 04 54 AM Mascot Quan 3 Click the Pen icon P and type the new name or the new description Importing Raw Data Files in Other Formats into a Workflow You can import raw data files that were saved as MGF MZDATA MZX
158. C drive e NTFS format Software e Microsoft Windows XP 32 64 Professional English version with latest service pack installed e Microsoft Windows 7 32 64 Professional English version Mascot Server e Mascot Server 2 1 Mascot servers running version 2 1 should be usable but retrieving the result files protein sequences from the servers can be a lengthy process because you can only retrieve the protein sequences one at a time Mascot servers running version 2 1 should have all available updates patches or both from Matrix Science installed In particular you must install a patch that enables MIME format for the result files otherwise the Proteome Discoverer application cannot receive the search results from the Mascot server e Mascot Server 2 2 Proteome Discoverer 1 4 does not support error tolerant searches e Mascot Server 2 3 Proteome Discoverer 1 4 does not support error tolerant searches Percolator based scoring or searches against multiple sequence databases Note Ensure that port 28199 is not blocked by firewalls xii Proteome Discoverer User Guide Thermo Scientific Preface Note Ensure that the Windows operating system first has the latest Microsoft NET Framework and Windows updates installed before installing the Proteome Discoverer application Special Notices Make sure you follow the precautionary statements presented in this guide Special notices appear in boxes
159. Compact icon Display Temporary option File Search Report Quantification Processing Workflow Editor Administrati n Tools Window Helo POE SEBS T SBRBRORH AIS BD KR seweth Gl Mascot JAAR KRG AZSBRARBARE 4 gt a db Add 2 Remove Cancel Refresh Compact Display Temporary Name File Size kB Sequences Residues Status LastModified gt c_elegans_100_021411 PWD 11590 22448 9603455 Available 05 19 2011 bovinefasta 670 1057 242627 Available 06 22 2006 6916 4939 1563388 Available 06 22 2006 yeastSprotmixfasta 3 5 2307 Available 04 16 2006 uniprot_sprot_2011_05fasta 243220 444175 186939477 Available 04 12 2011 102 Proteome Discoverer User Guide Thermo Scientific FASTA Files View Parameters 4 Searching for Data Using FASTA Databases Table 3 describes the options and columns in the FASTA files view in the Proteome Discoverer application Table 3 Options and columns in the FASTA files view Parameter ap Add Description Activates the Open dialog box so you can choose the FASTA database to import a Remove Cancel Deletes a FASTA database from the FASTA files view Cancels the addition or removal of a FASTA file Refresh Redisplays the view on the screen Compact Releases the storage space previously occupied by proteins that were imported from FASTA files and inserted during a Mascot search but subsequently deleted C Di
160. DR for determining peptide confidence Peptide Score Uses this filter to calculate the FDR for determining peptide confidence e Mascot Default Mascot Significance Threshold Uses this filter to calculate the FDR for determining peptide confidence Peptide Score Uses this filter to calculate the FDR for determining peptide confidence 3 Click Set Filter Type to apply the option that you selected in the Filter list to the settings in the Modest Confidence Filter Settings and the High Confidence Filter Settings panes Changing the Target Rate and Filter Settings Thermo Scientific You can change the filter settings on the Peptide Confidence page by changing the target rate or changing the filter settings If you change the target rate or the filter settings the application finds the actual relaxed FDR the strict FDR or both that come the closest to your target rate It displays this number under Actual Relaxed False Discovery Rate or Actual Strict False Discovery Rate It also displays the number of peptides and decoy peptides that pass the filters set in the Filter Settings area and changes the filter settings in the Filter Settings area Whether you change the target rate or the filter settings the Proteome Discoverer application updates the peptide confidence indicators in the MSF report As an example Figure 144 shows the results of entering a new target rate of 0 030 in the Target box of the Actual Relaxed Fa
161. Daemon from the Xcalibur data system 81 creating workflow for multiple raw files 53 creating workflow for processing 71 72 description 9 72 monitoring job execution in Discoverer Daemon 75 processing samples in Discoverer Daemon 69 74 97 Start Jobs page in Discoverer Daemon 72 73 using processing method 93 using Run Sequence dialog box 96 when to use to search for sample fractions 53 multiconsensus reports calculating and displaying protein ratios in mixed mode 330 calculating and displaying protein ratios in reports treated as replicates 326 328 calculating and displaying protein ratios in reports treated as treatments 326 328 calculating variability in reports treated as replicates 325 calculating variability in reports treated as treatments 923 setting up in Quantification Method Editor dialog box 328 treated as replicates 324 treated as treatments 324 when to use to search for sample fractions 53 Multidimensional Protein Identification Technology See MudPIT multiple MSF files 282 multiple searches 69 MZDATA files importing into Workflow Editor 65 input file type 12 29 44 output file type 13 66 MZML files input file type 12 29 44 65 output file type 13 66 Thermo Scientific MZXML files input file type 12 29 44 65 output file type 13 66 N terminus 39 40 Name column 103 131 National Center for Biotechnology Information NCBI 2 204 223 339 National Institute of Standards and Technology NIST 129
162. E E A A LEE E A A eee eee ee 0 0 0 1 0 2 03 0 4 05 06 07 08 0 9 1 0 Time min g Pause GB Resume j Abort amp Remove Refresh Open Report q o Relative Abundance 3 Z Li pitiriitirs 1 siili L iliris Job Queue Batchsample1 RA a w t p In this example the Proteome Discoverer application processes all three raw data files and places them in the directory that you set for the Discoverer Daemon application on the computer running the Proteome Discoverer application as shown in Figure 67 and Figure 68 92 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 67 Completed data processing Thermo Proteome Discoverer Figure 68 Storing the data in the Public Files directory C Discoverer PublicFiles DiscovererDa File Edit View Favorites Tools Help File and Folder Tasks a i 1 357 KB Te Mass Spec Format 9 10 2009 2 18 PM 22 417 KB RAW File 9 10 2009 2 17 PM Make Fold ee ae m 1KB XML Document 9 10 2009 2 17 PM Web EZ Share this Folder Other Places SpectrumFiles My Documents My Computer amp My Network Places Details Thermo Scientific Proteome Discoverer User Guide 93 3 Using the Proteome Discoverer Daemon Utility Running the Proteome
163. FASTA Databases These are the most important FASTA databases that the Proteome Discoverer application uses e NCBI e MSIPI e IPI e UniRef100 e SwissProt and TrEMBL e MSDB Follow the links given for each database if you would like to download the database and save it to your local machine Some databases are more time consuming to load than others NCBI NCBI is a non redundant database compiled by the NCBI National Center for Biotechnology Information as a protein database for Blast searches It contains nonidentical sequences from GenBank CDS translations Protein Data Bank PDB SwissProt Protein Information Resource PIR and Protein Research Foundation PRF http blast ncbi nlm nih gov Blast cgi ftp ftp ncbi nih gov blast db FASTA nr gz Thermo Scientific Proteome Discoverer User Guide 339 A FASTA Reference FASTA Databases A typical NCBI title line follows gt gi 70561 pir MYHO myoglobin horse_i 418678 pir MYHOZ myoglobin common zebra tentative sequence MASS 16950 FASTA ID e Accession gi70561 e Description myoglobin horse_i MSIPI MSIPI is a database derived from IPI that contains additional information about cSNPs N terminal peptides and known variants in a format suitable for mass spectrometry search engines MSIPI is produced by the Max Planck Institute for Biochemistry at Martinsried and the University of Southern Denmark It is distributed by the European Bioinformatics Institut
164. FILES 364 E ABRF_4FILES_2 7 __ amp ABRF_4FILES_3 7 E ABRF_4FILES_4 7 9 ABRF_4FILES_5 7 O ABRF_4FILES_6 7 l E ABRF_4FILES_7 7 E ABRF_4FILES_8 7 ABRF_4FILES_9 7 9 ABRF_AvsB_92_02 7 Aggregate _Elite_ProteinCenter prot 1987 E Blanken _UAPvsAMIst_ROC_Ohr 212 Book 257 E BRAHMS_ROC_4hrNEGVSHAM 124 BRAHMS_ROC_8hrNEGVSHAM 133 E9 BRAHMS_ROC_ShrNEGVSHAM_2 133 Brazil_QE2_060512 952 _ amp brazil_Test1 184 bsa_stdTOPS_ 02 07 prot 102 ecoli_A_01_neu prot 1353 Proteome Discoverer protXML Reference database NCBInr_CElegans fasta Trypsin Full is program Thermo Discoverer Analysis 1 4 0 175 Analysis time 2012 10 10T 15 38 59 9607163 07 00 E Properties Name Celegans_FT_6ITDDDT_01_01 msf_annotation prot Full path Incoming gt Celegans_FT_6ITDDDT_01_01 msf_annotation prot Created 2012 10 11 00 39 No No mappings mappings E Import sun Imported file Records 32 Proteins 32 Unique proteins Rejected records 0 Unique rejected records Unmatched peptides 0 Warnings 220 Proteome Discoverer User Guide Internet Protected Mode Off Thermo Scientific 6 Protein Annotation Accessing ProteinCards ProteinCenter Page Parameters Table 10 lists the parameters on the ProteinCenter page of the Options dialog box Table 10 ProteinCenter page parameters Command or Option Description Upload URL Specifies
165. Filter Add a Filter Peptide Grouping Enabled Settings Protein Grouping Enabled Settings Ready 602 602 Protein Group s 761 2496 Protein s 1228 9557 Peptide s 1456 11499 PSM s 4766 4766 Search Input s Set the options pertaining to the selected filter in the Filter or Grouping Settings area For example in Figure 113 you can set the Show Peptide Groups option and the Group Peptides By option If it is not already selected select the check box in the Active column it is selected by default To remove a filter before you apply it click pg To update the search results click in the Filter and Grouping Set area Note The Proteome Discoverer application might take several seconds to display the filtered data Filtering Peptides by Rank From the acquired MS MS spectra search engines like Sequest HT or Mascot create a list of poss spec spec The and Thermo Scientific ible peptides whose masses match the measured mass of the precursor ions of the MS MS trum and whose fragmentation patterns match the peaks detected in the MS MS trum The better the match the better the score of every peptide candidate considered Proteome Discoverer application ranks all considered peptide candidates by their scores reports a user specified number of peptide candidates per spectrum The default is Proteome Discoverer User Guide 157 5 Filtering Data Filtering the Search Results usu
166. Filters page to Sequence and not to Mass and Sequence This way the peptide groups created are similar to the protein groups created which are always based on peptide sequences Consider the example shown in Figure 135 where 10 different PSMs are identified for search input 3 The four PSMs ranked 1 through 4 all meet the specified protein grouping criteria They are of high confidence and their A Cn values are below the threshold of 0 4 so the protein group inference algorithm considers all three PSMs for grouping It does not consider the remaining PSMs of the spectrum which are ranked 3 through 10 and are of medium confidence when creating protein groups 182 Proteome Discoverer User Guide Thermo Scientific Figure 135 PSMs shown for search input File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OM 8 G B y o n OD Pasenumberl D a _ GK sequestT g Mascot i A ME a 5 Filtering Data Grouping Proteins Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x l Sequest TMT 6 plex Benchmark 1 4 0 175 msf x 4 E _ Result Filters Peptide Confidence Search Summary Quantification Summary Mass Analyzer Activation Type MS Order Identified Peptides Isolation Interference Ion Inject Time ms Precursor m z Da Precursor MH Da Precursor Charge Selected TT S PQD MS2 4 60 41 620 37958 1239 75188 2
167. For the light label the natural isotope dimethyl group is C Hg For the medium label the dimethyl group is C 7H H which is 4 Da more massive For the heavy label the dimethyl group is C H which is an additional 4 Da more massive You can use the dimethylation 3plex method to compare up to three samples You cannot apply labels to the C terminus nor to arginine 180 Labeling Method The 180 labeling method introduces 2 or 4 Da mass labels through the enzyme catalyzed exchange reaction of C terminal oxygen atoms with 180 Creating a Workflow for Precursor lon Quantification 246 To use a precursor ion quantification method you must use a workflow that includes the Precursor Ions Quantifier node To create a workflow for precursor ion quantification Note This procedure uses a SILAC 2plex example 1 Choose Workflow Editor gt New Workflow For instructions on creating a workflow with the Workflow Editor see Starting a New Search by Using the Workflow Editor on page 42 2 In the Workflow Editor drag the Spectrum Files node to the workspace Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Precursor lon Quantification 3 If you selected the Spectrum Files node as your input do the following a Drag the Spectrum Selector node and the Event Detector node to the workspace b Connect the Spectrum Selector node and the Event Detector node to the Spectrum Files node
168. LGGELGK Unambiguous 1 1 017214 CID 0 4570 0 0000 1 1 1455 7 KEGICLPDFFPLIK Unambiguous 1 1 044743 cD 0 2579 0 0000 1 1 I GAPTSSVNQLDEATLDLLR Unconsidered 4 0 ETD 0 0633 0 0000 1 1 M TIFTTTPGSEQVR Unambiguous 2 1 P34455 CID 0 4253 0 0000 1 1 M DANGFLIDGRR Unambiguous 1 1 017622 CID 0 2986 0 0000 1 1 I TIQETPFPSQSIAEALIK Unambiguous 2 1 A7LPD7 CID 0 4751 0 0000 1 1 I SFIDRFEKPIKNGQCVDS Unconsidered 1 0 ETD 0 2851 0 0000 1 1 LLFETATLR Unambiguous 1 1 Q22235 CID 0 5249 0 0000 1 1 I TTGVVLDSGDGVTHTVPIE Unambiguous 2 1 Q6A8K1 CID 0 5520 0 0000 1 1 M GDLKVPAINVNDSVTK Unambiguous 1 1 P27604 cID 0 5837 0 0000 1 1 LSDEEFDKLVAGQEDNGKK Unconsidered 1 0 ETD 0 0362 0 0000 1 1 KEEIFEWDEIK Unambiguous 1 1 Q9TZK6 CID 0 2308 0 0000 1 1 M FSELKDDFPSLVLLPR Unambiguous 1 1 B1Q254 CID 0 5818 0 0000 1 1 xl gt Ready 682 691 Protein Group s 6624 6624 Protein s 8628 8628 Peptide s 9571 9571 PSM s 4766 4766 Search Input s Note You can change the default confidence levels to alternative values on the Peptide Confidence page 188 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates Setting Up FDRs in Search Wizards and the Workflow Editor You can set up FDRs in both the search wizards and the Workflow Editor e Setting Up FDRs in the Search Wizards e Setting Up FDRs in the Workflow Editor Setting Up FDRs in the Search Wizards You can set the strict
169. Load Filter Set dialog box Load Filter Set EA Load Default Filter Set D Load 164 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results 2 In the Load Filter Set dialog box do the following a To load the default filter set select the Load Default Filter Set option To load another filter set click the Browse button and select the file containing the filter set that you want to load You can also type the name and path of the file to load in the box next to Load b Click OK in the Load Filter Set dialog box A Loading Filter Set confirmation box appears if you have already selected other filter settings 3 If the Loading Filter Set confirmation box appears click OK 4 If you are loading a filter set on the Results Filter page in an open MSF file click Apply Ifyou are loading a filter set on the Results Filter page during report loading the Proteome Discoverer application automatically applies the filters or sets The name and path of the selected filter set appear in the Filter and Grouping Set area of the page as shown in Figure 118 Figure 118 Loaded filter set File Search Report Quantification Processing Workflow Editor Administration Tools Window Help POUL gogor onnon A amp B DW DY BR SequestHT H Mascot Celegans_FT_6ITDDDT_01 Oi msf x WorkflowEditor x AA OK AB SRBPanre Proteins Peptides Search Input R
170. M Validator node description 15 parameters 191 setting up false discovery rates in Workflow Editor 190 target rate 197 198 Proteome Discoverer User Guide 361 Index U temporary FASTA files 105 third party nodes 46 time of flight mass spectrometer specifying in Reporter Ions Quantifier node 256 Tmap database 226 TMT 10plex quantification method as default 250 266 271 reporter ion masses 250 TMT 2plex quantification method as default 7 250 266 271 reporter ion masses 250 TMT 6plex quantification method as default 7 250 266 271 reporter ion masses 250 TMT quantification creating a workflow for 253 default methods available in 250 266 271 description 7 249 isotopic distribution values 308 on PQD and HC scans 256 performing 249 performing on HCD and CID scans 257 Reporter Ions Quantifier node 7 specifying label modifications 267 See also reporter ion quantification TMTe 6plex quantification method as default 250 266 271 reporter ion masses 250 transcription regulator activity GO Slim category 234 translation regulator activity GO Slim category 234 transport GO Slim category 239 transporter activity GO Slim category 234 treatments calculating protein ratios in multiconsensus reports 326 328 definition 324 in mixed mode 330 treating quantification results as 327 variability 324 variability in multiconsensus reports 325 TrEMBL database 340 342 triple quadrupole mass spectrometer 256 TXT
171. ML or MZML files into a workflow To import raw data as MGF MZDATA MZXML or MZML files 1 In the Workflow Editor drag the Spectrum Files node to the workspace pane and select it 2 In the Parameters pane click the Browse button next to the File Name s box Thermo Scientific Proteome Discoverer User Guide 65 2 Getting Started Starting a New Search by Using the Workflow Editor N WB WN A WH In the Select Analysis File s dialog box click Add Files Browse to the location of the MGF MZDATA MZXML or MZML file and select it Click Open In the Select Analysis File s dialog box click OK Continue with constructing the workflow according to the instructions in Creating a Search Workflow on page 44 Saving a Workflow as an XML Template To avoid losing any changes you might want to save a workflow file as an XML template if you intend to transfer it to another computer another software version or another person 1 bd 1 2 Exporting Spectra 66 To save a search workflow as an XML template Choose Workflow Editor gt Export Workflow to XML In the Export Workflow Template dialog box browse to the location where you would like to save the template type a file name in the File Name box and click Save By using the Spectrum Exporter node in your workflow you can export spectra in the following standard formats Data Archive DTA Places the exported spectra into DTA zip f
172. Methods License Management R Licenses Configuration a 3 4 Workflow Nodes amp 3 Annotation 53 Mascot Sg MSPepSearch S53 SEQUEST 53 Sequest HT amp SpectraST Server Settings amp FASTA Indexes Ready 80 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Creating a Parameter File That the Discoverer Daemon Application Uses In the Proteome Discoverer Daemon application you can create a parameter file that you can use to call the application from the Xcalibur data system The application automatically translates the options that you set in the Proteome Discoverer Daemon application interface and in the workflow used for the search into text commands in the parameter file To create a parameter file that calls the Discoverer Daemon application 1 Set up the search according to the instructions in Starting a Workflow on page 71 However you do not have to have files loaded to create a parameter file 2 Click the Export Parameter File tab shown in Figure 54 on the Start Jobs page Figure 54 Export Parameter File page Discoverer Daemon keka Start Jobs Configuration Job Queue Spectrum Files Workflow 5 Batch processing MudPIT BSA_ETD_CID_HIGH_HIGH pico_02 2 f Export Parameter File laadis PP Server Output Directory local connection
173. Only Unique Peptides option includes in the quantification peptides that do not occur in other proteins The Proteins Groups for Peptide Uniqueness option defines peptide uniqueness on the basis of protein groups rather than individual proteins 242 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Precursor lon Quantification When it determines peptide uniqueness for classification in the PSM Ambiguity column on the Peptides page the application only considers the PSMs that it considered when creating the protein groups if you select the Use Only Unique Peptides option For example it does not use for quantification a PSM of low confidence that it did not use to create the protein groups Performing Precursor lon Quantification In precursor ion quantification also called isotopically labeled quantification protein abundance is determined from the relative MS signal intensities of an isotopically labeled sample and an unlabeled control sample Stable isotope labeling by amino acid in cell culture SILAC is a proteomics identification and quantification technique that uses in vivo metabolic labeling to detect differences in the abundance of proteins in multiple samples It is a type of isotopically labeled quantification which uses stable nonradioactive heavy isotopes as labels You can also introduce the stable isotopes by chemical labeling at the protein or peptide level with the isotopomeric tags for example
174. PSMs with a A Cn larger than this value The minimum value is 0 0 and the maximum value is 1 0 Filtering Results by the Original Rank Assigned by the Search Engine If you apply PSM level result filters the Proteome Discoverer application dynamically recalculates the displayed ranks delta scores and A Cn values However you can also view the original rank assigned by the search engine for all PSMs and peptide groups by displaying the Search Engine Rank column on the Peptides page In addition you can filter by this rank For example you might find this feature helpful when you know that your raw data has a true mass accuracy below 5 ppm If you search this data with a precursor tolerance of 5 ppm and validate it by calculating FDRs you obtain false positive matches within this mass deviation tolerance You could find some of these incorrect matches if you searched the data with a larger precursor tolerance such as 50 ppm This step increases the chance of replacing incorrect matches with a mass deviation below 5 ppm by incorrect matches with a higher mass deviation When you review the results you can set a mass deviation filter of more than 5 ppm to remove all matches that have a mass deviation outside the true mass accuracy You can now find many of the remaining incorrect matches They have a Search Engine Rank worse than rank 1 because they were initially replaced by incorrect matches with a larger mass deviation Using Filter Sets
175. PTIK 390 5 1 1P100790783 1 Ti Phospho S3 Phospho 0 0000 T 1 100 0 S 3 100 0 T 7 14 0 00 3 28 7 TPLSFTNPLHsDDsDsDRR 11 1 1 1P100289082 2 S11 Phospho 14 Phosph 0 0000 T 1 0 0 S 4 0 0 T 6 0 7 10 0 00 3 W 29 7 GRLtPSsPDIIVLSDNEASSPR 10 1 1 1P100103554 1 T4 Phospho S6 Phospho 0 0000 T 4 100 0 S 6 100 0 S 7 05 0 00 3 E 30 I APEPHVEEDDDDELDK ia 5 1 1P100791712 1 0 0000 7 01 0 00 3 31 7 TPLSFTINPLHsDDsDSDR 37 1 1 1P100289082 2 S11 Phospho S14 Phosph 0 0000 T 1 0 0 S 4 0 0 T 6 0 6 99 0 00 3 32 7 RPDYAPMEssDEEDEEFQFK 9 1 1 1P100022790 1 S9 Phospho S10 Phospho 0 0000 Y 4 0 4 S 9 99 8 S 10 6 99 0 00 3 H 33 gt DSHssEEDEASSQTDLSQTIK 20 5 1 1P100012280 3 SA Phospho S5 Phospho 0 0000 S 2 52 1 S 4 52 1 S 5 6 98 0 00 2 cpm vamenn ne 2 x meena See ere Gren Ie RTE nH Ready 174 180 Protein Group s 5541 5541 Protein s 3919 3919 Peptide s 17835 17835 PSM s 3363 3363 Search Input s 3 In the Min Probability box select the probability that a modification will be found on the specified amino acid You can select values between 1 and 100 The default is 75 4 In the Target Acids box type the symbol or name of the amino acid You can use any lowercase or uppercase letters Thermo Scientific Proteome Discoverer User Guide 173 5 Filtering Data Grouping Proteins If you select a target amino acid all rows having a site pr
176. Processes Page e Diseases Page e External Links Page 4 Click OK to close the Protein Identification Details dialog box If the entire protein is not found in ProteinCenter but a protein with the same sequence exists the ProteinCard displays a warning that the displayed information is from a protein with different accession as shown in Figure 162 If there is more than one protein with the same sequence but from different organisms an additional list box appears so that you can select the correct species Figure 162 Warning displayed for protein with different accession The accession key could not be found but the protein sequence exists for these proteins Pan troglodytes 253 AA General Keys Features Molecular Functions Cellular Components Biological Processes Diseases External Links Pan troglodytes 7 PGAM2 phosphoglycerate mutase 2 muscle Gene Details Protein Details Entrez Gene record Description PREDICTED phosphoglycerate mutase 2 muscle ProteinCard Parameters The ProteinCard page of the Protein Identification Details dialog box contains the following pages e General Page e Keys Page e Features Page e Molecular Functions Page e Cellular Components Page e Biological Processes Page e Diseases Page e External Links Page 222 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation ProteinCard Parameters General Page The General page of t
177. Proteome Discoverer application does not add the library 2 In the Proteome Discoverer application choose Administration gt Maintain Spectrum Libraries or click the Maintain Spectrum Libraries icon mi on the toolbar 3 Click Add 134 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Searching Spectrum Libraries 4 In the Select a Spectrum Library dialog box do the following a In the list box in the lower right corner of the Select a Spectrum Library dialog box select All Spectrum Library Files gz msp zip or Zip archives gz zip b Browse to the location of the spectrum library where you downloaded and unpacked the _nist tar gz file c Select the filename gz file d Click Open When you add a spectrum library file the Proteome Discoverer application takes the following steps Extracts the archive file e Extracts spectra for visualization During library creation the job queue in the Administration view displays each step as shown in Figure 98 Figure 98 Adding a spectrum library for searching with the MSPepSearch node File Search Report Quantification Processing Workflow Editor Administration Tools Window Help SR BOVGDRDO SH LORD K sustit K Mascot ARLE AZEBTRIARE 2 Gi Pause GB Resume gf Abort BE Remove Refresh I Open Report Display Row Filter Job Queue Execution State Progress Name Spectrum Source Description Submitted a Pr
178. Rs the same filters are used to distribute the confidence indicators Peptide matches that pass the filter associated with the strict FDR are assigned a green confidence indicator peptide matches that pass the filter associated with the relaxed FDR are assigned a yellow confidence indicator and all other peptide matches receive a red indicator of low confidence Figure 137 gives an example of these confidence indicators Figure 137 Decoy search results File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BSBOTSTOOAH SAD K seqeth g Mascot AKA ACESTABPARE Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x Sequest TMT 6 plex Benchmark 1 4 0 175 msf x Celegans_FT_6ITDDDT_01_decoy msf x Tada Search Input Result Filters Peptide Confidence Search Summary Sequence PSM Ambiguity Proteins ProteinGroups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank SVEEILASLEK Unambiguous 1 1 Q19278 CID 0 6429 0 0000 T 1 M EIVYSIPANVVIKPGK Unambiguous 2 1 Q19289 2 CID 0 5848 0 0000 1 1 429 7 AYLLEELFNK Unambiguous 1 1 044502 cD 0 5580 0 0000 1 1 I QVSNNITEPVATPTSENLDA Unconsidered 0 ETD 0 1385 2 2 H 1431 NQVTGEYGAVPATAR Unambiguous 2 1 P34455 CID 0 3857 0 0000 1 1 1432 DPVLEELGNLK Unambiguous 3 1 Q9N5T2 CID 0 4664 0 0000 1 1 1
179. S MS processing using both the ETD and CID fragmentation methods for better confidence The example searches a FASTA database to determine how the worm s proteins are expressed Click the button below to view the demonstration Thermo Scientific Proteome Discoverer User Guide 51 2 Getting Started Starting a New Search by Using the Workflow Editor 52 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Creating a Search Workflow for Multiple Raw Files from the Same Sample Multidimensional Protein Identification Technology MudPIT experiments investigate complex proteomes by applying multidimensional chromatography to the samples before acquisition in the mass spectrometer Typically this process results in several dozen or even a few hundred fractions that are separately analyzed by LC MS resulting in one raw file per sample fraction Analyzing gel slices or performing in depth follow up acquisitions also results in multiple fractions Because all these fractions belong to the same sample the Proteome Discoverer application can process all raw files from these fractions as one contiguous input file and generates only one result file You have two ways to search for sample fractions e Search the sample fractions one at a time and open them in a multiconsensus report This method is appropriate for searching multiple samples When you open a multiconsensus report f
180. SM Validator Percolator lih Target Decoy PSM Validator E PTM Analysis ifs phosphoRS3 0 Quantification D Precursor Ions Area Detector m m Precursor Ions Quantifier 2 Reporter Ions Quantifier Data Export u Spectrum Exporter Annotation Annotation pee Ready 192 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 9 5 Filtering Data Calculating False Discovery Rates Click the Percolator node In the Maximum Delta Cn box in the parameters list specify the A Cn value For information on this parameter see Filtering Peptides by the Delta Cn Value on page 161 In the Target FDR Strict box set the target FDR for high confidence peptide hits In the Target FDR Relaxed box set the target FDR for medium confidence peptide hits In the Validation Based On box select either q Value or PEP posterior error probability to assign to the target and decoy PSMs For more information on these options refer to the Help Choose Workflow Editor gt Start Workflow or click the Start Workflow icon When you open results processed with the Percolator node each PSM and peptide group has two additional scores on the Peptides page a q value score and a posterior error probability PEP value as shown in Figure 141 Proteome Discoverer User Guide 193 5 Filtering Data Calculating False Discovery R
181. SMVAAGEKSGHLEKVLNR Unambiguous 1 1 P31743 PQD 0 1518 0 0000 1 10 J AAALLQHWATLEVGYRIPRMK Unambiguous 1 1 QawiT2 PQD 0 2588 0 0000 1 1 11 M ALLLILVFLESDCFLPNR Unambiguous 1 1 Q9wz35 PQD 0 1693 0 0000 i 1 12 I AAVGIEIPTPFPQAVNRR Unambiguous 1 1 Q9Z726 PQD 0 1811 0 0000 1 1 13 J IPKDSTTLQQWDEFTR Selected 1 1 Q922G7 PQD 0 1462 0 0000 1 1 14 M LLHEEPMTKLDLSTIR Unambiguous 1 1 013682 HCD 0 2174 0 0000 1 1 15 J LAGYKDALAQHGIALNK Selected 1 1 P24242 PQD 0 1080 0 0000 1 1 16 LGLGINAGHGLNYINRK Unambiguous 1 1 Q39UGO PQD 0 2880 0 0000 1 1 17 I VLHRLFGKWHEGLYR Selected 1 1 Q9BZF2 PQD 0 1165 0 0000 1 1 18 I FEGAGVAAIIYTDDR Unambiguous 5 1 Q2YQY9 PQD 0 2308 0 0000 1 19 M NGIVMLGSWIERGDLVGKL Unambiauous 1 O7YIXB POD 0 1967 0 0000 1 1 ate Quantification Spectrum ax C Program Files Proteome Discoverer source files Smoke_Test_Files myo_S8plex_hed_pqd raw 3408 RT 61 73 min FTMS HCD Precursor z 3 Mono m z 638 01855 Ds MH 1912 04111 Da Integrstion Most Confident Centroid Integration tolerance 20 ppm ee 114 11014 118 11089 6000 4 116 11 z 1 pb 117 11375 113 10564 zt 5000 4 z 1 121 12076 E 119 11432 E 115 10714 z1 amp 4000 4 zt 2 g 30004 2000 4 1000 4 o T T T T T T 112 114 116 118 120 122 mz E a m Ready 304 761 Protein Group s 1497 1497 Protein s 1241 1241 Peptide s 3827 3827 PSM s 1975 1975 Search Input s The Quantification Spectrum chart includes th
182. Save the workflow as a new workflow template a Choose Workflow Editor gt Save As Template b In the Save Processing Workflow Template dialog box type the name of the template in the Template Name box c Give a brief description of the template in the Template Description box d Click Save This newly created workflow template is now available in Discoverer Daemon 3 To start Discoverer Daemon follow the instructions in Starting the Proteome Discoverer Daemon Application in a Window on page 70 To select the server follow the instructions in Selecting the Server on page 70 Click the Start Jobs tab if it is not already selected Click the Load Files tab if it is not already selected N A W A Click Add 8 In the Open dialog box select Result Files msf from the list next to File Name 9 Browse to the MSF file that you want to save the new annotations in or type the name of the file in the File Name box and click Open 10 Repeat step 8 to add the names of multiple MSF files to reannotate 11 In the Spectrum Files area click Batch Processing 12 From the menu in the Workflow box select the reannotation workflow template that you saved in the Workflow Editor 13 Start the batch processing e Ifyou are connected to an instance of the Proteome Discoverer application running on the same computer click Start in Discoverer Daemon e Ifyou are connected to an instance of the Proteome Discover
183. Scientific To find a protein sequence or reference To filter a protein reference search To refine a filtered protein reference search To delete conditions in filtered protein reference searches To find a protein sequence or reference Choose Tools gt FASTA Database Utilities In the FASTA Database Utilities dialog box click the Find Protein References tab The Find Protein References page appears as shown in Figure 77 Proteome Discoverer User Guide 107 4 Searching for Data Using FASTA Databases Figure 77 Find Protein References page of the FASTA Database Utilities dialog box FASTA Database Utilities j Add Protein References Compile FASTA Database Find Protein References FASTA Database C Program Files Proteome Disboverer source files FASTA_Files bovine fasta l m Search for Searchin References Sequences Maximum number of matches reported 100 Start Search Click the Browse button next to the FASTA Database box to locate the FASTA file of interest In the Please Select a FASTA Database dialog box select the FASTA file and click Open In the Search For box of the Find Protein References page type an amino acid sequence or a protein reference search string In the Search In area specify whether the Proteome Discoverer application should search for the search string in the protein references or sequences e References Searches for the search string in the
184. See also Reporter Ions Quantifier node See also TMT quantification Reporter Ions Quantifier node creating a workflow 253 nodes not used with 253 performing TMT quantification on HCD and CID scans 257 reporter ion quantification 55 setting parameters 255 setting up quantification method 264 summarizing node settings 292 using to access Edit Quantification Method command 289 using to access Quantification menu 242 reproduction 239 Residues column 103 Restore FASTA indexes confirmation box 127 result filters 154 Result Filters page displaying 154 filtering data 153 filtering data in MSF file 154 filtering results 155 retrieving annotations from Pfam database 206 reverse dot score 139 ribosome GO Slim category 237 RNA 223 234 237 RNA binding GO Slim category 234 row filter menu 169 row filters clearing all 169 deleting individual 169 filtering PSMs and Peptides for site localization scores from phosphoRS 172 filtering search results 155 Run Sequence dialog box 86 90 96 Run Sequence icon 86 90 S sample fractions 53 sample types 84 Save a Parameter File dialog box 81 Save As Template icon 50 Save Filter Set dialog box 164 Save Processing Workflow Template dialog box 41 50 Scan Event Filter node used for Mascot or SEQUEST analysis 50 Scan Extraction Parameters page 33 Search Description page 40 search engine rank 163 search engines available 3 360 Proteome Discoverer User Guide Search Input page displaying filtered
185. The FASTA Indexes Options dialog box appears as shown in Figure 94 Figure 94 FASTA Indexes Options dialog box FASTA Indexes Options x FASTA index directory C ProgramData Thermo Discoverer Demo 1 4 FastaData New directory Maximum number of FASTA indexes 30 New maximum number of FASTA indexes 30 Note Another way to access these options is to choose Administration gt Configuration and click FASTA Indexes in the Server Settings area The FASTA Indexes Options dialog box contains two read only parameters e The FASTA Index Directory box displays the name of the current directory where the FASTA indexes are saved e The Maximum Number of FASTA Indexes box displays the current maximum number of FASTA indexes allowed 2 In the New Directory box browse to the directory where you want to store the FASTA indexes You can change the directory only if the server runs on the local machine 3 In the New Maximum Number of FASTA Indexes box type the new maximum number of FASTA indexes allowed 4 Click OK 5 In the FASTA index settings confirmation box click OK 128 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Searching Spectrum Libraries After you confirm the changes the Proteome Discoverer application saves them but the changes are only executed the next time that the server starts You can undo the changes made since the last time that the server started and before the n
186. The experimental results are down regulated e Blue The experimental results are up regulated e Red The experimental results exceed the setting in the Maximum Allowed Fold Change box These results are not used in calculations unless you select the Use Ratios Above Maximum Allowed Fold Change for Quantification option Setting Peptide Parameters Used to Calculate Protein Ratios Use the Protein Quantification page of the Quantification Method Editor dialog box to set the peptide parameters for calculating protein ratios To set the peptide parameters used to calculate protein ratios 1 Click the Protein Quantification tab shown in Figure 185 This page is the same for both precursor ion and reporter ion quantification 278 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Setting Up the Quantification Method Figure 185 Protein Quantification page of the Quantification Method Editor dialog box Quantification Method Editor SILAC 2plex Arg10 Lys6 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias V Show Peptide Ratio Counts V Show Protein Ratio Variabilities Consider Proteins Groups for Peptide Uniqueness Use Only Unique Peptides D Use All Peptides 2 If you want to display the number of peptide ratios that are used to calculate a protein ratio select the Show Peptide Ratio Counts check box The r
187. Unambiguous 5 2 494711 1942750 N Term iTRAQ8plex M13 PQD 0 2166 0 0000 i 1 15 I hPGDFGADAQAAmSk Unconsidered 1 2 494711 1942750 N Term iTRAQ8plex M13 PQD 0 2166 2 2 H 16 I7 hPGNFGADAQGAmTk Unconsidered 1 1 1942750 N Term iTRAQ8plex M13 PQD 0 3970 3 3 Thermo Scientific Go to the following sections e To group the proteins in your search results and set grouping options e To display other proteins belonging to the same protein group e To turn off protein grouping To group the proteins in your search results 1 Open the MSF file 2 On the Peptides or Proteins page of the MSF file right click a protein grid cell or row to access the shortcut menu and choose Enable Protein Grouping Proteome Discoverer User Guide 175 5 Filtering Data Grouping Proteins KD To group the proteins in your search results and set grouping options 1 Open the MSF file 2 Click the Result Filters tab 3 On the Results Filters page click Settings beneath Protein Grouping Protein grouping options appear in the Filter or Grouping Settings area as shown in Figure 131 Figure 131 Protein grouping options File Search Report Quantification Processing Workflow Editor Administration Tools Window Help GE SABSOlTSGPHOOSH g Do m i K seth Kost i A RARES ZGSBRABARB Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x Sequest TMT 6 plex Benchmark 1 4 0 175 msf x 4b Proteins Peptides Search Input R
188. Used 458 4 03 ng 7 663e4 Inconsistently Labeled NotUsed 337 4 03 Ot 1 87 2 540e5 Medium Redundant Not Used 0 801 0 966 189 4 01 Ot 1 46 5 971e6 Light Redundant Not Used 0 873 0 990 i 3 92 0 224 1 123e5 Heavy Unique l Used 0 807 0 848 535 3 85 Oly Oil Quantification Spectrum Ad C Documents and Settings ussjo prodeval2 My Documents Smoke_Test_Files SILAC Smoke Tests 081023_RR_BSA_1_1_1_2plus RAW RT 27 22 28 14 min Event Spectrum FTMS Quantified lon z 2 Mono m z 841 41315 Ds MH 1681 81902 Da 60 837 89460 1 z 2 833 86949 QueniD 87 841 1288 eo nid uoua QueniD 87 QuaniD 87 oRT 27 67 rimia Light eav 120 cRT 27 69 eRT 27 66 i 838 39568 834 36938 Lo i 1a oe QusniD 87 942 41446 Medium z2 a ra 1 Area counts sec 103 o 8 1 829 27090 cRT 28 00 830 27156 cRT 27 82 831 27004 cRT 27 75 PN 3 1 20 4 832 26845 cRT 27 66 830 835 840 845 lt an Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s The x axis of the chart displays the mass to charge ratio of the isotopes and the y axis displays the area of the extracted ion chromatogram for the isotopes Filled blue circles mark the isotope pattern peaks that were used for calculating the quantification values for the different quantification channels Unfilled blue circles mark the isotope pattern peaks that were identified but not u
189. Utility Starting a Workflow Figure 47 Start Jobs page of the Proteome Discoverer Daemon application for batch processing local connection BSA_ETD_CID_HIGH_HIGH _pico_02 C BSA_ETD_CID_HIGH_HIGH_pico_02 Output Filename test filename BSA_ETD_CID_HIGH_HIGH_pico_02 TAUsers Desktop iypmyo01 RAW Desktop trypmyo02 RAW CAUsers Desktop trypmyo03 RAW Output Filename test_filename 9 Click Start to execute the job 74 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Monitoring Job Execution in the Proteome Discoverer Daemon Application Monitoring Job Execution in the Proteome Discoverer Daemon Application You can use the Job Queue page in the Proteome Discoverer Daemon application window to monitor the execution of the jobs that you submit It performs the same function as the job queue in the Proteome Discoverer interface For information about the features of the job queue in the Proteome Discoverer interface refer to the Help A progress bar displays the progress of the overall batch processing This progress bar is only visible if you have started batch jobs To monitor the job execution e Click the Job Queue tab of the Proteome Discoverer Daemon application window Figure 49 shows the completed job for batch processing and Figure 50 shows the completed job for MudPIT processing
190. Workflow using SpectraST to search spectrum libraries de ceases l i Spectrum 1 Selector Fixed Value PSM Validator For a description of the parameters available in the SpectraST node refer to the Help The spectrum library search reports the three scores shown in Table 5 The dot score and the dot bias are secondary scores and their values are not shown by default Table 5 Scores generated by the SpectraST search node Sheet 1 of 2 Score Description F value Specifies the discriminant scoring function that the Proteome Discoverer application calculates from the dot score dot bias and the normalized difference between the best and second best hit AD The application uses the f value for FDR calculation For more information on the f value see F Value l Lam Henry et al Proteomics 7 2001 655 667 Thermo Scientific Proteome Discoverer User Guide 137 4 Searching for Data Searching Spectrum Libraries Table 5 Scores generated by the SpectraST search node Sheet 2 of 2 Score Description Dot score Specifies the spectral dot product as the primary similarity score For more information on the dot score see Dot Score Dot bias score Measures how much the dot score is dominated by only a few peaks which might indicate false positive hits For more information on the dot bias see Dot Bias Score Dot Score The dot score is the primary score from the spectral library sear
191. XCor value lower than this dont ass validation Minimum value 0 0 Maximum value 1000 0 s 9571 9571 PSM s 4766 4766 Search Input s iliz Filter target setting Ready 682 691 Protein Group s 6624 6624 Protein s 8628 8628 Peptidd If you used the Percolator node in the workflow you can set thresholds for the Percolator scores to separate PSMs of high confidence medium confidence and low confidence as shown in Figure 143 Thermo Scientific Proteome Discoverer User Guide 195 5 Filtering Data Calculating False Discovery Rates Figure 143 Setting thresholds for Percolator scores File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE SB BOTDOO OAH LORD g SeuestH g Mascot ARAM AEBSABIAREB Workflow Editor helaRVD1_1_1_1 _percolator a Administration X E_coli msf x helaRVD1_1_1_1 _percolator msf X 4d Proteins Peptides Search Input Result Filters High Confidence Filter Settings Percolator 2 FDR Setti Threshold 0 05 Threshold 0 01 Set Filter Type _ Apply Filters Apply FDRs Validation based on PEP Validation based on PEP Processing Node Name Workflow Name Short Display Name r Sequest HT helaRVD1_1_1_ A2 Validation based on Determines the value validation is based on Ready 378 398 Protein Group s 16500 16500 Pr
192. _2 12564 SEQUEST 2 400 800 800 result_3 7672 SEQUEST 2 1000 8000 8000 Ready 328 Proteome Discoverer User Guide By default the Proteome Discoverer application initially treats the quantification results in the single MSF files as if they were from different treatments of a sample You can change this treatment by selecting the Treat Quan Results as Replicates option on the Input Files page The application then initially treats the quantification data as if it were replicates of the same sample when it creates and opens the multiconsensus report If you do not select the Treat Quan Results as Replicates option and click Open on the Input Files page the application creates the multiconsensus report and calculates the quantification results the quantification ratios as specified in the quantification method for each of the three single results files separately In this example all three result files are TRAQ 4plex files so the application usually calculates ratios such as 115 114 116 114 and 117 114 for each of the files Because you did not select Treat Quan Results as Replicates the application reports them as if the three files represented different treatment states of a sample Thermo Scientific 7 Quantification Calculating and Displaying Protein Ratios for Multiconsensus Reports Calculating Protein Ratios in Multiconsensus Reports Treated as Treatments In the case of treatments the Proteome Discoverer application
193. a Precursor Charge Unambiguous FIMS l MS2 s 539 87262 1078 73796 2 None F S MS2 6 586 59283 2343 34951 None FIMS l MS2 10 642 35065 1925 03739 Unambiguous MS2 s 539 87262 1078 73796 None MS2 6 586 59283 2343 34951 Unambiguous MS2 642 35065 1925 03739 None MS2 559 84418 1118 68108 None MS2 573 86017 1146 71306 None MS2 559 84418 1118 68108 Unambiguous MS2 573 86017 1146 71306 None l MS2 467 30148 933 59569 None MS2 467 30148 933 59569 None MS2 459 79709 918 58690 None MS2 459 79709 918 58690 MS2 477 76236 954 51744 MS2 451 79852 902 58977 MS2 509 31293 1017 61858 MS2 477 76236 954 51744 451 79852 902 58977 MS2 666 86005 1332 71282 MS2 477 76410 954 52092 MS2 666 86005 1332 71282 MS2 477 76410 954 52092 MS2 503 33057 1005 65386 MS2 503 33057 1005 65386 MS2 459 79135 918 57543 MS2 459 79135 918 57543 477 76266 F r F r F r F F r r F r F r F r r INNNNNNNNNNNNNNNNNNNNNNYW BN YW To filter peptides by the A Cn value 1 Open the MSF file Refer to the Help 2 Click the Result Filters tab 3 Select Peptide Delta Cn in the Peptide Filters area of the Result Filters page The Peptide Delta Cn option appears in the middle of the Result Filters page 162 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results 4 Optional In the Maximum Delta Cn box specify a A Cn threshold that will filter out all
194. a iaa 203 Entrez Gene Database Annotation ics n cue chic eee h eK Rew ee 8 d8 204 UniProt Database Annomtiott 25 354 ci0 0s gt x s5 soe ins KOs eis Re eS 204 Configuring the Proteome Discoverer Application for Protein Annotation 204 Creating a Protein Annotation Workflow 000 cece eee eee ee 206 Displaying the Annotated Protein Results o oo don edvwiaeedentbeuo 208 Displaying GO Protein Annotation Results naana ananuna 208 Displaying GO Accessions aaaea rk cee e eee eee ees 212 Displaying Protein Family Pfam Annotation Results 214 Displaying Entrez Gene Identifications n 0 0 0 c eee eee eee 214 Displaying UniProt Annotation Data scsi 4scce nee snowed nes eree 215 Reannotating MSF Files 1 5 inka hee chew ederadastea hae eekenae ny 216 Uploading Results to ProteinCenter 242514 p02enes pes asiadesaseeex 218 Acc ssing ProteinCards ss oy hee ee Rey we ORE eRe Ed eee 221 ProteinCard Pariicieis4 coves es eet aehenesieieieeaes 222 General Pag pnd a Gert he ce Reet ee Renee Reese ernie Kees 223 Keys Page pias tanp e a E EE T Seek 224 Features Pages cand cane eee cH See SOATEST EOE ditas 225 Molecular Functions Page cicescd ened dedcces dtbecen dahacivses 227 Cellular Components Page ssc45 0ivs bse cios beta tee nee ee 228 Biological Processes Fates cis Veen Need ed ee nut d ede dee 229 Diseases Page sissies tech arent ay Meaen a iie Modem gt Asay Mooring E EEEE eee dad
195. aenorhab ACTin family member act 4 Caenorhabditis elegans F01G10 1 Caenorhabditis elegans Ribosomal Protein Small subunit family member rps 3 R05G6 7 Caenorhabditis elegans Temporarily Assigned Genename family member tac H28016 1d Caenorhabditis elegans Heat ShockProtein family member hsp 60 Caenorha Tubulin Beta family member tbb 1 Caenorhabditis d T22F3 3b Caenorhabditis elegans R11A5 4d Caenorhabditis elegans W05G11 6a Caenorhabditis elegans F59B8 2 Caenorhabditis elegans TPA TPA_exp ANC 1 Caenorhabditis elegans F01F1 12a Caenorhabditis elegans Ribosomal Protein Largesubunit family member p MYOsin heavy chain structural genes family member m Ribosomal Protein Largesubunit family member mp7 Y41E3 10a Caenorhabditis elegans Vacuolar H ATPase family member vha 11 Caenorha Elongation FacTor family member eft 3 Caenorhabdit Elongation FacTor family member eft 2 Caenorhabdit K02F2 2 Caenorhabditis elegans T25C8 2 Caenorhabditis elegans C16A3 10a Caenorhabditis elegans Patterned Expression Sitefamily member pes 9 Caen 139 42 67 19 59 45 59 36 58 10 45 81 44 29 43 66 43 16 39 95 37 69 36 72 35 21 34 91 34 83 32 55 31 78 31 41 30 80 30 55 29 83 29 81 29 67 29 21 29 16 28 69 28 04 27 66 27 11 26 96 25 83 25 80 25 38 25 13 24 66 24 63 24 30 23 89 23 76 ra BWR ENR NN EWEN de REN OW Sonen EN WDe ee e
196. ails dialog box see Accessing ProteinCards on page 221 You can display this information for the following proteins Proteins on the Proteins page of the MSF file Proteins associated with identified peptides Proteins shown in the Protein Group Members view You can access the ProteinCard for each protein by double clicking its row in the MSF report or clicking its row and choosing Search Report gt Show Protein ID Details and then clicking the ProteinCard tab of the Protein Identification Details dialog box The ProteinCard itself is split into separate tabs representing different aspects of that protein General Keys Features Molecular Functions Cellular Components Biological Processes Diseases and External Links You can display a ProteinCard for every identified protein whose accession is tracked in ProteinCenter For information on ProteinCard see Accessing ProteinCards on page 221 and ProteinCard Parameters on page 222 You can also upload protein results directly from the Proteome Discoverer application to ProteinCenter For information see Uploading Results to ProteinCenter on page 218 Gene Ontology GO Annotation The Gene Ontology GO database is a collaborative effort incorporating community input from database and genome annotation groups to address the need for consistent descriptions of gene products in different databases The GO project has developed three structured controlled v
197. ails dialog box 221 Protein Identification Details view displaying ProteinCard 202 ProteinCard page 202 PTM site localization scores 55 protein quantification 309 311 Protein Quantification page 278 protein ratios calculating 313 calculating for multiconsensus reports 326 calculating from peptide ratios 320 defining peptide uniqueness 279 displaying variability 279 setting up 275 setting up peptide parameters used in 278 variability used to calculate 324 protein references 106 107 protein sequences 106 107 protein uniqueness 242 ProteinCard accessing 202 221 accessing data in ProteinCenter 221 Biological Processes page 222 229 Cellular Components page 221 228 Diseases page 222 231 External Links page 222 231 Features page 221 225 General page 221 223 Keys page 221 224 Molecular Functions page 221 227 Pfam identification number 203 tabs in 202 ProteinCenter accessing annotation data in 208 221 description 2 201 retrieving annotations from GO database 206 retrieving information from Entrez gene database 202 retrieving information from GO database 202 retrieving information from Pfam database 202 retrieving information from UniProt database 202 uploading search results to 218 Thermo Scientific Index P Web server address 205 ProteinCenter page 221 proteins Unique Peptides column 184 accession keys 224 annotation See protein annotation biological processes 229 cellular components 228 determining which to i
198. alling the Proteome Discoverer application To create and save a filter set 1 For filters select the appropriate protein and peptide filters as described in To filter your search results with peptide filters on page 156 and To filter your search results with protein filters on page 155 and click Apply 2 In the Filter and Grouping Set area click i The Save Filter Set dialog box appears as shown in Figure 116 Figure 116 Save Filter Set dialog box Save Filter Set Save As Default Filter Set Save As C ProgramData Thermo Discoverer Demo 1 4 FilterSet 3 In the Save Filter Set dialog box do one of the following e To save the filter set or set of protein grouping settings as the default filter set select the Save As Default Filter Set option The Proteome Discoverer application automatically applies this filter set to the opened MSF results file or To save the filter set in a file select the Save As option Click the Browse button and browse to the file to save it in You can also type the name of a new file in the box next to Save As e Click OK in the Save Filter Set dialog box The saved filter set appears in the list in the Filter and Grouping Set area The default set is named Default in this list Toload a filter set 1 In the Filter and Grouping Set area click I Load The Load Filter Set dialog box appears as shown in Figure 117 Figure 117
199. ally 10 The rank of a peptide is its position in the reported list of identified peptide candidates per spectrum that is ordered from better to worse scores Peptides with a top ranking for example 1 or 2 are more likely to be the correct peptide than peptides with a lower ranking for example less than 2 The Proteome Discoverer application does not store the peptide rank in the results file but calculates it after loading the results file Only loaded peptides affect the peptide rank The Proteome Discoverer application loads peptides that pass all other peptide filters before applying the Peptide Rank filter It rejects those peptides that do not pass the Peptide Rank filter You can use the Peptide Rank filter to filter out peptides with a rank higher than the maximum rank that you specify with the Maximum Peptide Rank option Calculating Peptide Rank The Merge Results of Equal Search Nodes option in the Workflow Editor determines whether peptides and proteins identified by the same type of search engine are merged together If you select this option the Proteome Discoverer application ranks the peptides identified by the same search engine together Only one peptide can have rank 1 for each spectrum and search engine If you do not select the Merge Results of Equal Search Nodes option the Proteome Discoverer application ranks peptides identified by one search engine independently from the peptides identified by another search engine The
200. alue is 5 0 e For milli mass units the minimum value is 0 1 and the maximum value is 5000 e For parts per million the minimum value is 0 01 and the maximum value is 5000 The default is 10 0 In the Search Tolerances area specify the fragment mass search tolerance Select the Use Average Fragment Masses option to use the average mass for matching the fragments In the Fragment Mass Tolerance box specify the mass tolerance value used for matching fragment peaks in daltons Da or milli mass units mmu e For daltons the minimum value is 0 0001 and the maximum is 2 0 The default is 0 8 e For milli mass units the minimum value is 0 1 and the maximum value is 2000 In the Ion Series Calculated area specify the ion factors for a b c x y and z ions for your experiment type You can use a range of 0 through 1 0 for all ion factors For CID HCD and PQD activation types use b and y ion factors For ETD and ECD activation types use c y and z ion factors Note The Ion Series Calculated area does not appear in the Mascot wizard Optional Set up a decoy database by selecting the Search Against Decoy Database check box and setting the false discovery rate FDR parameters For detailed information about this procedure see Calculating False Discovery Rates on page 186 Proteome Discoverer User Guide 37 2 Getting Started Starting a New Search by Using the Search Wizards A decoy database gives a p
201. ample for iTRAQ 4plex the different reporter tags 114 115 116 117 are the four quantification channels of the TRAQ 4plex method The application calculates the ratios from the detected quantification values of the four quantification channels For precursor ion quantification a quantification channel is one of the different possible labeling states of a peptide corresponding to the different heavy amino acids used in the cell cultures For example the SILAC 2plex methods are normally used with two quantification channels named light and heavy The light quantification channel uses the natural isotopes of lysine 7C 4N gt and arginine 7C 4N In the heavy quantification channel arginine 10 8C N replaces all arginines and either lysine 6 1 Ge No or lysine 8 3C EN5 replaces all lysines To set up the quantification method 1 Set up a search by following the instructions in Starting a New Search by Using the Workflow Editor on page 42 2 In the workspace pane of the Workflow Editor add the Precursor Ions Quantifier node for precursor ion quantification or the Reporter Ions Quantifier node for reporter ion quantification 3 Click the Precursor Ions Quantifier node or the Reporter Ions Quantifier node and in the Quantification Method box click the Browse button that appears The Quantification Method Editor dialog box opens to the Quan Channels page Use this dialog box to set up the quan
202. an_2011_05_26 it 3 27PM 0 Spectrum LibraryCreator M Z Indexfile C PROGRA 2 Thermo DISCOV 2 4 SPECTR 1 2011_0 1 TAR human_2011_05_26_it_dec 3 27PM 0 Spectrum Library Creator Library file TEXT C PROGRA 2 Thermo DISCOV 2 4 SPECTR 1 2011_0 1 TAR human_2011_05_26_it 3 27PM 0 Spectrum Library Creator Library file BINARY C PROGRA 2 Thermo DISCOV 2 4 SPECTR 1 2011_0 1 TAR human_2011_05_26 3 04PM 0 Spectrum LibraryCreator Generating DECOYto C PROGRA 2 Thermo DISCOV 2 4 SPECTR 1 2011_0 1 TAR human_2011_05_26 3 04PM 0 Spectrum Library Creator SpectraST started at Fri Nov 09 15 04 02 2012 3 04PM 0 Spectrum LibraryCreator Start building decoy library 3 00PM 0 Spectrum LibraryCreator Preparing Spectrum Library 2011_05_26_human_spectrast tar gz for usage When the Proteome Discoverer application finishes adding the spectrum library the file name and the spectrum library Figure 97 properties appear in the Spectrum Libraries view as shown in Proteome Discoverer User Guide 133 4 Searching for Data Searching Spectrum Libraries Figure 97 Added tar gz file and the spectrum library properties in the Spectrum Libraries view File Search Report Report File Quantification Processing Workflow Editor Administration Tools Window Help Ba oeron va 7 2B DF BR SEQUEST GR Mascot 72 AAR LH AA PT A OBERABDA Administration x Workflow Editor C elegan SpectraST Benchmark 2 0 x 4
203. and Molecular Biology 228 230 InterPro database 226 iodo TMT 6plex quantification method as default 250 266 271 reporter ion masses 250 ion trap mass spectrometer processing PQD data from 334 335 specifying in Reporter Ions Quantifier node 256 IPI database 225 340 IRMPD activation type 9 347 Is filter 50 Is Not filter 50 isobaric tags for relative and absolute quantification See iTRAQ quantification isotope intensity 273 isotope patterns 332 isotope shift 273 isotopomers 243 iTRAQ 4plex quantification method as default 7 253 266 selecting in Quantification Method Editor dialog box 271 iTRAQ 8plex quantification method as default 7 253 266 selecting in Quantification Method Editor dialog box 271 iTRAQ quantification creating a workflow for 253 default methods available in 253 266 271 description 7 252 isotopic distribution values 308 performing 249 Reporter Ions Quantifier node 7 specifying label modifications 267 See also reporter ion quantification IUBMB Enzyme Nomenclature 228 230 J job queue in creating FASTA indexes 123 125 opening 31 Job Queue page 75 Thermo Scientific Index K Keys page 221 224 L Last Modified column 103 131 LC MS 9 LC MS MS 4 limitations 14 Load Files page 217 Load Filter Set dialog box 164 LTQ Orbitrap mass spectrometers adding a non fragment filter node 58 troubleshooting 334 workflow demonstration 51 lysine 244 Magellan server 70 76 Magellan storage files
204. and relaxed FDRs for every available search wizard To set up FDRs in a search wizard 1 Start your search by using the search wizards For information about using the search wizards see Starting a New Search by Using the Search Wizards on page 29 2 On the lt Wizard_name gt Search Parameters page select the Search Against Decoy Database option as shown in Figure 138 Thermo Scientific Proteome Discoverer User Guide 189 5 Filtering Data Calculating False Discovery Rates Figure 138 Setting up a decoy database search in a search wizard Setting up a decoy database search Sequest HT Search Wizard Sequest Search Parameters Please select the parameters for the SEQUEST search reed General Search Parameters Decoy Database Search Database human_ref fasta x Search Against Decoy Database 4 Enzyme Trypsin Full Target FDR Strict 0 01 Missed Cleavages 28 Target FDR Relaxed 0 05 Search Tolerances E Use Average Precursor Mass Precursor Mass Tolerance 10 0 E Use Average Fragment Mass Fragment Mass Tolerance 08 lon Series Calculated a lons Factor 0 x lons Factor 0 b lons Factor 1 y lons Factor 1 c lons Factor 3 In the Target FDR Strict box set the target FDR for high confidence peptide hits 4 In the Target FDR Relaxed box set the target FDR for medium confidence peptide hits 5 Click Next Setting
205. annotating MSF Files e Uploading Results to ProteinCenter e Accessing ProteinCards e ProteinCard Parameters e GO Slim Categories ProteinCenter ProteinCenter is a Web based application that you can use to download biologically enriched annotation information for a single protein such as molecular functions cellular components and biological processes from the GO database annotation information for protein families from the Pfam database gene identifications from the Entrez database and post translational modification information from the UniProt database The data in ProteinCenter is updated biweekly Thermo Scientific Proteome Discoverer User Guide 201 6 Protein Annotation Gene Ontology GO Annotation The Proteome Discoverer application gives you access to ProteinCenter in two ways e The Annotation node used in a search workflow retrieves GO Pfam Entrez and UniProt database information from ProteinCenter and stores it in the Proteome Discoverer results files This information is displayed in columns on the Proteins page of the MSF file For information on setting up an Annotation workflow to achieve these results see Configuring the Proteome Discoverer Application for Protein Annotation on page 204 and Creating a Protein Annotation Workflow on page 206 e The ProteinCard available for each protein displays the annotation data available in ProteinCenter and displays it on a page of the Protein Identification Det
206. anscript of collagen variants have been observed for this gene and a pseudogene of this gene is located on the Description short arm of chromosome 9 provided by RefSeq May 2011 Arsenic transactivated protein 3 Collagen binding protein 2 precursor Serpin H1 Serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 arsenic transactivated protein 3 cDNA FLJ16630 fis clone TESTI4019756 highly similar to Collagen binding protein 2 hypothetical protein XP_006220 proliferation inducing gene 14 rheumatoid arthritis related antigen RA A47 serine or cysteine proteinase inhibitor clade H heat shock protein 47 member 2 serpin H1 precursor serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 isoform CRA_a unnamed protein product uF Table 12 lists the parameters on the General page of the ProteinCard page Table 11 Parameters on the General page of the ProteinCard page Sheet 1 of 2 Command Description Top area Displays the protein name in bold font on the first line The second line in bold font is the official symbol of the gene that ultimately directs the synthesis of the protein through RNA and the text following it is the alternative name or names of the gene Top right area Displays the name of the species that contains the gene that ultimately directs the synthesis of this protein through RNA th
207. antification on page 259 You must attach the selected quantification node directly to the Event Detector node For information about the parameters that you can set for the quantification nodes see General Configuration Parameters on page 597 Creating an Annotation Workflow To create a workflow that uses the Annotation node to retrieve GO Pfam Entrez and UniProt database information from ProteinCenter and install it in the Proteome Discoverer results files see Creating a Protein Annotation Workflow on page 206 Creating a PTM Analysis Workflow Thermo Scientific If you want to focus on studying the biologically relevant post translational modifications of proteins you can create a workflow that includes the phosphoRS node refer to the Help This node calculates PTM site localization scores for phosphorylation and makes them available in the Proteins Identification Details view when you choose Search Report gt Protein ID Details View This view color codes the found phosphorylation modification above the amino acid sequences to indicate the probability of the modification being found on those portions of the amino acid The PTM Site Probabilities area to the left of the sequence table displays a legend explaining the color coding For more information o this view refer to the Help Proteome Discoverer User Guide 55 2 Getting Started Starting a New Search by Using the Workflow Editor You can use only one phosph
208. ascot node on the Mascot server as the search engine in a quantification workflow you can set the dynamic and static modifications as parameters For samples with isotopic labels and several PIMs you might need to specify several dynamic modifications usable within a single search but the current number that you can specify is limited to nine To avoid this limitation you can configure quantification methods on the Mascot server In a quantification method modifications are organized into groups classified as fixed variable or exclusive You can define modification groups as variable or exclusive at the component level where they usually characterize the component You can also define them at the method level but only as fixed or variable Defining modifications at the method level is convenient for modifications that are important to the method and saves having to choose them in the Workflow Editor Exclusive groups are effectively a choice of fixed modifications so the restrictions that apply to fixed modifications also apply to them With the Mascot node you can use the modification groups specified as part of a quantification method on the Mascot server You can use the node s From Quan Method parameter in the Parameters pane to select the dynamic modifications to search for rather than manually specifying each modification with a Dynamic Modifications parameter Proteome Discoverer User Guide 261 7 Quantification Searching for Qua
209. at potentially change the quantification values It applies the experimental bias correction after the first time that it calculates all peptide and protein ratios The application then determines the bias correction factor and applies it to every peptide and protein ratio Table 23 Calculating peptide ratios when quantification values are missing Sheet 1 of 2 Reject All Detected quantification Displayed used uae Quan values quantification values RE issing a Minimum Values If Minimum Quan Quan Not All detected Values Case Value z Quan quan with value Threshold Minimum Channels 114 115 116 114 115 116 setting Are Intensity setting Present setting All quan 33 0 No Irrelevant 100 50 300 100 50 300 valnes 33 0 Yes Irrelevant 100 50 300 100 50 300 detected 33 75 No Irrelevant 100 50 300 100 0 300 33 75 Yes Irrelevant 100 50 300 100 75 300 Thermo Scientific Proteome Discoverer User Guide 319 7 Quantification Calculating Peptide Ratios Table 23 Calculating peptide ratios when quantification values are missing Sheet 2 of 2 R Reject All Detected quantification Displayed used eplace Bee Missi Quan values quantification values ne issing ene Minimum Values If Minimum Quan Quan Not All detected Values Case Value z Quan quan with valie Threshold Minimum Channels 114 115 116 114 115 116 setting Are Intensity setting Present setting Quan 33 0 No No 100 0 300 100 0 300 val
210. ata for single or grouped scans Mascot Generic Format MGF files are mass spectral files produced during Mascot analysis They contain a list of precursor ions their fragments and the masses of the fragments MZDATA files are common data format files developed by the Human Proteome Organization Standard Initiative HUPO PSI for proteomics mass spectrometry data These files are in version 1 05 format They are exported with XML indentation enabled so that the different XML tags are broken into multiple lines instead of merged into one line Magellan storage MSF files contain the results of the searches conducted by the search wizards or the Workflow Editor Extensible Markup Language XML files contain workflow templates MZXML files are standard 2 x mass spectrometer data format files developed at the Seattle Proteome Center at the Institute for Systems Biology ISB that contain a list of precursor ions their fragments and the masses of the fragment MZML files are a combination of MZDATA and MZXML formats developed by the Human Proteome Organization Standard Initiative HUPO PSI and the Seattle Proteome Center at the Institute for Systems Biology ISB The Proteome Discoverer application supports version 1 1 0 of the MZML format ProtXML files contain protein identifications from MS MS derived peptide sequence data They are created by the File gt Export gt To ProtXML command PepXML files contain peptides that are inclu
211. ates Figure 141 PEP and q Value columns on the Peptides page of results processed with Percolator File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OU SS BOF SATO TH ZORA K sweat Q Mso ARAM GEBZASARB Workflow Editor helaRVD1_1i_1_1_percolator x Administration X E_coliimsf x helaRVD1_1_1_1 percolator msf x dp Search Input Result Filters Peptide Confidence Search Summary A2 Sequence PSMs Proteins Protein Groups Protein Group Accessions ACn q Value PEP Charge MH Da AM ppm RT min a I TQTSDPAMLPTMIGLLAEAG 2 1 1 23308577 0 0000 0 7 97e 06 6 19 3 2272 18082 5 77 79 18 I TALLDAAGVASLLTTAEVW 1 1 1 31542947 0 0000 0 2 72e 06 5 94 3 2482 41574 5 71 91 84 M AQEALLQLSQALSLMETVK 2 1 1 20127486 0 0000 0 0 000106 5 84 2 2073 13701 5 25 77 98 M TGAVDQIQLTQAQLER t 1 1 42734430 0 0000 0 5 21e 05 5 76 2 2025 11064 6 45 60 13 I7 NEGSESAPEGQAQQR 1 1 1 34098946 0 0000 0 0 000124 5 54 2 1587 70452 3 20 34 70 LLEDGEDFNLGDALDSSNSM 2 1 1 4557888 0 0000 0 0 000133 5 54 3 2740 27842 6 02 64 42 I IVENSDAVTEILNNABLK 1 1 1 4826952 0 0000 0 3 81e 05 5 54 2 2085 11992 5 96 70 58 Cle 2 1 1 32189394 0 0000 0 0 000124 5 49 2 2119 06230 6 93 78 94 I _IAIPGLAGAGNSVLLVSNLNP 2 3 1 14165466 0 0000 0 0 000479 5 49 2 2275 29057 5 97 69 81 M ILTATVDNANILLQIDNAR i 1 4557701 0 0000 0 0 000265
212. ates Replicates are repeated measurements of the same sample You repeat measurements to obtain better statistics Without replicates you cannot be sure that something that you observed is real that is statistically significant and not a result of an error in the sample preparation the liquid chromatography the acquisition and so forth To generate replicates you can 324 Proteome Discoverer User Guide Thermo Scientific Treatments Ratio Count Ratio Variability 7 Quantification Calculating Ratio Count and Variability repeat the sample preparation or use the same sample and measure it multiple times This data highlights the variance within the different steps For example if you measure a difference of 17 percent between two samples representing different treatments with a new drug but the variance between the different replicates of the same treatment is already 28 percent the observed difference might not be significant Treatments are samples that are brought to different states For example they might be different samples representing different exposure levels to a certain drug or cultures of the same cells exposed to different levels of stress such as radiation salts or heat The Ratio Count or the Heavy Light Count column displays the number of peptide ratios that were used to calculate a particular protein ratio If only one ratio was reported for example the Heavy Light ratio for SILAC data the displayed cou
213. ation box shown in Figure 119 appears Figure 119 Restore Factory Filter Set confirmation box Restore Factory Filter Set e Do you really want to restore default factory filter set OK Cancel 2 Click OK The confirmation box shown in Figure 120 appears Figure 120 Loading Filter Set confirmation box Loading Filter Set The current filters will be removed as 3 Click OK Removing and Deactivating Filters You can remove or deactivate filters to alter the search results e To remove a filter e To deactivate a filter To remove a filter 1 Open your search results 2 Click the Result Filters tab 3 Select the filter in the list of filters in the Peptide Filters or Protein Filters area 166 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results 4 Click x The filter is removed from the list of filters 5 Click Apply to update the Proteins Peptides or Search Input page To deactivate a filter 1 Open your search results 2 Click the Result Filters tab 3 Clear the check box in the Active column 4 To update the Proteins Peptides or Search Input page click Apply The filter is deactivated but not removed from the Result Filters page Filtering Results with Row Filters The following procedures describe how to set and clear basic row filters display filtered out rows use row filters to filter precursor masses and filter peptid
214. ation with TMT tags is no different from quantification with iTRAQ described in TRAQ Quantification on page 252 except that it uses the following default mass tags by Proteome Sciences PLC e TMT 2plex e TMT 6plex e iodo TMT 6plex e TMTe 6plex e TMT 10plex Note If you are installing the Proteome Discoverer application for the first time the TMT 6plex quantification method is no longer available The TMTe 6plex method replaces it You can use these default methods to create your own quantification templates For information on adding quantification methods see Changing a Quantification Method on page 288 Table 20 lists the masses of the reporter ions of the tags available in the different TMT kits The masses for the original TMT reagents which but no longer available are included for reference Table 20 Monoisotopic masses of the reporter ions after CID or HCD fragmentation of the tags in the different TMT kits Sheet 1 of 2 TMT 2plex TMT 6plex Original TMTe 6plex Current TMT 10plex iodo TMT 6plex Tag Mass Tag Mass Tag Mass Tag Mass Tag Mass 126 126 127725 126 1216 127725 126 126 127725 126 126 127725 126 126 127725 127 127 131079 127 127 131079 127 127 124760 127_N 127 124760 127 127 124760 127_C 127 131079 250 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Reporter lon Quantification Table 20 Monoisotopic masses of the reporter ions after CID or HCD fragmentati
215. ats into a Workflow on page 65 2 Proteome Discoverer User Guide Thermo Scientific Search Engines Thermo Scientific 1 Introduction Features e The ability to export protein and peptide reports in standard spectrum data formats such as MZDATA DTA MZML and MGE You can also export search results to XML and tab delimited TXT files In addition you can export annotated spectra for selected peptides into a ZIP file that includes an HTML page with peptide information and links to spectrum images The Help describes how to export your data to these and other formats e The ability to merge filtered or unfiltered search results For information refer to the Help e A number of protein and peptide filtering and grouping options to help you sort and filter your data For information on Proteome Discoverer s filtering capabilities see Filtering Data on page 153 For information on grouping see Grouping Proteins on page 174 and Grouping Peptides on page 185 The Proteome Discoverer application includes the Sequest HT SEQUEST and Mascot search engines each produces complementary data The Sequest HT and SEQUEST search engines are distributed by Thermo Fisher Scientific Mascot is a protein identification search engine created by Matrix Science The Mascot search engine uses mass spectrometry data to identify proteins from primary sequence databases The Sequest HT and SEQUEST search engines can analyze different data
216. aw file or files that you want to load and click Open The selected spectrum file or files appear on the Load Files page To remove a file from the Load Files page select the file and click Remove 5 To specify the type of processing select the Batch Processing or MudPIT option e Batch processing the default Executes the workflow once for each spectrum file e MudPIT Feeds all spectrum files into one workflow When you select the MudPIT option the Output Filename box becomes available 6 In the Workflow list select the workflow template that you want to import e Select the workflow from the Workflow list if it resides on the server that the Proteome Discoverer Daemon application is connected to This workflow must be the one that was saved with the search parameters to be used with the given searches You cannot modify parameters from the Proteome Discoverer Daemon application itself Workflow templates that are missing more than the Spectrum File Names parameter do not appear in the Workflow list because the Proteome Discoverer Daemon application cannot complete them or 72 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Starting a Workflow e Select a valid workflow by clicking the Browse button to select the workflow from your local machine If you add workflow templates to the Proteome Discoverer application while the Proteome Discoverer Daem
217. braries LysC K 1 Full a Chemical Modifications TodosoBenzoate W 1 Full z Gluc ED 1 Full 3 Elastase Tryp Ch ALIVKRWFY Full A Quantification Methods Elastase ALIV 1 Full Cyanogen_Bromi M 1 Full Clostripain R 1 Full License Management Chymotrypsin F FWY 1 ChTr Full R Licenses Chymotrypsin FWYL 1 ChyTr Full AspN D 0 Full Configuration Workflow Nodes amp amp Annotation amp 3 Mascot 5 MSPepSearch E SEQUEST 4 Sequest HT amp 3 SpectraST 4 E Server Settings 3 Discoverer Daemon amp FASTA Indexes Ready Adding a Cleavage Reagent To add anew cleavage reagent 1 Click the Name column cell and click Click Here To Add a New Record 2 Modify the default values in the row of that new reagent 3 Click Apply Thermo Scientific Proteome Discoverer User Guide 151 4 Searching for Data Customizing Cleavage Reagents Deleting a Cleavage Reagent To delete a cleavage reagent 1 Click the box in the column next to the row that you want to delete 2 Click Delete 3 Click Yes in the confirmation box that appears Modifying a Cleavage Reagent To modify a cleavage reagent 1 Click in the column for the reagent you want to modify select the current contents and enter the new information 2 Click Apply Filtering Cleavage Reagent Data To filter cleavage reagent data 1 Click the Funnel icon Y next to the header of the column 2 Select one of the following e Al
218. can manually specify the label modification which then triggers the validation of the peptides and save the change in the quantification method in the MSF file When you do not set the label modifications on the Quan Channels page the Proteome Discoverer application does not perform the validation The process of specifying quantification channels for precursor ion quantification is slightly different from the process of specifying label modifications for reporter ion quantification Thermo Scientific Proteome Discoverer User Guide 267 7 Quantification Setting Up the Quantification Method To specify quantification channels for precursor ion quantification 1 Click the Quan Channels tab of the Quantification Method Editor dialog box shown in Figure 178 if it is not already selected Figure 178 Quan Channels page of the Quantification Method Editor dialog box for precursor ion quantification Quantification Method Editor SILAC 2plex Argl0 Lys6 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias None E Label Name None Modification Target Side Chain Modification N Teminal Modification C Teminal Modification Modification None z x p m ucts aan ok cca He 2 In the top list select the name of the labeling method to use When you create a new workflow and first acce
219. cation channels Thermo Scientific Proteome Discoverer User Guide 307 7 Quantification Displaying the Quantification Spectrum Chart Figure 207 Expected but unsuitable peaks in the Quantification Spectrum chart File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 648 88 oF BOOT SH g amm K suetht g Macot ARAM ASB GRaRvPaAaRE Sequest SILAC Dimethylation Search 1 3 0 297 msf x E Quan Channel Quan Info Quan Usage Heavy Light Medium Light Peptides Matched 1 Inconsistently Labeled NotUsed 458 1 7 663e4 Inconsistently Labeled NotUsed 337 4 03 o 1 87 2 540e5 Medium Redundant NotUsed 0 801 0 966 189 4 01 ine 1 46 5 971e6 Light Redundant Not Used 0 873 0 990 vd 3 92 0 1 6 2 583e5 Light Unique Used 1 064 1 1 0 1 284 1 108e6 Heavy Unique 0 982 0 899 18 3 80 o 1 557 2 239e5 Heavy Unique 0 782 1 175 628 3 74 0 1 400 1 676e6 Heavy Unique 0 915 0 905 560 3 68 Ot 1 48 7 754e5 Medium Redundant 0 786 0 985 19 3 68 ne 1 400 1 650e6 Medium Redundant 0 915 0 905 590 3 61 ne 1 5 1 016e6 Medium Redundant 1 094 1 192 200 3 57 On 1 3 870e5 Inconsistently Labeled 715 3 57 o 1 3 870e5 Inconsistently Labeled 665 3 57 o 1 3 870e5 Inconsistently Labeled 463 3 57 0 0 1 187 2 2 262e5 Medium Excluded by Method 329 3 56 o 1 46 5 90426 Medium Redundant 0 873 0 990 14 3 53 0 1 642 3 672e5 Light Unique 0 767 0 986
220. cause the peaks lie outside of any integration window or because the setting of the Integration Method parameter specified in the Reporter Ions Quantifier node determined that only one peak per integration window should be chosen from any integration window A different peak was picked for this integration window according to the criterion specified by the Integration Method setting Missing Reporter Peaks in the Quantification Spectrum If reporter ions are missing in the quantification spectra you can use settings on the Ratio Calculation page of the Quantification Method Editor dialog box to influence how the Proteome Discoverer application handles this problem For example if all six intensities of an TMTe 6plex are missing or if the reference ion is missing for example in the TMTe 6plex method shown in Figure 201 the 126 ion is missing the corresponding spectrum is always excluded from the protein quantification In the Quan Info column of the Peptides page these peptides are marked No Quan Values as shown in Figure 201 The protein ratios were calculated according to the settings displayed in Figure 202 on page 302 300 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart Figure 201 Quantification results with missing reporter ions iva BB BOF SAGTAH g BD K suth g Mascot ARAM RESSRPARB X58989 serum albumin Bos taurus OVALBUMIN PLAKALBUMIN
221. ccur only if you have a sufficient number of peptides to use for protein quantification 302 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart In the example in Figure 201 on page 301 the protein is identified with 30 peptides For 19 of the peptides the corresponding quantification spectra show no reporter tag at all These peptides are never considered in calculating the protein ratios and are marked as No Quan Values in the Quan Info column of the Peptides page Two additional spectra are missing individual reporter ions Although two peptides are marked as Used for quantification in the Quan Info column their extreme ratios are not considered in the protein ratio calculation with the settings in Figure 202 on page 302 If at least one of the reporter intensities is present see Figure 201 you can use the Replace Missing Reporter Intensities With Minimum Intensity option on the Ratio Calculation page of the Quantification Method Editor dialog box to replace the missing intensities with the minimum intensity detected among all spectra on all reporter channels Reporter intensities are missing because they fall under the detection limit so replacing them with an intensity estimate that is close to the detection limit might make sense Figure 203 shows the same protein as in Figure 201 on page 301 after the selection of the Replace Missing Re
222. ch To calculate the dot score the Proteome Discoverer application splits the reference spectrum into equal bins It then adds the product of the normalized intensities of each bin up to the dot score as shown in the following formula D gt ibrary I query j where flibrary j and Iquery j are normalized intensities of the j bin of the spectra D is the dot score The application reports the dot score together with the dot bias Dot Bias Score The application calculates the dot bias score as follows 2 f library j ety Ail D DB where library j and query j are normalized intensities of the jh bin of the spectra D is the dot score A high dot bias DB value indicates that the dot score results from only a few peaks F Value The Proteome Discoverer application calculates the AD value in the F value formula as follows D AD The application calculates the F value F as follows 0 6D 0 4AD b 138 Proteome Discoverer User Guide Thermo Scientific where D is the do 0 12 0 12 b 0 18 0 24 0 4 Searching for Data Searching Spectrum Libraries t score and 6 is the following DB is the dot bias if DB lt 0 1 if 0 35 lt DB lt 0 4 if 0 4 lt DB lt 0 45 if DB gt 0 45 for all other values of DB Searching Spectrum Libraries with the MSPepSearch Node Thermo Scientific Figure 101 shows the basic workflow for searching spectrum libraries with the MSPepSearch nod
223. cot ARAM AEBTABARE Administration X Sequest TMT 6 plex Benchmark 1 4 0 175 msf x dp Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Quan Spectra Mass Analyzer Activation Type MS Order Isolation Interference Ion Inject Time ms Precursor m z Da Precursor MH Da RT min First Scan Spectrum File 127 126 128 126 129 12 130 12 131 126 8 352 75751 704 50774 0 958 1 129 1 043 1 008 1 188 HCD MS2 150 547 29248 1093 57768 40 04 2481 10mixTMT_45CE_1us RAW HCD MS2 150 612 83215 1224 65703 40 09 2486 10mixTMT_45CE_ius RAW HCD MS2 150 403 90256 1209 69312 40 11 2487 10mixTMT_45CE_1us RAW HCD MS2 28 373 89212 1119 66181 40 15 2491 10mixTMT_45CE_ius RAW 0 665 1241 0 721 0 645 0 441 HCD MS2 150 380 20255 759 39781 40 17 2492 10mixTMT_45CE_ius RAW 0 977 1 020 1 130 0 810 0 518 HCD MS2 150 526 31232 1051 61736 40 19 2493 10mixTMT_45CE_1us RAW 1344 0 599 1 092 0 909 HCD MS2 99 448 25201 895 49675 40 24 2498 10mixTMT_45CE_lus RAW 1 491 1 995 1 274 1 652 2015 HCD MS2 150 415 86948 1245 59388 40 26 2499 10mixTMT_45CE_ius RAW HCD MS2 150 553 59808 1658 77970 40 27 2500 10mixTMT_45CE_1us RAW HCD MS2 16 48 409 72714 818 44701 40 32 2505 10mixTMT_45CE_ius RAW 0 750 0 859 0 757 0 834 1 033 HCD MS2 48 150 388 18500 775 36272 40 36 2508 10mixTMT_45CE_ius RAW HCD MS2 51 300 337 97824 1348 89113 40 38 2509 10mixTMT_45CE_1us RAW HCD MS2 18 300 678 30200 1355 59673 40 40 2510 1
224. coyDatabase True Ready Thermo Scientific The Workflow Failures pane contains three columns e Error Information Displays information about the problem that the application encountered in the workflow e Parameter Displays the name of the node parameter that has an erroneous setting e Value Displays the erroneous setting of the node parameter When a warning symbol is attached to a node the Proteome Discoverer application automatically updates the node with the correct version preserving the previous parameter values in the updated node It does not include any node parameters that are no longer available and adds any new parameters set to their defaults If the Parameter and Value columns indicate a problem with the parameter settings enter the correct parameter settings in the Parameters pane of the Workflow Editor Proteome Discoverer User Guide 63 2 Getting Started Starting a New Search by Using the Workflow Editor Opening an Existing Workflow from an XML or MSF File You can open a workflow from an existing MSF or XML file 1M kod To open an existing workflow from an XML or MSF file 1 Choose Workflow Editor gt Import Workflow 2 In the Import Workflow dialog box browse to the XML or MSF file containing the workflow to import and click Open The selected workflow now opens in the Workflow Editor The Proteome Discoverer application validates parameter settings and uses warning symbols to
225. ct the charge number of the precursor ions From the data in the raw file the Proteome Discoverer application evaluates the spectrum and uses an algorithm to determine the charge state of the spectrum It cannot calculate the mass without knowing the charge state of the spectrum If the algorithm cannot determine the charge state of the evaluated spectrum the application assigns the charge state that you select to the spectrum You can assign the following charge number e Automatic Assigns a charge number of 2 and 3 to the spectrum e 1 through 8 Assigns a charge number of from 1 through 8 to the spectrum The default is Automatic In the Intensity Threshold box enter an intensity value below which to filter out ions The Proteome Discoverer application filters out low intensity ions which are ions that are most likely chemical noise and serve only to slow down the analysis without improving the results The default is 0 0 In the Minimum Ion Count box enter a value for the minimum ion count or use the increment or decrement buttons The minimum ion count is the minimum number of ions that must be present in an MS MS spectrum for it to be included in a search The default is 1 In the S N Threshold box enter a value for the signal to noise threshold setting This setting specifies the intensity of the signal to the intensity of the background noise It filters out low intensity ions that function as noise The defaul
226. ctory Defaults iTRAQ 4plex Thermo Scientific gt 3 In the New Method Name box type the name of the quantification method that you want to create 4 Select one of the following methods of creating a quantification method e Clone From Existing Method Uses the same settings as those of the existing quantification method that you select from the list The list of methods is the same as that given at the beginning of Setting Up the Quantification Method on page 264 e New Empty Quan Method Uses one of the following templates so that you can build a new processing method from scratch e Reporter Ion Quan Method Provides a template for reporter ion quantification Precursor Ion Quan Method Provides a template for precursor ion quantification Default Create From Factory Defaults Creates a new method using the same settings from one of the default settings that appear when the Proteome Discoverer application is newly installed 5 Click Create The Quantification Method Editor dialog box appears as shown in Figure 181 on page 272 through Figure 186 on page 280 The Quan Channels page and the Ratio Reporting page are blank if you selected the New Empty Quan Method option In this case the Quan Channels page resembles Figure 193 Proteome Discoverer User Guide 287 7 Quantification Changing a Quantification Method Figure 193 Empty quantification method template
227. ctrum Selector nodes added to a workflow File Search Report Quantification Processing Workflow Editor Administration Tools Window Help JARA ACEBBRBARB Name Based on template i Spectrum Files Description E Spectrum amp Feature Retrieval A Event Detector Spectrum Selector Spectrum Processing i Noise Peak Filter ig Non Fragment Filter V Merge results of equal search nodes iQ Spectrum Grouper i Spectrum Normalizer i Top N Peaks Filter Spectrum Filters mw Scan Event Filter Ty Spectrum Confidence Filter Ty Spectrum Properties Filter Sequence Database Search Q Mascot Q SEQUEST Q Sequest HT C Spectral Library Search W MsPepSearch Q SpectrasT PSM Validation Fixed ValuePSM Validator Percolator Target Decoy PSM Validator PTM Analysis phosphors E Quantification 2 Precursor Ions Area Detector D Precursor Ions Quantifier fd Reporter Ions Quantifier Data Export iu Spectrum Exporter Annotation Annotation Ready ja4 a0 8 OT ooon amp B BW GD R SequestHT GR Mascot Workflow Editor x Workflow Nodes a Data Input a Parameters q 4 Show Advanced Parameters 9 Depending on your data needs drag the appropriate nodes from the Workflow Nodes pane to the workspace pane For a description of the nodes that you can select refer to the Help T
228. ctrum with the fragment match settings refer to the Help Figure 102 Mirror plot in the Peptide Identification Details view Load fy Save Factory Defaults Display Options Charge Detail Level AIl Annotation Threshold 5 0 of base peak 0 absolute E Show legend V Show reference spectrum E Use search settings Match Tolerances Mass analyzer ITMS Match tolerance 2100000 Fragments Activation Type CID lon Series Neutral Losses a x v H0 Yb My 7 NH c 1 Iz c z 1 Oc 22 PTMs Other v H3PO P Immonium 140 Proteome Discoverer User Guide Extracted from C Smoke_Test_Files Celegans_FT_6ITDDDT_01 RAW 4446 RT 54 82 ITMS CID z 2 Mono m z 1288 16174 Da MH 2575 31621 Da Match Tol 2 Da Reference Spectrum R NLQIAQGTPGGLITYGAIDTVNCAK Q 2 MH 1288 9667 Da Replicates Total Used 231 100 All Spectra Median dot Product 0 877 0 050 Match to theoretical Spectrum 0 93 bis ye 1263 46 692 44 4 yz bs H O 807 29 Yio 1048 35 M 2H gt NH H O Measured spectrum 1270 60 Yis 1652 55 Intensity counts Reference spectrum T T T ro 1200 1400 1600 1800 miz The Proteome Discoverer application displays the reference spectrum using intensities multiplied by 1 in the same plot as the measured spectrum In the reference spectrum it also labels peaks of the
229. culating Peptide Ratios Figure 213 Peptide Ratio Distributions Chart for precursor ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH SBS SOF BOO DD BH LIB WD BD MR SequetHT MB Mascot A LMA RBEBSERRBAARE PQD with TMT 6 plex 10 175 min msf X Sequest SILAC Dimethylation Search 1 3 0 297 msf x ne El Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Edl Accession Description ZCoverage V Z Proteins Z UniquePeptides z Peptides Z PSMs Area A7 Heavy Light A7 Heavy Light Count A7 Heavy Lit S 1 7 1P100708398 2 49 92 1 19 32 125 1 161 0 893 24 2 T 1P100706427 2 Cationictrypsin 20 73 2 3 4 10 3 040 0 834 4 3 7 1P100691212 1 Alpha 1 acid glycoprotein 14 85 2 2 3 5 1 674 0 861 2 a 4 7 1P100823795 1 Vitamin D binding protein 8 65 1 1 5 8 6 412 0 779 5 7 1P100867199 1 KTN1protein 6 61 1 1 9 15 2 262 2 Peptide Ratio Distributions AX IP100708398 2 Serum albumin HeavyiLight Medium Light 1 0 4 R 0 16 0 89 10 4 R 0 02 0 99 IQR 0 30 0 18 IQR 0 26 0 18 o o 1 o 054 05 4 a o z T 8 8 z fa o o 3 ta 8 g a 0 0 li 0 0 El S i E g 3 g L 5 T 8 8 a o 8 054 054 o o o o o a Ready 5 6 Protein Group s 2911 2911 Merged Protein s
230. d a quantification ratio follow these steps For precursor ion quantification a In the Numerator list select the Light or Heavy label b In the Denominator list select the Light or Heavy label that you did not select in the Numerator box For reporter ion quantification a In the Numerator list select the fragmented mass tag of the sample b In the Denominator list select the name of the mass tag of the reference sample You now see the specified numerator and denominator in the Ratio Name box which is read only 4 Continue setting up the quantification method by following the instructions in Setting Up the Ratio Calculation on page 275 Setting Up the Ratio Calculation The Ratio Calculation page of the Quantification Method Editor dialog box controls how peptide and protein ratios are calculated from the raw quantification values of each quantification channel and how they are displayed on the Proteins and Peptides pages For background information on the options available on this page see Missing Reporter Peaks in the Quantification Spectrum on page 300 To set up the ratio calculation 1 Click the Ratio Calculation tab shown in Figure 184 This page is the same for both precursor ion and reporter ion quantification Thermo Scientific Proteome Discoverer User Guide 275 7 Quantification Setting Up the Quantification Method Figure 184 Ratio Calculation page of the Quantification Method Editor dialog b
231. d box to an appropriate setting For example you could set this option to 1 5 Thermo Scientific 7 Quantification Searching for Quantification Modifications with Mascot For other parameters that you can optionally set for the Spectrum Selector node refer to the Help 13 Click the search engine node for example SEQUEST and set the parameters for it in the Parameters pane a In the Protein Database box select an appropriate FASTA database b In the Dynamic Modifications area select the dynamic modifications For example you might select the Oxidation 15 995 Da M dynamic modification If you do not find this label you can enable it by following the instructions in Updating Chemical Modifications on page 141 c Inthe Static Modifications area select the static modifications For example you might select Carbamidomethyl 57 021 Da C in the Static Modification box d Set any other parameters as needed 14 Set the parameters for all other nodes in the Parameters pane For information about all the parameters that you can set for each node refer to the Help For information on the parameters that you can set for the Precursor Ions Quantifier node see step 14 of Creating a Workflow for Precursor Ion Quantification 15 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon g Searching for Quantification Modifications with Mascot Thermo Scientific When you use the M
232. d from another computer To import a quantification method 1 Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g either on the toolbar or on the Administration page The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification 2 Click Import 3 In the Import Quan Method dialog box select the method file containing the method that you want to import and click Open e Ifthe new method is valid the Quantification Method Editor dialog box opens showing the new method e Ifthe new method is not valid a message box appears that describes the error 4 If the new method is valid click OK in the Quantification Method Editor dialog box 5 Change the name of the imported quantification method by changing it in the table of the Quan Method Manager Exporting a Quantification Method You can save a quantification method to use on another computer To export a quantification method 1 Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g either on the toolbar or on the Administration page The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification 2 Select the method that you want to e
233. d isotope pattern Event list view Find most suited monoisotopic event i Collect all event candidates that deviate from the monoisotopic event or theoretical pattern by m z intensity or a centroid retention time less than three times the standard deviation Y Event candidates for isotope pattern Y Find optimal pattern events that minimize the error in m z intensity and centroid RT Identified isotope pattern 334 Proteome Discoverer User Guide Thermo Scientific Troubleshooting Quantification 7 Quantification Troubleshooting Quantification The following procedures can help you obtain optimal results when performing quantification To troubleshoot reporter ion quantification e Ifyou obtain unexpected quantification results verify that all settings of the nodes in your processing workflow are reasonable Thermo Scientific Make sure that the Integration Tolerance parameter of the Reporter Ions Quantifier node fits the data that you are processing The default is 20 ppm which is too low if you are processing PQD data from an ion trap Make sure that the settings of the Mass Analyzer MS Order and Activation Type parameters of the Reporter Ions Quantifier node are correct for the data that you are processing Figure 228 shows the typical settings to use if you want to quantify HCD scans from the Orbitrap Figure 228 Typical settings for quantifyi
234. database For a description of static and dynamic modifications see Updating Chemical Modifications on page 141 38 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards a Ifyou are searching for dynamic modifications select the modifications and the amino acids on which they can occur in the Dynamic Side Chain Modifications area In the boxes on the left select the modifications In the boxes on the right select the amino acids on which the modifications occur In the Sequest HT wizard delta masses appear next to the names of the modifications in the modification lists to clearly identify the modification as shown in Figure 20 Figure 20 Modifications with identifying delta masses Dynamic Side Chain Modifications Acetyl 42 011 Da x Carbamidomethyl 57 021 Da f Carbamyl 43 006 Da amj Carboxymethyl 58 005 Da f Deamidated 0 984 Da x Dimethyl 28 031 Da f Dimethyl 2H 4 32 056 Da X Dimethyl 2H 6 13C 2 36 076Da Formyl 27 995 Da X ICAT C 227 127 Da ICAT C 13C 9 236 157 Da piTRAQ4plex 144 102 Da iTRAQ8plex 304 205 Da Label 13C 6 6 020 Da Label 13C 6 15N 2 8 014 Da x Label 13C 6 15N 4 10 008 Da f Label 180 1 2 004 Da X Label 2H 4 4 025 Da i Methyl 14 016 Da Oxidation 15 995 Da Palmitoyl 238 230 Da Phospho 79 966 Da Propionamide 71 037 Da Sulfo
235. de in a sample to the ion intensity of the peptide in the control sample that exceeds a certain maximum enter this maximum number in the Maximum Allowed Fold Change box The minimum value is 1 and the maximum value is 100 000 Proteome Discoverer User Guide 277 7 Quantification Setting Up the Quantification Method The default is 100 With the default setting calculated ratios above 100 are set to 100 and calculated ratios below 0 01 are set to 0 01 For example if you set Maximum Allowed Fold Change to 10 the Proteome Discoverer application excludes any peptide ratios showing a greater than a ten fold change in ion intensity for an experiment compared to the control 10 To report larger ratios than you have indicated in the Maximum Allowed Fold Change box select the Use Ratios Above Maximum Allowed Fold Change for Quantification check box This option reports the quantification ratios based on the maximum values Values greater than the value selected in the Maximum Allowed Fold Change box are replaced by the maximum or minimum value By default this check box is clear 11 Continue setting up the quantification method by following the instructions in Setting Peptide Parameters Used to Calculate Protein Ratios on page 278 The settings of the options on the Ratio Calculation page govern the appearance of the experimental results in the columns in the MSF report The data can appear in the following colors e Pink
236. ded in the results of searches performed by the Sequest HT SEQUEST and Mascot search engines They are in PepXML format version 1 14 which is an open data format developed by SPC Institute for Systems Biology for storing exchanging and processing peptide sequence assignments from MS MS scans PepXML files are created by the File gt Export gt pepXML command The Proteome Discoverer application supports version 1 14 Tab delimited TXT files are in a simple text format that stores tabular data and is widely used to exchange data between different computer programs Proteome Discoverer User Guide 13 1 Introduction Limitations Limitations This release of the Proteome Discoverer application has the following limitations e The spectra count is not directly available in the application results report However the number of identified peptides is displayed for each protein This number should be similar to the spectra count for that protein e The Proteome Discoverer application supports peptide quantification methods that use reporter ions Examples of these methods are TMT and iTRAQ The application also supports peptide quantification methods that measure precursor ion abundances Examples of these methods are SILAC ICPL 80 N and label free methods New Features in This Release The Proteome Discoverer application version 1 4 adds the following new features Sequest HT Search Engine The new Sequest HT search engine is
237. deleted FASTA index To delete a FASTA index Be sure that the Auto Remove check box is selected for the index that you want to delete Select the index that you want to delete by clicking the first cell to the right of the plus sign The cell now changes to the Right Arrow icon i Click the Right Arrow icon 126 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases 4 Click the Remove icon Remove 5 Click OK in the Remove FASTA Indexes confirmation box The name of the deleted index disappears from the FASTA Indexes table and reappears in a separate table called Deleted FASTA Indexes as shown in Figure 93 It no longer appears in the FASTA Indexes table However because the FASTA index might be used in some calculations its removal from the application only takes place the next time that the server starts Figure 93 Deleted FASTA Indexes table File Search Report Quantification Processing Workflow Editor Administration Tools Window Help POE SEBSOTSRRTSH SADR smetht g Mascot ALARMS OBERSTE Administration x 4p 4 id Add 3 Remove Apply W Restore Refresh g Options Process Management 2 EE Auto Remove Indexed FASTA File Enzyme Index Size kB Last Access Time 3 E4 eA ol Vv _elegans_100_021411_FWD_combined fa_ Trypsin Full 58310 10 03 2012 04 05 PM k CEL V uniprot_sprot_2011_05 fasta Trypsin Full 886116 10 04 201
238. des page of the results report select the Show Peptide Groups check box 5 Select the method of grouping peptides from the Group Peptides By list e Sequence Groups peptides by sequence e Mass and Sequence Groups peptides by mass and sequence Calculating False Discovery Rates The false discovery rate FDR or the false positive rate is a statistical value that estimates the number of false positive identifications among all identifications found by a peptide identification search It is a measure of the certainty of the identification You can use the Proteome Discoverer decoy database search feature to determine FDRs You can use FDRs to validate MS MS searches of large data sets but they are not effective on searches of a small number of spectra or searches against a small number of protein sequences because the number of matches will likely be too small to give a statistically meaningful estimate A decoy database gives a probability value to identifiers and the percentage of false discoveries that you can expect A one percent FDR is a typical target for searches 186 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates A good decoy database should contain entries that look like real proteins but do not contain genuine peptide sequences The simplest approach to achieving such a decoy database is to reverse all protein sequences which is the scheme that the Proteome Disco
239. dmap view of the Xcalibur Home Page window opens 2 In the Roadmap view do one of the following e Choose GoTo gt Processing Setup e Click the Processing Setup icon eet The Processing Setup window opens 3 Open the processing method that you want to modify as follows a Choose File gt Open b Browse to the location of the processing method file and select the file c Click Open The selected processing method opens in the Processing Setup window 4 Open the Programs view of the Processing Setup window as follows a Choose View gt View Bar The view bar appears on the left side of the dialog box b On the view bar click the Programs icon By The Programs view of the Processing Setup window opens as shown in Figure 55 Figure 55 Programs view with an empty table Programs Enable Std QC Unk Other Action Program or Macro Name Sync Parameters Nes Yes Yes Yes Run Program Yes 82 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System 5 Ifthe Programs view contains an empty table right click the table and choose Insert Row from the shortcut menu A new row appears above the placeholder row as shown in Figure 56 An asterisk to the left side of a table row defines the row as a placeholder row Figure 56 Programs view with an unedited table row Programs fevers Sample type
240. e EBD ftp ftp ebi ac uk pub databases IPI msipi current A typical MSIPI title line follows gt MSIPI IPI00000001 2 Gene_Symbol STAU1 Isoform Long of Double stranded RNA bin ding protein Staufen homolog 1 Ing 577 CON 595 R 359 A FASTA ID e Accession IPI00000001 2 e Description Isoform Long of Double stranded RNA bin ding protein Staufen homolog 1 Ing 577 CON 595 R 359 A IPI The International Protein Index IPI is compiled by the European Bioinformatics Institute EBI to provide a top level guide to the main databases that describe the human and mouse proteomes SwissProt TrEMBL NCBI RefSeq and Ensembl http www ebi ac uk IPI ftp ftp ebi ac uk pub databases IPI current A typical IPI title line follows gt IPI IPI00685094 1 SwISS PROT 2K1IJ2 ENSEMBL ENSBTAPO0000028878 REFSE NP_001073825 XP_593190 Tax_Id 9913 Gene_Symbol MGC137286 LOC515210 Uncharacterized protein Clorf156 homolog FASTA ID e Accession IP100685094 1 e Description Uncharacterized protein Clorf156 homolog 340 Proteome Discoverer User Guide Thermo Scientific UniRef100 A FASTA Reference FASTA Databases UniRef also known as UniProt NREF is a set of comprehensive protein databases curated by the Universal Protein Resource consortium UniRef100 contains only nonidentical sequences and UniRef90 and UniRef50 are non redundant at a sequence similarity level of 90 percent and 50 percent respectively http www eb
241. e MSDB is compiled by the Clinical and Biomedical Proteomics group at the University of Leeds using the PIR TrEMBL GenBank SwissProt and NRL3D source databases http proteomics leeds ac uk bioinf msdb html ftp ftp ncbi nih gov repository MSDB A typical MSDB title line follows gt CBMS Ubiuinol cytochrome c reductase EC 1 10 2 2 cytochrome b mouse mitochondrion FASTA ID e Accession CBMS e Description Ubiuinol cytochrome c reductase EC 1 10 2 2 cytochrome b mouse mitochondrion Custom Database Support The Proteome Discoverer application has three general parsing rules to support custom sequence database formats The generic parsing rules are applied only if no other parsing rule matches the given FASTA title line e Custom Parsing Rule A e Custom Parsing Rule B e Custom Parsing Rule C Custom Parsing Rule A The application uses custom parsing rule A if the FASTA ID the accession number and the description are separated by a pipe symbol A typical FASTA title line that matches this parsing rule would look like this one gt tr 18FC3 18FC3_HALWD 1IS1341 type transposase Halouadratum walsbyi strain DSM 16790 FASTA ID 18FC3_HALWD e Accession 18FC3 e Description 1S1341 type transposase Halouadratum walsbyi strain DSM 16790 342 Proteome Discoverer User Guide Thermo Scientific A FASTA Reference Custom Database Support Custom Parsing Rule B The application uses custom parsi
242. e You can use this node as an alternative to a search node such as SEQUEST Figure 101 Workflow using the MSPepSearch node to search spectrum libraries o CQ Spectrum Files 0 A Spectrum W Selector Fixed Value PSM Validator The Fixed Value PSM Validator is the only possible peptide validator for the MSPepSearch node It is impossible to perform a decoy search because there is no proper decoy spectrum library For a description of the parameters available in the MSPepSearch node refer to the Help The spectrum libr ary search reports the three scores shown in Table 6 Dot score and reversed dot score are secondary scores and their values are not shown by default Table 6 Scores generated by the MSPepSearch node Score Description MSPepSearch Is the main score of MSPepSearch Dot score Is the score from a cross correlation computed between two spectra Reverse dot score Is the reversed spectral dot product Proteome Discoverer User Guide 139 4 Searching for Data Searching Spectrum Libraries Visually Verifying Spectrum Library Matches You can visually verify matches between measured spectra from your experiment and the reference spectra in the spectrum library for peptides identified with the SpectraST or the MSPepSearch node In the Peptide Identification Details view you can display a mirror plot of the matching peptides as shown in Figure 102 You can use the reference spe
243. e 141 Dynamic Modifications 2 4 42 9d ae ped a owed we Sed OL ae eR OS eee 141 Static Modifications e esteses cete or acannon tna iaka EE ae Sill Seasons 141 Opening the Chemical Modifications View 0000 cc eee eee 142 Adding Chemical Modifications v coe oeedlo ty anniek teen eae 144 Adding Amino Acids eien ieiti an tla ie ae aha iae paia i E 145 Deleting Chemical Modifications i042 ip ev da aes ie tte thie hae eee 146 Importing Chemical Modifications 0 e eee ee eee ee eee 146 Deleting Amino Acids o clio very itabead pba vee ob dope EREA 148 Using the Qual Browser Application 3 506 shay wearin bee ees 149 Customizing Cleavage Reagents tba ots Latin DA Re oe a 150 Adding a Cleavage Reagent ass conde needy Meanie nah cen te wanoauete ath 151 Deleting a Cleavage Reagent hati dal epics an eae Sam Calpe aa at 152 Modifying a Cleavage Reagent sot neni cndea keene in tae 152 Filtering Cleavage Reagent Data ic weeinass aes eae A a 8 152 Filtering Data cearr exert tated oe he a a we barnett eet 153 Res lt Filters Paces serisi uayt ia ae tenat e gerd a ei Enara E ALETE dodo wie 153 Filtering the Search Results ov sy shaie es Gd wes Bake se teow eae 154 Filtering Results with the Filters on the Result Filters Page 155 Wsitig Filter Sete igcnsce eG eww ution t Ree Aa w hs BEKO aU 163 Removing and Deactivating Filters stscjc cp ssce sey se asaceones dea galing 166 Filtering Results with Row Filters ccnouade
244. e Discoverer Daemon Application on the Command Line e p parameterFile rawFi le Processes the specified raw data file with all the parameters given in the parameter file including the connection to the server parameterFi le Specifies the name of the parameter file rawFile Specifies the name of the raw file In the following example of the p syntax the Proteome Discoverer Daemon application processes the 9mix_LysC_monolith raw file with the parameters given in the parameter file called c Xcalibur methods batchprocessing param DiscovererDaemon p C Xcalibur methods batchprocessing param 9mix_LysC_monolith raw f foldername On a remote server this option creates a user named folder in the PublicFiles folder of the server where the local version of the raw file and the result files are stored If the directory already exists the Proteome Discoverer Daemon application issues an error message and the process returns with exit code 1 standard exit code 0 If you attempt to create a file other than in the PublicFiles folder in the Current File Directory box Discoverer Daemon issues a message informing you that the Proteome Discoverer application will apply the change the next time that you start it This option performs the same function as the c foldername option except that you cannot use the name of the folder more than once On a local server this option does nothing Examples The following are some
245. e Irestriction enzymeHindVIIP Rprotein OS Haemophilus influenzae GN HI1285 PE 3 SV 1 gt sp 005074 HLDE_HAEIN Bifunctional protein hidE OS Haemophilus influenzae GN hldE PE 3 SV 1 gt sp 086224 KDKA_HAEIN 3 deoxy D manno octulosonic acid kinase OS Haemophilus influenzae GN kdkA PE 1 SV 1 gt sp P10324 PAL_HAEIN Outermembrane proteinP6 OS Haemophilus influenzae GN pal PE 1 SV MNKFVKSLLV AGSVAALAAC SSSNNDAAGN GAAQTFGGYS VADLQQRYNI VYFGFDKYDI IGEYVQILDA HAAYLNATPA AKVLVEGNID ERGIPEYNIA LGQRRADAVK GYLAGKGVDA GKLGIVSYGE EKPAVLGHDE AAYSKNRRAV LAY 13 Optional To specify any information that you want to exclude from the extracted results follow these steps a Click gi above the Step 2 String s to Exclude From the Results of Step 1 box on the Compile FASTA Database page A line enabling you to specify the first set of conditions now appears in the box b Click the first line in the Select Operator column and select the operator to apply to the information from the list You can choose from the following e Starts With Excludes information that begins with this string e Does Not Start With Excludes information that does not begin with this string e Ends With Excludes information that ends with this string e Does Not End With Excludes information that does not end with this string Thermo Scientific Proteome Discoverer User Guide 115 4 Searching for Data Using FASTA Databases 14 15 e Contains Excludes infor
246. e Parameters pane 9 Click the Spectrum Selector node and set the parameters for it in the Parameters pane a Change the setting in the Total Intensity Threshold box to an appropriate setting For example for TMTe 6plex quantification you could set this option to 20 000 b Change the setting in the Minimum Peak Count box to an appropriate setting For example for TMTe 6plex quantification you could set this option to 200 For other parameters that you can optionally set for the Spectrum Selector node refer to the Help 10 Click the search engine node for example SEQUEST and set the parameters for it in the Parameters pane a Inthe Protein Database box select the FASTA database b In the Dynamic Modifications area select the dynamic modifications Use the following modifications for a Sequest HT search e TMT 2plex seldom used TMT 2plex for lysine and N terminal you can use these as static or dynamic modifications 254 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Reporter lon Quantification Dynamic TMT 2plex for threonine e TMTe 6plex or TMT Gplex TMT 6plex for lysine and N terminal you can use these as static or dynamic modifications Dynamic TMT 6plex for threonine e TMT 10plex the same modifications as for TMT 6plex e iodo TMT 6plex iodo TMT 6plex for cysteine you can use these as static or dynamic modifications For example for TMTe 6plex qua
247. e following features e The light blue boxes represent the integration windows for the reporter tags The boxes are centered on the masses of the reporter tags as specified in the quantification method The width of the boxes is the integration window used for extracting the reporter tags It is 0 3 Da as specified by the settings of the parameters in the Reporter Ions Quantifier node you can look up all these values on the Quantification Summary page The height of the line in the box represents the actual tag intensity used for calculating the peptide ratios The height of the box represents the corrected tag intensity The height depends on the setting of the Integration Method parameter specified in the Reporter Ions Quantifier node It is always the value that results from correction for isotopic impurities as specified in the Reporter Ion Isotopic Distribution area of the Quan Channels page of the Quantification Method Editor dialog box shown in Figure 181 on page 272 e To calculate the actual intensity of a particular tag the Proteome Discoverer application chooses the blue fragment peaks from the spectrum and considers only peaks in the integration window Thermo Scientific Proteome Discoverer User Guide 299 7 Quantification Displaying the Quantification Spectrum Chart e The black fragment peaks represent peaks that are present in the spectrum but that are not chosen for calculating the tag intensities They might not be chosen be
248. e number of the chromosome that the gene resides on and the location of the chromosome that the gene resides on The name of the species is linked to the National Center for Biotechnology Information NCBI taxonomy browser Thermo Scientific Proteome Discoverer User Guide 223 6 Protein Annotation ProteinCard Parameters Keys Page Table 11 Parameters on the General page of the ProteinCard page Sheet 2 of 2 Command Gene Details area Description Displays information about the gene that directs the synthesis of the protein If no information about the gene is available a link to the Entrez database Web site is given Protein Details area Lists the keywords that produce this protein in a database search the functions of the protein and a description of the protein The Keys page of the ProteinCard shown in Figure 164 lists all the accession keys for a given protein Figure 164 Keys page of the ProteinCard page Deta Coverage ProteinCard General Features Molecular Functions Cellular Components Biological Processes Diseases External Links p Keys Primary Key Sre Secondary Key Sre Description ENSP00000434657 ESBL ENSP00000434412 ESBL ENSP00000350894 ESBL 333360851 GI NP 001193943 1 REF serpin H1 precursor hsa 871 KEGG 193783823 GI BAG53805 1 DBJ unnamed protein product 119595385 GI EAW74979 1 GB serpin peptidase inhibitor clade H heat shock protein
249. e of your results report appears as shown in Figure 111 Figure 111 Result Filters page File Search Report Quantification Processing Workflow Editor Administration Tools Window Help ua aago oocom omm R SequethT K Mso AA LHe RBEBERIARE Celegans_FT_6ITDDDT_01 O1 msf x 4 Proteins Peptides Search input Result Filters Peptide Confidence Search Summary Filter and Grouping Set E loa A Save A Factory Defauts Based on Fiter Set Filter or Grouping Settings Peptide Filters Active Filter V Peptide Rank IV Peptide Confidence Rx Add a Filter Protein Filters Active Filter Add a Filter Peptide Grouping Enabled Settings Protein Grouping Enabled Settings Ready 602 602 Protein Group s 761 2496 Protein s 1228 9557 Peptide s 1456 11499 PSM s 4766 4766 Search Input s Filtering the Search Results You can use Proteome Discoverer application filters to selectively hide and sort the visible results of the matched search results You have two methods of filtering your search results data e Results filters on the Result Filters page exclude peptides and proteins from the results on the Proteins and Peptides pages Applying these filters to filter out peptides does the following Changes the number of identified peptides and the percentages shown in the Coverage column of the Proteins page Affects the numbers of filtered
250. eanalyze the quantification results 10 Proteome Discoverer User Guide Thermo Scientific 1 Introduction Workflow Figure 3 The Proteome Discoverer workflow Experiments produce raw data Download a FASTA database Proteome Discoverer application Choose search wizard or define workflow in the Workflow Editor Identify raw file Upload FASTA database if you intend to use Sequest Select your search parameter settings Search the database Sort and filter search results view graphs and interpret search results Optional Review quantification results and change parameters Optional Re analyze quantification results Export search results and data to other applications Thermo Scientific Proteome Discoverer User Guide 11 1 Introduction Inputs and Outputs Inputs and Outputs The Proteome Discoverer application can accept several different file formats as input and can export data in several formats FASTA Databases The Proteome Discoverer application includes FASTA databases including multiple example FASTA databases and example raw files Use these files when exploring and learning how to use the application For a detailed description of the different types of FASTA databases and their purpose see Using FASTA Databases on page 101 Inputs The Proteome Discoverer application accepts the following file types as input Xcalibur raw files contain raw data collected from a mass spectrometer
251. ect the FASTA file that you want to process and then click Open The FASTA file that you selected appears as a job in the job queue To cancel the addition of this file click i Abort When you see the Completed in the Execution State column the database has finished downloading To add another FASTA file wait until the Execution State column indicates that the addition of the FASTA file is completed click FASTA Files in the left pane of the Administration page under Content Management and then click Add to add the next file The amount of time that it takes to process a FASTA file depends on the file size When a FASTA file finishes processing the Status column displays the Available status The FASTA file is now available to use for a protein or peptide search with the Proteome Discoverer application Deleting FASTA Files You can delete a FASTA file from the application bd I To delete a FASTA file Choose Administration gt Maintain FASTA file The Administration page appears with the FASTA files view shown in Figure 74 on page 102 Click gt at the beginning of a row to select the row Click x Remove In the Remove FASTA databases dialog box click OK 104 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases The FASTA file that you selected appears as a job in the job queue After you start the deletion of the file you cannot cancel the deletion You can
252. ecursor ion and one peptide that is contained in one protein The quantification result is unique for this protein The Proteome Discoverer application can mark peptide A Unique in the Quan Info column of the Peptides page if the quantification result meets other criteria Figure 214 Case 1 Quantification result associated with one identification spectrum one peptide and one protein Quantification result A Unique Thermo Scientific Unique Proteome Discoverer User Guide 321 7 Quantification Calculating Protein Ratios from Peptide Ratios Case 2 Two Quantification Results Associated with Two Spectra One Peptide and One Protein Case 2 shown in Figure 215 is a variant of case 1 Each of two different quantification results is associated with a different identification spectrum Both identification spectra identify peptide A which is a peptide with the same sequence Peptide A is only contained in one protein Each of the two different quantification results is unique for just one protein The peptides are redundantly identified and quantified and you could use both for the quantification of protein A Figure 215 Case 2 Two different quantification results associated with two identification spectra one peptide and one protein Quantification result A Peptide A 1 Unique P Unique Case 3 Quantification Result Associated with Two Spectra Two Peptides and One Protein Case 3 shown in Fi
253. ed browser Go Slim Specifies the basic GO Slim category for the GO term GO Slim categories are reduced versions of the GO ontologies containing a subset of the terms in the entire GO database They give a broad overview of the ontology content without the detail of the specific fine grained terms Table 19 provides the Go Slim categories for biological components Name Describes the biological process for a GO term This description is created by the GO consortium Enzymes with an EC number for IUBMB Enzyme Nomenclature are displayed with links to detailed information at the International Union of Biochemistry and Molecular Biology 230 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation ProteinCard Parameters Diseases Page The Diseases page of the ProteinCard page shown in Figure 169 lists the diseases that the selected protein is associated with Figure 169 Diseases page of the ProteinCard page Coverage ProteinCard General Keys Features Molecular Functions Cellular Components Biological Processes External Links Diseases Defects in SERPINH1 are the cause of osteogenesis imperfecta type 10 O110 MIM 613848 A connective tissue disorder characterized by bone fragility low bone mass bowing of limbs due to multiple fractures short limb dwarfism and blue sclerae External Links Page The External Links page of the ProteinCard page shown in Figure 170 lists the Web links
254. elp BH BEBOP STD SH g BBB g sequestht MK Mascot A A Alii a Celegans_FT_6ITDDDT_01 01 msf x Work lowEditor x Proteins Peptides Search Input Result Filters Pilptide Confidence Search Suil mary Protein Group Accessions m m CiS 2 cID 1 0000 0 0000 1 1 ic 2 J GHYTEGAELVDNVLDVR Unambiguous 2 CID 1 0000 0 0000 1 1 3 J7 KADADLTAISNDSSLSVQAK Unambiguous 1 cD 1 0000 0 0000 1 1 H 4 J7 NLITSVSSGAGSGPAPAAAA Unambiguous 1 CID 1 0000 0 0000 1 1 5 I NKLETELSTAQADLDEVIK Unambiguous 2 cD 1 0000 0 0000 1 1 6 J HTDAVAELTDQLDQLNK Unambiguous i CID 1 0000 0 0000 1 1 7 J7 LVAQKPINDAPAIDLHVGSK Unambiguous 1 CID 1 0000 0 0000 i 1 I re EA REEE in 602 602 Protein Group s 761 2496 Protein s 1228 9557 Peptide s 1456 11499 PSM s 4766 4766 Search Input s Figure 122 Logic operator and command menu File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 6H SB BOTBRTD SH LBBB K sewetht K Mascot Celegans_FT_6ITDDDT_01 01 msf x Workflow Editor x 1 J GHYTEGAELVDNVLDVR 2 GHYTEGAELVDNVLDVR 3 KADADLTAISNDSSLSVQAK 4 J7 NLITSVSSGAGSGPAPAAAA 5 NKLETELSTAQADLDEVIK 6 J HTDAVAELTDQLDQLNK 7 J7 LVAQKPINDAPAIDLHVGSK 8 J
255. ent Centroid Integefontoemn FTMS HCD Precursor z 3 Mono m z 672 02185 Da MH 2014 05100 Ds Integration Most Confident Centroid Integrati 9000 5 8000 J 131 13855 s000 4 7000 z 1 7000 7 6582 6000 129 13843 z 6000 4 7 a z1 2 sooo 4 pom u 128 13483 REA E 4000 4 3736 E s E 3000 3000 4 zow pon 127 13141 2000 4 1778 126 89038 1000 1000 4 bhi ie o at o 4 e x l i 126 127 128 123 130 131 132 126 127 128 131 mz a Quan Channels 7 z Ready 1021 3819 Protein Group s 9007 9007 Protein s 16804 16804 Peptide s 22705 22705 PSM s 2504 2504 Search Input s You exclude spectra with one or more missing reporter peaks from the protein ratio calculation by selecting the Reject All Quan Values If Not All Quan Channels Are Present option on the Ratio Calculation page Displaying the Quantification Spectrum Chart for Precursor lon Quantification For precursor ion quantification the Quantification Spectrum chart displays a quantification spectrum for each peptide It also displays the different abundances of the identified Light Medium and Heavy isotopic peak patterns used to quantify a peptide The abundances are measured by calculating the area of the extracted ion chromatogram of each isotope of a pattern The chart highlights the corresponding isotope pattern peaks and labels them with the quantification channel names as shown in Figure 204 It also includes any peaks that are not part of an isotope pa
256. eonine T Thr 101 04768 101 1051 C4H NO Cysteine C Cys 103 00919 103 145 C3H NOS Isoleucine I Ile 113 08407 113 1598 CH NO Leucine L Leu 113 08407 113 1598 CH NO Asparagine N Asn 114 04293 114 1039 C4H6N203 Aspartic Acid D Asp 115 02695 115 0885 C4H NO3 Glutamine Q Gln 128 05858 128 13091 Cs5HgN 202 Lysine K Lys 128 09497 128 1745 C6H N O Thermo Scientific Proteome Discoverer User Guide 345 B Chemistry References Enzyme Cleavage Properties Table 25 Amino acid mass values Sheet 2 of 2 Amino acid One letter Three letter Monoisotopic Average Sum code code mass mass formula Glutamic Acid E Glu 129 0426 129 1156 C H7NO3 Methionine M Met 131 0405 131 1994 C5H NOS Histidine H His 137 05891 137 1414 C6H7N30 Phenylalanine F Phe 147 06842 147 1772 CyH NO Arginine R Arg 156 10112 156 188 CoH 2N O Tyrosine Y Tyr 163 06332 163 17661 CHNO Tryptophan Ww Trp 186 07932 186 2141 C11H10N20 Table 26 Special amino acids One letter Three letter Monoisotopic Average Sum Amino acid code code mass mass formula Avrg N D B Bnd 114 53494 114 5962 C H5NO3 Avrg E Z Ze 128 55059 128 62326 C5H7NO Unknown acid X X Xxx 110 110 N A Pyrrolysine O Pyl 237 14772 237 29874 Ci2H19N302 Seleno Cysteine U Sec 150 95309 150 0369 C3H NOSe Enzyme Cleavage Properties Table 27 lists the enzymes and reagents with cleavage properties Table 27 Cleavage properties of enzymes and reagents Sheet 1 of 2 Cleaves
257. eptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Filter and Grouping Set Active Filter IV Peptides Per Pr O mw E oa Seve Factory Defauts Based on Filter Set Filter or Grouping Settings Peptide Filters Peptides Per Protein Active Filter Add a Filter Minimal number of peptides 18 Protein Filters V Count only rank 1 peptides V Count peptide only in top scored proteins Add a Filter Peptide Grouping Enabled Settings Protein Grouping Enabled Settings Ready 41 103 Protein Group s 204 2266 Protein s 169 3286 Peptide s 617 7721 PSM s 1541 1541 Search Input s Set the options pertaining to the selected filter in the Filter or Grouping Settings area For example in Figure 112 you can set the Minimal Number of Peptides and also select the Count Only Rank 1 Peptides and the Count Peptide Only in Top Scored Proteins options If it is not already selected select the check box in the Active column The check box is selected by default To remove a filter before you apply it click ge To update the search results click in the Filter and Grouping Set area Note The Proteome Discoverer application might take several seconds to display the filtered data Filtering Search Results with Peptide Filters Follow this procedure to apply peptide filters to your search results
258. er When you examine the quantitative differences between two measurements all these single factors combine to create an overall variability of the quantitative values under investigation for example the expression levels of certain proteins This overall variability can be quite significant To minimize the variability when comparing two samples such as different treatments or disease states and to calculate a statistical measure of the inherent variability you must measure replicates In this process you repeatedly measure a sample multiple times and calculate the average values for the quantitative values under investigation You perform these measurements for all states of the sample and then compare the calculated average values You can then calculate whether a detected difference between two states of a sample is statistically significant or is only due to the inherent variability of the sample In the Proteome Discoverer application you can load multiple result files containing quantification results and treat the single results as replicates of the same sample or as different treatments of a sample You determine whether the single results of an open multiconsensus report should be treated as replicates or treatments and you can change them from replicates to treatments and vice versa As an example assume that you have three result files from measuring and processing a yeast sample result_1 msf result_2 msf and result_3 msf Assu
259. er Ion Isotopologues with Isobaric Masses Analytical Chemistry 2012 Volume 84 7469 7478 2 Werner T Becher I Sweetman G Doce C Savitski M M and Savitski B M High Resolution Enabled TMT 8 plexing Analytical Chemistry 2012 Volume 84 7188 7194 Proteome Discoverer User Guide 251 7 Quantification Performing Reporter lon Quantification Figure 173 Structures of the TNT reagents contained in the TMT 10plex quantification method TMT 1265 okei TEn ii Fh A Pele Tht st TAQ dQ PAA PRA KAN Recent research concludes that avoiding the application of any correction for isotopic impurities improves quantification results for the TMTe 6plex TMT 10plex and iodo TMT 6plex kits so the default methods for these kits turn off the purity correction iTRAQ Quantification iTRAQ is a protein quantification technique that uses isobaric amine specific stable isotope reagents to label all peptides in up to eight different samples simultaneously The labeled peptides from each sample are combined separated by two dimensional liquid chromatography and analyzed with tandem mass spectrometry MS MS The same peptide from each sample appears as a single peak in the MS spectrum In single MS mode the differentially labeled versions of a peptide are indistinguishable In tandem MS mode which isolates and fragments peptides each tag generates a unique reporter ion Protein quantification compares the peak intensity
260. er activity Mediation of the transfer of a signal from the outside to the inside of a cell by means other than the introduction of the signal molecule itself into the cell Structural molecule activity The action of a molecule that contributes to the structural integrity of a complex or assembly within or outside a cell Transcription regulator activity Activity that plays a role in regulating transcription it might bind a promoter or enhancer DNA sequence or interact with a DNA binding transcription factor Translation regulator activity The initiation activation perpetuation repression or termination of polypeptide synthesis at the ribosome Transporter activity Activity that enables the directed movement of substances such as macromolecules small molecules ions into out of within or between cells GO Slim Categories for Cellular Components Table 18 describes the GO Slim categories for cellular components Table 18 GO Slim categories for cellular components Sheet 1 of 4 GO Slim cellular component Cell surface Description Proteins that are attached to the external part of the cell wall cell membrane or both Chromosome A structure composed of a very long molecule of DNA and associated proteins for example histones that carry hereditary information Cytoplasm All of the contents of a cell excluding the plasma membrane and nucleus but including other subcellular structures
261. er application running on a remote machine specify in the Server Output Directory box the name of the folder where you want the original output files placed on the server and then click Start Thermo Scientific Proteome Discoverer User Guide 217 6 Protein Annotation Uploading Results to ProteinCenter By default the Proteome Discoverer Daemon application places this folder in the c Documents and Settings All Users DiscovererDaemon SpectrumFiles directory You can specify a different folder by choosing Administration gt Configuration in the Proteome Discoverer application clicking Discoverer Daemon in the Server Settings section and browsing to the location in the New Directory box Figure 158 shows MSF files being processing in batch mode in Discoverer Daemon Figure 158 Reannotating MSF files in batch mode in Discoverer Daemon Discoverer Daemon kaka Start Jobs Configuration Job Queue Spectrum Files Workflow WF_Re Annotation x 2 b Load Files Export Parameter File Server Output Directory Add Remove Start local connection WF_Re Annotation C Program Files Proteome Discoverer source files 10mixTMT_45CE_1us_Ratio_1 Output Filename test_filename Processing completed workflow 10mixTMT_45CE_1us_Ratio_1to1 Sequest Processing workflow 10mixTMT_45CE_1us_Ratio_1to 1 Sequest ID 35 Registering C Program Files Proteome Discoverer source files 10mixTMT_45CE_1us_Ratio_1t
262. er proteins Proteins are grouped according to the peptide sequences identified for the proteins A protein group consists of the following e One master protein that is identified by a set of peptides that are not included all together in any other protein group e All proteins that are identified by the same set or a subset of those peptides The Proteins column on the Proteins and Peptides pages of the results report displays the number of identified proteins in the protein group of a master protein It should match the number of proteins that are displayed in the Protein Group Members view when you choose Search Report gt Show Protein Group Members see Figure 132 on page 178 Protein groups can overlap because proteins might be included in several master proteins Each of two compared master proteins must have at least one peptide that is not contained in the other master protein However if you do not select the Apply Strict Maximum Parsimony Principle option in the Protein Grouping area of the Result Filters page the peptides that distinguish these two master proteins could be contained in other master proteins A master 174 Proteome Discoverer User Guide Thermo Scientific Figure 130 Expanding an identified peptide 5 Filtering Data Grouping Proteins protein does not have to contain a unique peptide unless you select the Apply Strict Maximum Parsimony Principle option A unique peptide is only contained in the proteins of
263. ere you downloaded and unpacked the _consensus_final_true_lib tar gz file c Select the filename msp file d Click Open If you want to add a gz or zip file to the Proteome Discoverer application a Browse to the location of the spectrum library where you downloaded the _spectra tar gz file b Select the filename_spectra tar gz file c In the list box in the lower right corner of the Select a Spectrum Library dialog box select All Spectrum Library Files msp gz zip or Zip archives gz zip d Click Open When you add a spectrum library file the Proteome Discoverer application takes the following steps e Constructs the library from the filename msp file or extracts the archive file e Creates a decoy spectrum library and other files needed for the actual search e Extracts spectra for visualization During library creation the job queue in the Administration view displays each step as shown in Figure 96 132 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Searching Spectrum Libraries Figure 96 Adding a spectrum library for searching with the SpectraST node File Search Report Quantification Processing Workflow Editor Administration Tools Window Help Thermo Scientific 64 88 alo y aaora Z omm R SequethT Q Maso i A R AE REBERIARE Administration x 4b 1 G Pouse GB Resume jf Abort JE Remove Refresh I O
264. ernal Links Biological Processes GO Id Evidence codes PMIDs Go Slim Name GO 0006950 IEA TAS response to stimulus response to stress GO 0006986 IEA TAS 10023073 1309665 response to stimulus response to unfolded protein GO 0010951 ISS IBA regulation of biological process negative regulation of endopeptidase activity metabolic process GO 0030162 ISS IBA regulation of biological process regulation of proteolysis metabolic process GO 0030198 TAS cell organization and biogenesis extracellular matrix organization GO 0030199 IEA cell organization and biogenesis collagen fibril organization GO 0032964 IEA metabolic process collagen biosynthetic process GO 0051604 IEA metabolic process protein maturation Table 16 lists the parameters on the Biological Processes page of the ProteinCard page Table 16 Parameters on the Biological Processes page of the ProteinCard page Command Description GO Id Lists the GO code for each of the protein s molecular functions Each code is linked to the QuickGO browser of the EBI which hosts a number of databases and services Evidence Codes Lists the evidence codes for each of the protein s biological processes for GO annotation Evidence codes describe how the GO information was proven for example by computer prediction or by experiment PMIDs Lists the biological process codes in the PubMed database which is maintained by the NLM and the NIH Each code is linked to the PubM
265. ers Peptide Confidence Search Summary Search name Sequest SILAC Dimethylation Search 1 3 0 297 Search description 081023 _RR_BSA_1_1_ 1 2plus RAW ipi BOVIN v3 50 fasta Dimethylation 3plex C2H6 C2H2D4 13C2D6 Search date 04 27 2011 09 24 37 Mass Precision S N Threshold 1 m Quantification Method Dimethylation 3plex C2H6 C2H2D4 13C2D6 2 Isotope Pattern Identification RT Tolerance of Isotope Pattern Multiplets min 0 2 Single Peak Missing Channels Allowed Quantification Method Dimethylation 3plex C2H6 C2H2D4 13C2D6 Method Descripti methylation 3plex C2 13C2D4 Method Light Dimethyl K Dimethyl 28 031 Da K Dimethyl N Term Dimethyl 28 031 Da Any N Terminus Medium Dimethyl K Medium Dimethyl 2H 4 32 056 Da K Dimethyl N Term Medium Dimethyl 2H 4 32 056 Da Any N Terminus Heavy Dimethyl K Heavy Dimethyl 2H 6 13C 2 36 076 Da K Dimethyl N Term Heavy Dimethyl 2H 6 13C 2 36 076 Da Any N Terminus Ratio Calculation Show the Raw Quan Values False Minimum Quan Value Threshold 0 Replace Missing Quan Values With Minimum Intensity False Use Single Peak Quan Channels False Apply Quan Value Corrections True Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s 292 Proteome Discoverer User Guide Thermo Scientific
266. ertain search parameters for the Sequest HT SEQUEST and Mascot search engines To configure search parameters 1 Choose Administration gt Configuration or click the Edit Configuration icon The Administration page changes to the Configuration view shown in Figure 5 Figure 5 Configuration view of the Administration page File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BSE S S TIBOR SH Z BB WL 0 Seth MmMaxt Ak LHe ABEBRRBSARE C18 2mgPBMC Sample A 200uLbeads 01 PhosPhoNodemsf xX Administration x 4p Apply A Reset Factory Defaults x Process Management 1 ProtenCorter Server a js ProteinCenter URL ha Nebservice proteincenter proxeon com ProXweb Job Queue Number of attempts to submit the annotation request Time interval between attempts to submit the annotation request sec a Timeout of the annotation request min 15 a FASTA Files B FASTA Indexes m ectral Librari iu SP al Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management L R Licenses Configuration a 48 Workflow Nodes E Mascot E MSPepSearch E SEQUEST od Sequest HT E FASTA Indexes ProteinCenter URL The URL of ProteinCenter Ready 2 Follow these procedures e Configuring the SEQUEST Search Engine e Configuring the Mascot Search Engine e Configuring the Seq
267. es Strings check box Click above the Step 1 String s to Include box A line enabling you to specify the first set of conditions now appears in the box Click the first line in the Select Operator column and select Contains if it is not already selected Leave the first line in the Condition column blank Click gi above the Step 2 String s to Exclude From the Results of Step 1 box A line enabling you to specify the first set of conditions now appears in the box Click the first line in the Select Operator column and select Contains 116 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases 10 In the first line of the Condition column type the protein reference or sequence that you want to remove 11 Click Compile Database The compiling process creates the target FASTA file that excludes protein entries that match the condition Managing FASTA Indexes A FASTA index is a type of lookup table containing masses theoretical peptide sequences and associated proteins which minimizes search time The index lists all possible amino acid sequences that can be produced when an enzyme digests a protein or peptide The peptide fragments are listed by molecular weight The index stores information about every nominal mass every peptide that has that mass every protein that contains this peptide and the location of its protein description in the FASTA file Rather than read al
268. es and proteins by site localization scores from phosphoRS e Setting and Clearing Row Filters e Displaying Filtered Out Rows e Filtering Precursor Masses e Filtering PSMs and Peptides for Site Localization Scores from phosphoRS e Grouping Proteins Setting and Clearing Row Filters You can use row filters on the Proteins Peptides and Search Input pages to set up simple filter criteria that only consist of a single filter statement such as number is greater than 5 or text contains kinase To filter your search results using row filters 1 Open your search results 2 Select the Proteins Peptides or Search Input page 3 Ungroup the peptides by right clicking and choosing Show Peptide Groups 4 Right click to access the shortcut menu and choose Enable Row Filters Thermo Scientific Proteome Discoverer User Guide 167 5 Filtering Data Filtering the Search Results A filter row appears beneath the column header that contains the icons shown in Figure 121 For a description of these icons see the Help You can select an operator enter the filter value clear the currently set filter or open the Enter filter criteria for header_name dialog box for more complex transactions Figure 121 Row filter icons Logic operator and command menu icon Down arrow icon Logic operator menu icon Clear filter criteria icon File Search Report Quantification Processing Wijrkflow Editor Administratic i Tools Window H
269. es of the protein If you want to recalculate the peptide ratio you must ensure that all peptides are displayed By default the application considers only unique peptides in the calculation so that only peptides that have no other protein references are considered You can activate a chart of the peptide ratios This graph shows the distribution of peptide ratios for the selected protein displaying the ratios of the peptides associated with the selected protein as a log2 fold change To calculate peptide ratios 1 Click the row of the peptide or protein that you are interested in 2 Choose Quantification gt Show Peptide Ratios or click the Show Peptide Ratios icon H The Peptide Ratio Distributions chart shown in Figure 210 appears The following sections describe the pages available in this view 314 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Calculating Peptide Ratios Figure 210 Peptide Ratio Distributions chart File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OH BEB OTSAAOAH BAT K sme Kur ARABS GEBTAFARE PQD with TMT 6 plex 10 175 min msf X 4 gt Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary fe Accession Description Score F Coverage Proteins UniquePeptides Peptides PSMs 131 126 131 126 Count 131 126 Variabili m 1 J ALBU_BOVIN
270. esult Filters Peptide Confidence Search Summary Filter and Grouping Set O aw amp load H Save KA Factory Defauts Based on Filter Set C ProgramData Thermo Discoverer Demo 1 4 FilterSets FilterSet 1 filters Filter or Grouping Settings Peptide Delta Cn Peptide Filters Active Filter V Peptide Confidence Vv Vv Peptide Mass Deviation Maximum delta Cn 0 1 Add a Filter Protein Filters Active Filter V Protein Score x Add a Filter Peptide Grouping Enabled Settings Protein Grouping Enabled Settings Ready 602 602 Protein Group s 761 2496 Protein s 1228 9557 Peptide s 1456 11499 PSM s 4766 4766 Search Input s To delete a filter set L e Click next to the peptide filters protein filters or grouping sets that compose the set Thermo Scientific Proteome Discoverer User Guide 165 5 Filtering Data Filtering the Search Results To clear the default filter set 1 Remove all the peptide and protein filters from the Result Filters page by clicking 8 next to the peptide filters protein filters or grouping sets that compose the set 2 Click See 3 In the Save Filter Set dialog box shown in Figure 116 on page 164 select the Save As Default Filter Set option 4 Click OK To restore the default filter set in effect after installing the Proteome Discoverer application 1 Click Factory Defaults The confirm
271. esult Filters Peptide Confidence Search Summary Quantification Summary Filter and Grouping Set iv E Load E Save iA Factory Defaults Based on Filter Set Filter or Grouping Settings Peptide Filters Protein Grouping Options Active Filter Add a Filter 7 Enable protein grouping Protein Filters Consider only PSMs with confidence at least Medium m Active Filter Consider only PSMs with delta Cn better than 0 15 j Add a Filter V Apply strict maximum parsimony principle Peptide Grouping Disabled Settings Protein Grouping Enabled Settings an Ready 292 1120 Protein Group s 2266 2266 Protein s 3286 3286 Peptide s 7721 7721 PSM s 1541 1541 Search Input s 4 If you want to group homologous proteins select the Enable Protein Grouping check box if it is not already selected by default 5 To specify the type of PSMs that the Proteome Discoverer application considers for inclusion in protein grouping set the Consider Only PSMs with Confidence at Least parameter to the desired setting e Low Considers all low medium and high confidence PSMs for inclusion in protein grouping e Default Medium Considers medium and high confidence PSMs for inclusion in protein grouping e High Considers high confidence PSMs for inclusion in protein grouping 6 If you want the Proteome Discoverer application to consider only PSMs with values lower
272. esults appear in the Heavy Light Count column of the Proteins page of the MSF report for precursor ion quantification and in the Ratio Count columns of the Proteins page for reporter ion quantification For more information on ratio counts see Ratio Count on page 324 This option is selected by default If you want to show the variability of the peptide ratios used to calculate the protein ratios select the Show Protein Ratio Variabilities check box The results appear in the Heavy Light Variability column of the Proteins page of the MSF report for precursor ion quantification and in the Ratio Variability columns of the Proteins page for reporter ion quantification For more information on protein variability see Ratio Variability on page 324 This option is selected by default If you want to define peptide uniqueness on the basis of protein groups rather than on individual proteins select the Consider Proteins Groups for Peptide Uniqueness check box This option is selected by default Choose the type of peptides for the Proteome Discoverer application to use in the quantification e Default Use Only Unique Peptides Includes peptides that do not occur in other proteins Proteome Discoverer User Guide 279 7 Quantification Setting Up the Quantification Method e Use All Peptides Includes all detected peptides whether or not they also occur in other proteins 6 Continue setting up the qua
273. esults of the single searches are simply displayed side by side and the variabilities are the same as those of the single reports For more information on how the Proteome Discoverer application calculates protein ratios when treating quantification results in multiconsensus reports as treatments see Calculating Protein Ratios in Multiconsensus Reports Treated as Treatments on page 328 Calculating Variability in Multiconsensus Reports Treated as Replicates For multiconsensus reports that treat quantification data as replicates the Proteome Discoverer application calculates the protein ratios for single searches and then calculates a classical coefficient variation for these ratios It calculates the variability of the protein ratio calculated from WN replicates from the protein ratio r 7 of the single searches std dev r 7 variability r r CV 100 x hace a Using the protein ratios rather than their logarithms is reasonable because in contrast to the peptide ratios which are at least approximately log normally distributed the protein ratios of the single searches should be normally distributed at least for larger values of n For more information on how the Proteome Discoverer application calculates protein ratios when treating quantification results in multiconsensus reports as replicates see Calculating Protein Ratios in Multiconsensus Reports Treated as Replicates on page 328 326 Proteome Discoverer
274. etector Spectrum Selector E Spectrum Processing ig Noise Peak Filter ooma 2B WD g equetH g Mascot AA LUG RABEBTERBPARGE Workflow Editor x 4 Workflow Nodes a v Parameters va A Data Input pS A m EB Hide Advanced Parameters aia Spectrum Files Description ug Non Fragment Filter ug Spectrum Grouper i Spectrum Normalizer ig Top N Peaks Filter E Spectrum Filters T Scan Event Filter Ti Spectrum Confidence Filter i Spectrum Properties Filter Sequence Database Search SEQUEST Q Sequest HT E Spectral Library Search Q MsPepSearch Q SpectrasT PSM Validation Jih Fixed ValuePSM Validator Percolator Target Decoy PSM Validator PTM Analysis if phosphors Quantification Precursor Ions Area Detector Precursor Ions Quantifier 2 Reporter Ions Quantifier E Data Export i Spectrum Exporter Q Mascot m Thermo Scientific Proteome Discoverer User Guide 57 2 Getting Started Starting a New Search by Using the Workflow Editor The following instructions show you how to create the simple parallel workflow shown in Figure 35 To create a parallel workflow 1 Drag the Spectrum Files node to the workspace pane and specify the name and path of the raw data file in the Parameters pane 2 Drag the Spectrum Selector node to the workspace
275. examples of the Proteome Discoverer Daemon command line syntax This example constructs the spectrum file collection called Rawfiles adds the TrypMyo raw file to the collection and executes the SequestEquine workflow using the raw file in the Rawfiles directory DiscovererDaemon c Rawfiles a Rawfiles c Rawfiles TrypMyo raw e Rawfiles ANY c workflows SequestEquine xm In the following example the Proteome Discoverer Daemon application evaluates several fractions in a single workflow DiscovererDaemon c Fractions DiscovererDaemon a Fractions c rawfiles fractionl raw DiscovererDaemon a Fractions c rawfiles fraction2 raw DiscovererDaemon a Fractions c rawfiles fractionN raw DiscovererDaemon e Fractions ANY c wfs fractions xml The next example demonstrates that you can start several workflows with one invocation of the Proteome Discoverer Daemon application DiscovererDaemon Thermo Scientific Proteome Discoverer User Guide 99 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application on the Command Line 100 c RawFile a RawFile c Rawfiles TrypMyo raw e RawFile ANY c wfs SequestEquine xml c RawFile a RawFile c Rawfiles BSADigest raw e RawFile ANY c workflows SequestEquine xml The following example runs the Proteome Discoverer Daemon application on a remote host called protlab2 uploads the TRA_BSA_3ITMS2_3HCD raw spectrum file to the server execute
276. ext time that the server starts even though you clicked OK in the FASTA Indexes Options dialog box and closed it For example when you change the location of the directory in the FASTA Indexes Options dialog box click OK and close the dialog box the server moves all FASTA indexes to the new target directory when the server restarts But if you reinvoke the dialog box and click Reset before restarting the server the changes that you made previously are deleted and the directory reverts to its previous location To reset the changes made in a previous FASTA index session 1 Click the Options icon g Options The FASTA Indexes Options dialog box appears as shown in Figure 94 on page 128 2 Click Reset Removing FASTA Indexes When a FASTA File Is Deleted When you delete a FASTA file in the Proteome Discoverer application it removes the FASTA indexes belonging to the deleted FASTA file the next time that the server starts Searching Spectrum Libraries Thermo Scientific Spectrum library search is a different search approach from the sequence database search ubiquitously used in shotgun proteomics The main difference between a database search and a spectrum library search is in the origin of the spectra that the measured spectra from your experiments are compared to Sequence database searches use theoretical spectra generated from peptide sequences but spectrum libraries are libraries of measured consensus spectra from actual previ
277. f each label applied to at least one amino acid unless you chose None for a modification e Each amino acid labeled only once in a channel Labels must have an elemental composition defined e Each label mass used only once label masses vary by at least 1 0 Da You cannot apply changes to a quantification method unless the method meets all these criteria Restoring Quantification Method Template Defaults If you have altered one of the quantification method templates listed at the beginning of Setting Up the Quantification Method on page 264 you can restore the original template Thermo Scientific Proteome Discoverer User Guide 281 7 Quantification Setting Up the Quantification Method To restore the original template 1 Choose Administration gt Maintain Quantification Methods or click on the Maintain Quantification Methods icon The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification 2 To open the Quantification Method Editor dialog box click Add in the Quantification Methods view The Create Quantification Method dialog box opens as shown in Figure 192 on page 287 3 Select the appropriate template from the Create from Factory Defaults list 4 Set up the quantification method according to the instructions in Setting Up the Quantification Method on page 264 Setting Up the Quantifica
278. f you have not already done so See Adding FASTA Files on page 104 e Make spectrum source files available as RAW MGF MZDATA MZXML or MZML files Creating a Search Workflow 44 You can use the following procedure to process one raw file from one sample multiple raw files from one sample or multiple raw files from multiple samples For additional details on creating a workflow for multiple raw files from one sample see Creating a Search Workflow for Multiple Raw Files from the Same Sample on page 53 For a demonstration showing how to create a new workflow see Demonstrating How to Create a Workflow on page 51 To create a new workflow 1 Choose Workflow Editor gt New Workflow or click the New Workflow icon The Workflow Editor opens as shown in Figure 24 on page 43 2 In the Name box in the workspace pane type a name for the workflow 3 Optional In the Description box type a description of the workflow 4 To perform two searches using the same search engine node and then merge the search results in the output MSF file select the Merge Results of Equal Search Nodes check box 5 From the Data Input area of the Workflow Nodes pane drag the Spectrum Files node to the workspace pane 6 Select the Spectrum Files node if it is not already selected 7 Select the data input file a In the Input Data section at the top right of the Parameters pane click the File Name s row see Figure 24 o
279. ffect the method in the selected results report Removing a Quantification Method You can delete a quantification method if it is no longer useful or make a quantification method temporarily unavailable to new workflows To remove a quantification method 1 Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification 2 Click the box to the left of the method that you want to remove The Remove icon Remove now becomes available 3 Click 8E Remove 4 In the Delete Methods dialog box click OK To deactivate a quantification method 1 Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g The Quantification Methods view opens as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification 2 Clear the check box in the Is Active column on the line containing the quantification method that you want to render inactive To make the quantification method active again select the same check box 290 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Importing a Quantification Method Importing a Quantification Method You can import a new quantification metho
280. fic ee i Introduction This chapter introduces you to the Proteome Discoverer application and describes its features and functionality Contents e Features e Workflow e Inputs and Outputs e Limitations e New Features in This Release Features The Proteome Discoverer application identifies proteins from the mass spectra of digested fragmented peptides It compares the raw data from mass spectrometry to the information from the selected FASTA database You can use this application to analyze spectral data from all Thermo Scientific and other mass spectrometers Specifically the Proteome Discoverer application does the following e Works with peak finding search engines such as Sequest and Mascot to process all data types collected from low and high mass accuracy mass spectrometry MS instruments The peak finding algorithm searches the raw mass spectrometry data and generates a peak list and relative abundances The peaks represent the fragments of peptides for a given mass and charge Produces complementary data from a variety of dissociation methods and data dependent stages of tandem mass spectrometry e Combines filters and annotates results from several database search engines and from multiple analysis iterations The search engines correlate the uninterrupted tandem mass spectra of peptides with databases such as FASTA See Using FASTA Databases on page 101 Thermo Scientific Proteome Discoverer Use
281. fication Spectrum chart 304 identifying isotope patterns 332 setting up protein ratios 278 setting up quantification method 264 specifying label modifications 267 specifying quantification channel names 273 specifying quantification channels 266 268 summarizing settings for 292 troubleshooting 336 See also Precursor Ions Quantifier node Precursor Ions Area Detector node nodes not used with 259 performing peak area calculation quantification 7 55 259 using to access Quantification menu 242 Precursor Ions Quantifier node description 247 nodes not used with 247 performing precursor ion quantification 55 246 247 setting parameters 249 setting up quantification method 264 summarizing node settings 292 using to access Edit Quantification Method command 289 using to access Quantification menu 242 PrediSi database 226 PRF database 339 processing methods 82 85 Processing Setup icon 82 Processing Setup window 82 product limitations 14 Programs icon 82 Programs view 83 protein annotation configuring 204 creating workflow 206 Entrez gene database 202 206 Thermo Scientific GO database 202 206 Pfam database 202 203 206 UniProt database 202 206 protein binding GO Slim category 233 protein complex GO Slim category 237 protein databases 105 Protein Group Members view displaying 175 177 Is Master Protein column 178 matching number of proteins displayed to Proteins column 174 Protein Identification Det
282. files See tab delimited TXT files Type column 131 U U S National Library of Medicine NLM 227 229 230 UNIMOD importing chemical modifications 146 updating chemical modifications 141 UniProt database 362 Proteome Discoverer User Guide accession key 225 displaying annotation results from ProteinCenter in MSF file 215 retrieving annotations from ProteinCenter 206 retrieving information from ProteinCenter 202 208 specifying in ProteinCard 226 UniProt NREF database 341 UniRef database 341 UniRef50 database 341 UniRef90 database 341 Universal Protein Resource consortium 341 uploading search results to ProteinCenter 218 V vacuole GO Slim category 237 variability displaying 279 inherent in experiments 326 multiconsensus reports 325 W Welcome to the Search Wizard page 31 Workflow Editor aligning nodes 48 207 annotation workflow 206 attaching Fixed Value PSM Validator node to search engine nodes 46 attaching Percolator node to search engine nodes 46 changing names and descriptions of workflow templates 65 color of nodes in 46 creating an annotation workflow 55 creating new search workflow 44 51 creating parallel workflows 57 creating PTM analysis workflow 55 creating quantification workflow 55 creating workflow for MudPIT samples 53 deleting workflow templates 64 description 5 exporting spectra in multiple formats 66 features 2 importance of understanding nodes 42 importing from MSF or XML file 64 importing work
283. filters 167 pepXML files 13 Percolator node attaching to search engine nodes 46 setting thresholds for scores 195 setting up false discovery rates in Workflow Editor 190 very small searches 192 Pfam database accession identifier 226 computational enrichment 226 description 203 displaying annotation results from ProteinCenter in MSF file 214 features 203 Hidden Markov Model 203 retrieving information from 202 retrieving information from ProteinCenter 206 208 Pfam IDs column 203 206 214 phosphoRS node creating PTM analysis workflow 55 description 15 filtering PSMs and peptides for site localization scores from 172 phosphorylation calculating PTM site localization scores with phosphoRS node 55 PIR database 339 342 Please Select a FASTA Database dialog box 108 Position column 145 post acquisition method 89 post translational modifications PTMs determined by dynamic modifications 141 356 Proteome Discoverer User Guide PQD activation type description 9 ion factors 37 selecting in search wizards 33 specifying in Reporter Ions Quantifier node 256 334 precursor ion quantification calculating peptide ratios 313 317 checking the quantification method 281 controlling protein and peptide ratios 275 correcting experimental bias 280 correcting for isotopic impurities 277 creating workflow for 246 default methods available in 243 265 268 description 243 displaying quantification channel values 295 296 displaying Quanti
284. flows in MGF format 65 in MZDATA format 65 in MZML format 65 in MZXML format 65 incorrect parameter node settings 62 inputs 42 job queue See job queue joining nodes 47 Thermo Scientific opening 42 opening existing workflow 61 organizing nodes 47 outdated nodes 61 panes 42 parameter filters 50 renumbering nodes 48 207 saving workflow as template 50 saving workflow as XML template 66 setting node parameters 48 setting up false discovery rates Peptide Validator node 190 Percolator node 190 specifying raw file 44 starting a new search 42 starting workflow 51 using third party nodes 46 warning symbols 61 Workflow Failures pane 62 workspace definitions 6 Workflow Failures pane 62 Workflow Nodes pane 6 workflow templates changing names and descriptions of 65 deleting 64 opening 61 saving 50 Workspace pane 6 X x fragment ions definition 7 Xcalibur data system creating a parameter file 81 Qual Browser 149 running injections sequence that starts the Discoverer Daemon application 85 starting Discoverer Daemon in 2 0 7 78 starting Discoverer Daemon in 2 1 0 78 85 Xcalibur Home Page window 82 XCorr Confidence Thresholds parameter 22 24 XCorr Score Versus Charge peptide filter 197 XML files input to Proteome Discoverer application 12 64 output by Proteome Discoverer application 13 XML template 66 Y y fragment ions 347 348 y fragment ions abstracting proton from precursor 7 Thermo Scientific Index X
285. format see Saving a Workflow as an XML Template on page 66 2 In the Save Processing Workflow Template dialog box shown in Figure 32 do the following a Type a template name in the Template Name box b Type a description in the Template Description box c Click Save 50 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 32 Save Processing Workflow Template dialog box Save Processing Workflow Template Template Name elegans_sequest_flow Template Description c_elegans_100_021411_FWD_combined fasta basic SEQUEST workflow To perform the search 1 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon lt The job queue appears showing the status of your search 2 Use the job queue to check the status of your search as the search progresses For information about the job queue refer to the Help 3 Choose File gt Open Report to display your search results Refer to the Help a Filter and sort your results See Filtering Data on page 153 b Use different views to aid in your analysis Refer to the Help Demonstrating How to Create a Workflow The following demonstration shows you how to set up a workflow In this example a sample containing a trypsin digest of Caenorhabditis elegans a nematodal worm was submitted to an LTQ Orbitrap XL mass spectrometer at a resolution of 60 000 for M
286. g 24 2 O Oa O ODO o N ai ommmmm O O onma COO OO ENT COND A O o N 1 eae Oomen COCO O o N 1 CMD O OOD O OT Understanding the Peptide Ratio Distributions Chart The Peptide Ratio Distributions chart shows the distribution and spread of the ratios of all peptides belonging to a particular protein Figure 211 shows an example for the albumin protein Thermo Scientific Proteome Discoverer User Guide 315 7 Quantification Calculating Peptide Ratios Figure 211 Peptide Ratio Distributions chart Peptide Ratio Distributions ALBU_BOVIN P02769 Serum albumin precursor Allergen Bos d 6 BSA MASS 69293 log2 Fold Change 131 126 130 126 129 126 128 126 127 126 R 0 17 0 89 R 0 03 0 98 R 0 05 1 04 R 0 18 0 88 R 0 05 0 97 8 4 IQR 0 52 0 31 8 IQR 0 53 0 37 84 IQR 0 48 0 35 8 IQR 0 37 0 23 8 4 IQR 0 31 0 20 s a O CMa OO b o N ai ro ail Lani o ammo O OOUE O O o N a 1 1 amano cd OO NN O o 2 a JH diaman OO NED CO o N P 1 Cn o fa h a h The chart shows the distribution of peptide ratios for each of the ratios reported as defined in the quantification method for this search Each of the ratio distribution charts displays the peptide ratios as the binary logarithm The logarithmic form is common for such displays because it provides a reasonable display
287. g Workflow Editor Administration Tools Window Help Oa 8s8 8 e Administration _ gt 7AaDRRaS 2B DW DL 0 SequethHT MB Mascot A A LHC AZEBRBRBAR i Process Management py x Job Queue Content Management a FASTA Files E B FASTA Indexes Z p a PAS il Libr i Spectral Libraries a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management R Licenses Configuration B 43 Workflow Nodes amp Annotation 9 Mascot 3 MSPepSearch SEQUEST 53 Sequest HT 3 SpectraST B Server Settings a amp FASTA Indexes Apply A Reset Current File Directory C ProgramData Thermo Discoverer 1 4 PublicFiles New Directory mm Ready Thermo Scientific 3 Start the Proteome Discoverer Daemon application on the local machine A message box informs you that the Proteome Discoverer Daemon application cannot connect to the server 4 Click OK in the message box The Proteome Discoverer Daemon application opens with the Configuration page selected 5 In the Host box type the name of the remote computer 6 In the User box type the login name of the remote server 7 Click Apply Proteome Discoverer User Guide 71 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Running the Proteome Discoverer Daemon Application from the Xcalibur Data System You can u
288. gans_FT_6ITDDDT_01 RAW 25 S 20 oO F E 145 3 g 10 c 2 94 61 105 64 5 F 476 18 41 i 116 56 1 19 11 98 24 00 20 40 60 80 100 120 Time min Lower RT Limit min 0 007 Upper RT Limit min 119 981 5 Set the basic search parameters a 32 Proteome Discoverer User Guide In the Rawfile box click the Browse button to search for the raw file in the Open Analysis File s dialog box Note The Workflow Editor can accept multiple input raw data files but the search wizards cannot For information about creating a workflow for multiple input raw data files see Starting a New Search by Using the Workflow Editor on page 42 A base peak chromatogram for the raw data file appears on the page as shown in Figure 16 Select the range of data to use by choosing either of these methods e Hold down the CTRL key and drag the cursor over the range e Enter the beginning of the range in the Lower RT Limit min box Enter the end of the range in the Upper RT Limit min box Thermo Scientific Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards You might want to exclude the first few minutes of collected data in the raw data file because they contain no peptides or exclude the last few minutes because of cleanup at the end of the data collection 6 Click Next The Scan Extraction Parameters page appears as shown in Figure 17 Figure 17 Scan Extraction Para
289. ge Sheet 1 of 2 Command Description GO Id Lists the GO code for each of the protein s molecular functions Each code is linked to the QuickGO browser of the European Bioinformatics Institute EBI which hosts several databases and services Evidence Codes Lists the evidence codes for each of the protein s molecular functions for GO annotation Evidence codes describe how the GO information was proven for example by computer prediction or by experiment PMIDs Lists the molecular function codes in the PubMed database which is maintained by the U S National Library of Medicine NLM and the National Institutes of Health NIH Each code is linked to the PubMed browser Thermo Scientific Proteome Discoverer User Guide 227 6 Protein Annotation ProteinCard Parameters Table 14 Parameters on the Molecular Functions page of the ProteinCard page Sheet 2 of 2 Command Description Go Slim Specifies the basic GO Slim category for the GO term GO Slim categories are reduced versions of the GO ontologies containing a subset of the terms in the entire GO database They give a broad overview of the ontology content without the detail of the specific fine grained terms Table 17 on page 233 provides the Go Slim categories for molecular functions Name Describes the molecular function for a GO term This description is created by the GO consortium Enzymes with an EC number for IUBMB Enzyme Nomenclature are displayed wi
290. ges To set up the quantification channels for ratio reporting 1 Click the Ratio Reporting tab shown in Figure 182 for precursor ion quantification and in Figure 183 for reporter ion quantification Thermo Scientific Proteome Discoverer User Guide 273 7 Quantification Setting Up the Quantification Method Figure 182 Ratio Reporting page of the Quantification Method Editor dialog box for precursor ion quantification In precursor ion quantification the quantification ratios left box displays the ratio of the amino acids using heavy isotopes to the amino acids using normal isotopes Figure 183 Ratio Reporting page of the Quantification Method Editor dialog box for reporter ion quantification iTRAQ 4plex Fale Pepa 444 116 114 117 114 274 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Setting Up the Quantification Method In reporter ion quantification the quantification ratios box to the left displays the name of the fragmented mass tag of a sample over the name of the mass tag of the reference sample 2 To add any new quantification ratios click beneath the quantification ratios box A new quantification ratio with the default name of New number appears in the quantification ratios pane To remove the quantification ratio select a quantification ratio and click beneath the label type box 3 If you adde
291. gory colors Molecular Function antioxidant activity catalytic activity DNA binding enzyme regulator activity metal ion binding motor activity nucleotide binding protein binding receptor activity RNA binding signal transducer activity structural molecule activity transcription regulator activity translation regulator activity transporter activity Cellular Component cell surface chromosome cytoplasm cytoskeleton cytosol endoplasmic reticulum endosome extracellular golgi membrane mitochondrion nucleus organelle lumen proteasome ribosome spliceosome vacuole Biological Process cell communication cell death cell differentiation cell division cell growth cell organization and biogenesis cell proliferation cellular component movement cellular homeostasis coagulation conjugation defense response development metabolic process regulation of biological process reproduction response to stimulus transport When you hold the cursor over the GO Slim category box the category name appears in a ToolTip as shown in the Molecular Function column in Figure 152 Figure 152 ToolTip identifying the annotation category File Search Report Quantification Processing Workflow Editor Administration Tools Window Help LERLE aljo F aonni S g omm o sequetht g Maso i A R KHHRe AQEBRRIAREB Celegans_FT_6ITDDDT_01 01 msf x Workflow Editor Celegans_FT_6ITDDDT_01_01 msf_annotation x Admini
292. gs because of impurities in the tags themselves 7 If you add a correction factor do the following a Inthe Name box to the right of the list of correction factors enter the name of the new correction factor For the name Thermo Fisher Scientific recommends that you use a plus or a minus symbol and the preferred shift number b In the Isotope Shift box enter the isotope shift of the new correction factor Isotope shift is a change in the spectral lines caused by different isotopes in an element It often reflects impurities in the sample and you must remove its corresponding mass to charge ratio from the calculations c In the Isotope Intensity box enter the isotope intensity of the new correction factor as a percentage Isotope intensity is the intensity of the different isotopes in an element often from impurities in the sample Note The sum of the isotope intensities for each tag should add up to 100 8 Continue setting up the quantification method by following the instructions in Setting Up Quantification Channels for Ratio Reporting on page 273 Setting Up Quantification Channels for Ratio Reporting The Ratio Reporting page of the Quantification Method Editor dialog box specifies the names of the quantification channels for precursor ion quantification or mass tags for reporter ion quantification for the reporting of ratios that appear in the Ratio columns of the Proteins and Peptides pa
293. gt Sequest TMT 6 plex Benchmark 1 4 0 175 msf x 4b Accession Description Score 5 Coverage Proteins UniquePeptides Peptides 127 126 127 126 Count 127 F E gt 100 Tim X58989 serum albumin Bos taurus 166 85 41 19 12 3 35 0 969 2 J 418694 serum albumin precursor bovine MASS 69270 151 74 39 54 12 cl 33 0 948 The following example shows how to use the row filter menu opened by the down arrow icon in the MSF report columns and the Enter Filter Criteria for Header_name dialog box This example sets a precursor mass filter To clear all filter conditions set by the row filter menu e Click the Clear Filter Criteria icon il if you want to clear all filter criteria set by the commands on the row filter menu opened by clicking xD To clear an individual filter set by the row filter menu 1 In the appropriate column move your cursor over the row with the filter set by the commands on the row filter menu opened by clicking xD 2 Click and choose Custom The Custom Filter dialog box appears For information on the parameters in the Custom Filter dialog box see the Help 3 In the dialog box click in the first column in the row of interest The condition is activated as shown in Figure 125 Thermo Scientific Proteome Discoverer User Guide 169 5 Filtering Data Filtering the Search Results Figure 125 Deleting filter condition Fitterbasedon All _ of the following
294. gure 188 You can access these two pages by selecting the individual MSF file from the list at the top of the dialog box Figure 188 Applying Quan Channel page settings to an individual MSF file when multiple MSF files are loaded File Search Report Quantification Processing Workflow Editor Administration Tools Window Help iW AaB OFTIBDD OAH gomm MR seth g Maso AL ARE ACEBTZABARE Sequest SILAC Dimethylation Search 1 3 0 297 msf x Multi Report from 2Reports x Accession LYSC_CHICK CYC_HORSE P00004 Cyto I ALBU_BOVIN P02769 Seru E I MYG_HORSE P68082 Myo E TRFELHUMAN P02787 Seroi El 7 CAH2_BOVIN P00921 Carb H 7 LALBA_BOVIN P00711 Alph 8 7 OVAL_CHIX P01012 Oval B 9 I G3PPIG P00355 Glyc 10 7 CASB_BOVIN P02666 Beta f 1P100708398 2 Serum albumi D 1P100706427 2 Cationic trypsi 1P100691212 1 Alpha 1 acid g 1P100710783 2 Hemoglobin s 1P100823795 1 Vitamin D bin P 1P100696956 4 similar to EGF Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary 400 On 2 A TMT 6plex 12 1 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Residue Modification None z N Terminal Modification None Tag Name 126 Monoisotopic m z Average m z
295. gure 216 is similar to case 2 but varies from it in a slight but important way In case 3 the two identification spectra are associated with the same quantification result rather than with two different quantification results For example you might obtain these results if you trigger the same precursor two times for MS MS It does not matter whether peptide A and peptide B are the same peptides redundantly identified or different peptides that are accidentally contained in the same protein It also does not matter whether they are identified by the same search engine or by two different search engines for example a CID spectrum and an ETD spectrum The quantification result is still unique for just one protein However you cannot use the quantification ratio of both peptides A and B to calculate the quantification ratio of protein A because it is the same quantification result and you do not want to use the same quantification result multiple times for the same protein In this case the Proteome Discoverer application marks peptide A the peptide with the better identification as Unique and the other peptide as Redundant for quantification rather than redundant for identification Figure 216 Case 3 Quantification result associated with two identification spectra two peptides and one protein Peptide A ID spectrum A w Quantification result A j Unique Protein A ID spectrum B Unique Peptide
296. h For a list of all operators refer to the Help 2 In the line below the operator that you selected type the search string or condition that you want the operator to apply to The example in Figure 79 filters out those protein references that contain fragment Thermo Scientific Proteome Discoverer User Guide 109 4 Searching for Data Using FASTA Databases Figure 79 Filtering out protein references containing fragment Add Protein References Compile FASTA Database Find Protein References FASTA Database C Program Files Proteome Discoverer source files FASTA_Files bovine fasta Search for ASE Searchin References Sequences 1000 G i 1m Does notcontain gt gi 100120 pir S09415 proteinase inhibitor cowpea gt gi 10120915 pdb 1FGX B Chain B Crystal Structure Of The Bovine Beta 1 4 Galactosyltransferase B4galt1 Catalytic Domain Complexed WithUmp gili gt gi 1042206 gb AAB34886 1 purine nucleosidephosphorylase PNP purine nucleoside orthophosphateribosyltransferase EC 2 42 1 cattle spleen Pep gt gi 1071846 pir A55277 hexokinase EC 2 7 1 1 1 bovine gt gi 1071864 pir A27053 lipoprotein lipase EC 3 1 1 34 bovine gi 163305 gb AAA30624 1 lipoprotein lipase EC 3 1 134 gt gi 1082982 pir A48719 3 5 cyclic GMP phosphodiesterase EC 3 1 4 35 5A bovine gt gi 1083022 pir A33650 dopamine beta monooxygenase EC 1 14 17 1 precursor membrane bound bov
297. hat given in Creating a Search Workflow on page 44 except that you select multiple files to load with the File Name s parameter of the Spectrum Files node Note The following method is not appropriate for batch processing different sample data files because the process generates a single result file Thermo Scientific Proteome Discoverer User Guide 53 2 Getting Started Starting a New Search by Using the Workflow Editor KD To load multiple raw files from the same sample 1 In the Workflow Editor drag the Spectrum Files node from the Data Input section of the Workflow Nodes pane to the workspace pane 2 Select the Spectrum Files node 3 In the Parameters pane click File Name s and click the Browse button The Select Analysis File s dialog box appears as shown in Figure 33 Figure 33 Select Analysis File s dialog box Select Analysis File s E Selected Files Add Folder Remove annB aain 4 To add new input files click Add Files and in the Add Analysis File s dialog box select the raw data files to load and click Open or To add all the raw data files in a specific folder click Add Folder and in the Browse for File dialog box click OK To remove a file or folder from the Selected Files area of the dialog box select the file and click Remove 5 In the Select Analysis File s dialog box click OK 6 Drag the Spectrum Selector node to the work
298. hat you want to display in the FASTA Indexes view The Proteome Discoverer application instantly makes the selected columns visible and the cleared columns invisible For a description of these columns refer to the Help Figure 89 Column Chooser dialog box in the FASTA Indexes view Column Chooser V IV Index Size kB V Last Access Time Max Missed Cleavage Sites Max Precursor Mass Da Min Precursor Mass Da Use Average Precursor Masses Automatically Creating a FASTA Index The Proteome Discoverer application automatically creates FASTA indexes for a full enzymatic digestion during a Sequest search if an adequate FASTA index does not already exist You can manually create a FASTA index for a semi enzymatic or non specific digestion see Manually Creating FASTA Indexes on page 125 You can only create a specific FASTA index once 1 To automatically create a FASTA index 1 Choose Administration gt Maintain FASTA Indexes or click the Maintain FASTA Indexes icon FE f 2 Click the Add icon amp Add Thermo Scientific The FASTA Index Creator dialog box appears as shown in Figure 90 Proteome Discoverer User Guide 121 4 Searching for Data Using FASTA Databases Figure 90 FASTA Index Creator dialog box FASTA Index Creato 4 1 General Auto Remove True Create Additional Decoy Database Index False 4 2 Input Data FASTA File Enzyme Name Trypsin Full Maximum Missed
299. he Maintain FASTA Indexes icon to display the FASTA Indexes view 9 In the FASTA Indexes view click the Refresh icon Refresh The new FASTA index appears in the FASTA Indexes view on the Administration page as shown in Figure 92 124 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases Figure 92 FASTA Indexes view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OF BEB STI SBBOOBH LB BD SequethT Q Maso AA LRP AZEBEABARGE Administration x 4 a i dR Add Remove Apply W Restore Refresh p Options io i d Auto Remove Indexed FASTA File Enzyme Index Size kB Last Access Time R Job Queue H gt c_elegans_100_021411_FWD_combined fa Trypsin Full 58310 10 03 2012 04 05 PM H Vv ecoli fasta Trypsin Full 9996 10 04 2012 11 31 AM ZE Vv uniprot_sprot_2011_05 fasta Trypsin Ful 886116 10 04 2012 11 50 AM a Name FASTA Database uniprot_sprot_2011_05 fasta Enzyme Trypsin Full File Size kB 886116 Last Access Time 10 4 2012 11 50 45 AM Max Missed Cleavage Sit 2 Spectral Libraries Precursor Mass Range Da 350 5000 tn Use Average Precursor M_ False BD crerica mocircaton EE Auto Remove indexed FASTA File Enzyme __ IndexSize k _ Last Access Time g Vv bovine fasta Trypsin Full 1278 10 04 2012 11 51 AM 3 Cleavage Reagents Name FASTA Database bovine fasta ipa Enzyme Try
300. he Keys page of the ProteinCard page Table 12 Parameters on the Keys page of the ProteinCard page Command Primary Key Description Lists the accession key of the database that the sequence was imported from It is linked to the original database records in the source database such as Ensembl SGD NRDB IPI or UniProt The preferred type of accession is emphasized Src Specifies the abbreviation of the primary source database Secondary Key Lists the secondary accession key which is either an alternative key used in the source database or the key of the original database Src Specifies the abbreviation of the secondary source database Description Displays the original description for the original database entry An exclamation mark flags outdated protein keys and the keys are linked to the outdating history in their respective source database The Features page of the ProteinCard page shown in Figure 165 includes a selection of sequence features from UniProt from various conserved domain predictions and from the computational enrichment undertaken by ProteinCenter Computational enrichment refers to information that has no experimental evidence but was found by using a computer prediction program The features are sorted according to their start positions in the protein sequence Proteome Discoverer User Guide 225 6 Protein Annotation ProteinCard Parameters Figure 165 Featu
301. he ProteinCard shown in Figure 163 displays information about the protein its name its description its function the keywords that produce it in a database search and the gene that ultimately directs the protein s synthesis through RNA Figure 163 General page of the ProteinCard Keys Features Molecular Functions Cellular Components Biological Processes Diseases External Links serpin H1 precursor Homo sapiens 11 11q13 5 SERPINH1 CBP1 0110 SERPINH2 RA A47 CBP2 AsTP3 PIG14 HSP47 PPROM gp46 serpin peptidase inhibitor clade H heat shock protein 47 member 1 collagen binding protein 1 Gene Details Protein Details Entrez Gene record Keywords This gene encodes a member of the serpin superfamily of serine proteinase inhibitors The Reference proteome Endoplasmic reticulum Osteogenesis imperfecta encoded protein is localized to the endoplasmic reticulum and plays a role in collagen Glycoprotein Signal Chaperone Stress response Polymorphism Complete biosynthesis as a collagen specific molecular chaperone Autoantibodies to the encoded proteome protein have been found in patients with rheumatoid arthritis Expression of this gene may bea Functions marker for cancer and nucleotide polymorphisms in this gene may be associated with preterm Binds specifically to collagen Could be involved as a chaperone in the biosynthetic pathway birth caused by preterm premature rupture of membranes Alternatively spliced tr
302. he nodes in each section of the Workflow Nodes pane appear in unique colors for example the Data Input nodes are blue the quantification nodes are pink and the Spectrum Processing nodes are yellow When you use any of the search engine nodes in the workflow you must attach the Fixed Value PSM Validator or the Percolator node to it You can also add third party nodes that are in your installation that are not documented in this manual For further information on those nodes consult the third party documentation 46 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 10 11 2 Getting Started Starting a New Search by Using the Workflow Editor You cannot drag workflow nodes into the workspace pane that cannot logically be added at that point For example if you add the Target Decoy PSM Validator node you cannot connect it to the Percolator node Organize the nodes to reflect a procedural order from top to bottom so that the Spectrum Files node remains on top as the root node Delete a node by selecting the node in the workspace pane and pressing DELETE or by right clicking the node and choosing Cut or CTRL X from the shortcut menu You can use the Cut command and the Paste or CTRL P command on the shortcut menu to move a node to another place in the workspace or use the Copy or CTRL C and Paste commands to duplicate a node in the workspace You can paste copied or cut nodes into other workfl
303. he protein peptide or fragment ion 6 In the Static Modifications area specify the static modifications that occur on the amino acid e Peptide N Terminus Select the static modification that occurs on the N terminus of the peptide e Peptide C Terminus Select the static modification that occurs on the C terminus of the peptide e Static Modification Select the static modification that occurs on the amino acid side chain 7 Click OK The Proteome Discoverer application starts creating the FASTA index and the job queue appears as shown in Figure 91 Thermo Scientific Proteome Discoverer User Guide 123 4 Searching for Data Using FASTA Databases Figure 91 Creating a FASTA index SESON ERIA E E E PA EEE OT EN va n T SBDRDOBB oom g SequethT Q Mso LA ALR ABEBSRRBPARE Administration x 4p a Gj Pause Resume jf Abort 2 Remove amp Refresh I OpenReport Display Row Filter Job Queue Execution State Progress a Time 1 37PM_ 0 FASTA Index Creator FASTAindexing completed in3 s 1 37PM_ 0 FASTA Index Creator Building new target FASTA index This may take up to several hours 1 37PM 0 FASTA Index Creator Looking for existing target FASTA index amp SpectraST B Server Settings amp Discoverer Daemon SG FASTA Indexes 8 When the job finishes choose Administration gt Maintain FASTA Indexes or click t
304. he substitution the group that it is leaving the position or the abbreviations of the modifications For chemical modifications that you add yourself you can edit any column except the Unimod Accession No column The Unimod Accession No column identifies these modifications by a zero For chemical modifications that you import from UNIMOD you can edit only the Modification and Abbreviation columns UNIMOD chemical modifications are identified by a number greater than zero in the Unimod Accession No column Columns that you can edit activate an edit button when you click them Columns that you cannot edit display a gray background 4 To accept the changes click the Apply icon Apply Adding Amino Acids You can add amino acids to a modification that has been set up for any position 1 Thermo Scientific To add an amino acid to a modification Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 Click to the left of the modification row that you want to update The row must display Any in the Position column The list of classifications now appears as shown in Figure 104 on page 143 Click the Add a Modification line below the list of amino acids Proteome Discoverer User Guide 145 4 Searching for Data Updating Chemical Modifications Figure 106 shows this line Figure 106 Adding an amino acid to a modification
305. her atoms For example the 116 label would not consist only of label molecules having three 13C atoms but might also contain label molecules with only one or two C atoms or even four or five C atoms As a result these impurities lead to an observed peak of 116 m z which is smaller than might be expected if the tag were 100 percent isotopically pure and to additional peaks at positions 2 1 1 2 Da apart from 116 m z The intensities of the latter peaks are proportional to the amount of the described isotopic impurities When the 116 label and the 114 115 and 117 labels are used these latter three labels contribute to the peak at 116 m z because of their isotopic impurities The intensity of the peak at 116 m z effectively includes the following contributions observed intensity 116 true intensity 116 intensity loss because of 116 impurities intensity gain because of other label impurities To obtain the true intensity value of the 116 label that is the amount of the substance initially labeled with the 116 tag you must correct the experimentally observed peak for the impurity of the labels Proteome Discoverer User Guide 309 7 Quantification Excluding Peptides from the Protein Quantification Results For a 4plex sample there are four formulas that use the equation just given for each of the labels and the proper correction would consider both contributions in the formula by solving the system of coupled
306. i ac uk uniref ftp ftp uniprot org pub databases uniprot current_release uniref uniref100 A typical UniRef100 title line follows gt UniRef100_4U9M9 Cluster 104 kDa microneme rhoptry antigen precursor n 1 Theileria annulata Rep 104 kDa microneme rhoptry antigen precursor Theileria annulata FASTA ID e Accession 4U9M9 e Description Cluster 104 kDa microneme rhoptry antigen precursor n 1 Theileria annulata Rep 104 kDa microneme rhoptry antigen precursor Theileria annulata SwissProt and TrEMBL Thermo Scientific The SwissProt database is developed by the SwissProt groups at the Swiss Institute of Bioinformatics SIB and the European Bioinformatics Institute EBI TrEMBL is a computer annotated supplement of SwissProt that contains all the translations of EMBL nucleotide sequence entries not yet integrated into SwissProt http www expasy org sprot ftp ftp expasy org databases uniprot knowledgebase uniprot_sprot fasta gz ftp ftp ebi ac uk pub databases uniprot knowledgebase uniprot_trembl fasta gz A typical SwissProt title line follows gt 43495 108_SOLLC Protein 108 precursor Solanum lycopersicum Tomato Lycopersicon esculentum FASTA ID 108_SOLLC e Accession 43495 e Description Protein 108 precursor Solanum lycopersicum Tomato Lycopersicon esculentum Proteome Discoverer User Guide 341 A FASTA Reference Custom Database Support MSDB The Mass Spectrometry Protein Sequence Databas
307. ick synchronous processing of the same workflow you can use the Proteome Discoverer Daemon application as a post acquisition method in the Run Sequence dialog box Note Using the post acquisition method with the Proteome Discoverer Daemon application does not work with the Xcalibur data system 2 1 0 It only works with the Xcalibur data system 2 0 7 which runs on Windows XP Thermo Scientific Proteome Discoverer User Guide 89 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System To use the Proteome Discoverer Daemon application in the Run Sequence dialog box you do not need a processing method Figure 63 shows the sequence setup without a processing method Figure 63 Sequence used to start batch processing in the Run Sequence dialog box File Name Path Inst Meth Position Proc Meth Sample Type BatchS ample1 C calibursData C XcalibursmethodsStop50ph H6 Unknown BatchS ample2 C calibursData C Xcalibur methods top50ph H7 Unknown BatchSample3 C XcalibursData C XcalibursmethodsStop50ph H8 Unknown To perform batch processing by using the Run Sequence dialog box 1 To start the sequence click the Run Sequence icon Ep 2 In the Run Sequence dialog box shown in Figure 64 enter the following in the Post Acquisition box C Program Files Thermo Discoverer System Release discovererdaemon exe p C Xcalibur methods BatchProcess
308. ide ratios by the median protein ratio The median protein ratio should be 1 after the normalization 280 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Setting Up the Quantification Method e Manual Normalization Specifies a user defined normalization number When you select the Normalize on Protein Median setting the Minimum Protein Count box appears When you select the Manual Normalization setting the Normalization Factor box appears 3 Do one of the following e In the Minimum Protein Count box which appears when you selected Normalization on Protein Median enter the minimum number of proteins that must be observed to allow normalization or e In the Normalization Factor box which appears when you select Manual Normalization enter the normalization factor The default for the Minimum Protein Count option is 20 and the default for the Normalization Factor option is 1 0 Normalization cannot work if there are too few proteins in a sample 4 Click OK Checking the Quantification Method The Proteome Discoverer application checks the parameters that you have set for the quantification method For reporter ion quantification it verifies that the method has at least two channels For precursor ion quantification it checks for the following e At least one quantification channel e At least one label for each quantification channel e Unique label names in a channel e The modification o
309. identification number The Pfam annotation system is an alternative to GO annotations You might want to use the Pfam system to filter your proteins when you want the results to be traceable scored and uniformly grouped You might also consider its computationally based data more reliable However it might be easier to use the hierarchy and grouping of the GO system to help you interpret results Table 9 compares the features of the GO and Pfam databases Table 9 Comparison of GO and Pfam features GO features Pfam features Proteins grouped in biologically meaningful Proteins grouped by similarity categories Deep hierarchical order of terms Few hierarchies Data input by experts with different Computational data input with no human confidence levels and differing opinions influence or expert knowledge Thermo Scientific Proteome Discoverer User Guide 203 6 Protein Annotation Entrez Gene Database Annotation Entrez Gene Database Annotation The Proteome Discoverer application can retrieve the Entrez gene identifications from ProteinCenter The Entrez gene identification is a unique identification assigned to the genes in the Entrez database maintained by the National Center for Biotechnology Information NCBI The database assigns an identifier to all proteins transcribed from the corresponding gene The Proteins page of the results report displays these identifications in the Gene IDs column You can use this information to gro
310. ifficult because they might have changed in the different workflows 184 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Grouping Peptides Grouping Peptides Thermo Scientific In the results report you can turn peptide grouping on or off with the Show Peptide Groups command on the shortcut menu or with the settings in the Peptide Grouping Enabled area on the Result Filters page Using the latter method you can select more options in grouping Grouping is turned on by default In the Peptide Grouping area of the Result Filters page you can specify whether you want to group peptides only by sequence or by mass and sequence The Mass and Sequence setting of the Group Peptides By option separates the differently modified forms of a peptide into different peptide groups This setting is the default The number of peptides displayed in the status bar is always the number of distinct sequences The number of peptide groups on the other hand depends on the peptide grouping settings If you group peptides by sequence only the two numbers are the same If you group peptides by sequence and mass the number of peptide groups is normally larger than the number of peptides displayed in the status bar unless the peptides have no modifications To group the peptides in your search results 1 Open the MSF file 2 On the Peptides or Proteins page of the MSF file right click a peptide grid cell or row to access the shor
311. ification Calculating Peptide Ratios Table 23 and Table 24 do not include cases that arise as a result of peptide uniqueness and protein grouping They focus on cases that arise where one or both of the quantification channels that are used for calculating peptide ratios are zero In these cases the application detects nothing on a channel because the spectrum does not contain one of the reporter peaks the heavy or light isotope pattern is missing a quantification value falls below a specified minimum threshold or the calculated ratios are very high or very low Table 23 and Table 24 list the different possible cases exemplified by arbitrary values The values in the tables have counts as units if the cases are presented for reporter ion quantification For precursor ion quantification 114 and 115 are replaced by Light and Heavy and the quantification values have counts x min as units In addition to the options listed in the tables the handling of quantification values is also affected by the Apply Quan Value Corrections option on the Ratio Calculation page of the Quantification Method Editor dialog box and by the options on the Experimental Bias page of the same dialog box For reporter ion quantification the Apply Quan Value Corrections option determines whether to apply the purity correction for the detected quantification values The Proteome Discoverer application applies the purity correction after it applies the other settings th
312. ifications area select the dynamic modification that occurs on the C terminus of the peptide In the N Terminus list in the Static Peptide Modifications area select the static modification that occurs on the N terminus of the peptide In the C Terminus list in the Static Peptide Modifications area select the static modification that occurs on the C terminus of the peptide Click Next The Search Description page opens as shown in Figure 21 Figure 21 Search Description page Sequest HT Search Wizard ca Search Description Please give this search a name and provide an optional additional description Search Name Celegans_FT_6ITDDDT_01 Search Description 11 Give your search a name and a brief description Proteome Discoverer User Guide In the Search Name box type a name for your search In the Search Description box type a brief description of the search Click Next The Completing the Wizard_name Search Wizard page appears as shown in Figure 22 Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards Figure 22 Completing the Wizard_name Search Wizard page Completing the Sequest HT Search Wizard Review your settings in the summary paragraph below and start the search by clicking the Start button earch name Celegans_FI_6ITDDDT_01 Search description File Name s C Program Files Proteome Discoverer source file Precursor Selection Use MS1 Precurso
313. igurations 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 Release A December 2012 e Software version Thermo Proteome Discoverer version 1 4 Microsoft Windows XP 32 64 Professional English version Microsoft Windows 7 32 64 Professional English version Mascot Server 2 1 For Research Use Only Not for use in diagnostic procedures B Contents it ee a ee ere er er re ret xi Related Docutiientation i sca 200 seks iO Gees be See dee RS es Cee OES xi System Requirements 0 0 cece eee eee eens xii Special Notices ei cel eee tedeRea AEA xiii Contacting Us sirier tierse Mie dew Sha eds Cae A a xiii Chapter Introduction cccscacdietire dareacndeddieadineisderddadecseeadedacsecees 1 POACULES 92 socom ts eesti Ea EE E ott s sleds ae eee teste lo brie ase nee 1 Search Eng
314. ii ayat E od i t ene 258 Performing Peak Area Calculation Quantification 0 0 00005 259 Searching for Quantification Modifications with Mascot 261 Setting Up the Quantification Method cs d sc eu ea heen dane een eee 264 Specifying the Quantification Channels 0 n nie dase ucr nese wie 266 Setting Up Quantification Channels for Ratio Reporting 273 Setting Up the Ratio Calculation axis ceaeiadans ees oe wake mere 275 Setting Peptide Parameters Used to Calculate Protein Ratios 278 Correcting Experimental Biasstiiec2k beak ei Ak eke we hire ees 280 Checking the Quantification Method 0 0 0 0 cc eee eee eee 281 Restoring Quantification Method Template Defaults 281 Setting Up the Quantification Method for Multiple Input Files 282 Adding a Quantification Method 32 2 nbd x ee ee eek 285 Changing a Quantification Method nid praia koe naee toe aaid Vena aies 288 Removing a Quantification Method 0 eee e cee eee ee 290 Importing a Quantification Method sii ides ua Lie Nee han Mee es 291 Exporting a Quantification Method oad cue eg cee daca ete alae eee 291 Summarizing the Quantification oaae rn alo nleth ad saree lantnce a etaonntne land 292 Displaying Quantification Spectra aise is owe bt ware oe ay BERANE mes 293 Quan Spectra Page Parameters 0 eee eee eee 294 Displaying the Quantification Channel Values Chart 00
315. ile FASTA Database page appears In the Original box browse for the FASTA file that you are taking the information from or type its path and name In the Please Select a FASTA Database dialog box click Open In the Target box browse for the FASTA file that you are placing the extracted information into or type its path and name In the Save Add to FASTA File dialog box select the file verify that the file extension is fasta and click Save In the Target Database Options area select one of the following options to indicate what you want to do with the extracted information e Create Replace Creates a new FASTA file for storing the information or overwriting an existing FASTA file This option is the default e Append Adds the extracted information to an existing FASTA file In the Search In area specify whether the Proteome Discoverer application should search for the search string in the protein references or sequences e References Searches for the search string in the protein references e Sequences Searches for the specified amino acid sequence within the protein sequences To disregard the case of the information to be extracted select the Ignore Case of Reference Strings check box 112 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 4 Searching for Data Using FASTA Databases 10 Specify the information to be extracted a Click af above the Step 1 Stri
316. iled description of these nodes refer to the Help You can access the Workflow Editor through the Workflow Editor menu in the Proteome Discoverer application or through the Workflow Editor icons on the main toolbar After you choose a menu command or click an icon the application opens a Workflow Editor page in the main window The three pane layout of the Workflow Editor page provides a pane for node selections a workspace for placing the nodes and a pane where you can choose parameters for each node as shown in Figure 24 42 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 24 Workflow Editor workspace Select to merge search results of identification nodes in complex workflows File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BE BlOT SHOOT LOAD K sett Kmart ARAM GCEBTABAREB Workflow Nodes pane Workspace pane Parameters pane To create a workflow see Creating a Search Workflow on page 44 Thermo Scientific Proteome Discoverer User Guide 43 2 Getting Started Starting a New Search by Using the Workflow Editor Before Creating a Workflow As with the search engines follow these steps before using the Workflow Editor to create a workflow e Download a FASTA file if necessary i
317. iles which are files containing MS data for single or grouped scans Mascot Generic Format MGF Places the exported spectra into MGF files which are mass spectral files produced during Mascot analysis They contain a list of precursor ions their fragments and the masses of the fragments MZDATA Places the exported spectra into MZDATA files which are common data format files developed by the Human Proteome Organization HUPO for proteomics mass spectrometry data These files are in version 1 05 format They are exported with XML indentation enabled so that the different XML tags are broken into multiple lines instead of merged into one line MZXML These files are standard 2 x mass spectrometer data format files developed at the Seattle Proteome Center at the Institute for Systems Biology ISB that contain a list of precursor ions their fragments and the masses of the fragment MZML These files are a combination of MZDATA and MZXML formats developed by the Human Proteome Organization Standard Initiative HUPO PSI and the Seattle Proteome Center at the Institute for Systems Biology ISB The Proteome Discoverer application supports version 1 1 0 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor You can select only one format for each Spectrum Exporter node To export to multiple formats in a single workflow you must add more than one Spectrum Exporter node
318. indicate outdated nodes It displays error information in the Workflow Failures pane as shown in Figure 40 on page 63 Ifyou selected an MSF file and this file was created with an older version of the Proteome Discoverer application the message box shown in Figure 41 appears Figure 41 Message box Result File Update Required Ds The result file was created with an older version of the software To open the file C Program Files abc msf it needs to be updated first The update can take some time Should the file be updated cee es 3 Click Yes to update to the current version The Proteome Discoverer application validates parameter settings and displays the selected workflow in the Workflow Editor using warning symbols to indicate outdated nodes and displaying error information in the Workflow Failures pane If the Proteome Discoverer application cannot load the selected MSF file it displays a message box with information about the issue It cannot load files that are read only or invalid could not be updated or were created with a newer version of the Proteome Discoverer application Deleting an Existing Workflow Template 64 You can delete an existing workflow template 1 1 To delete an existing template Choose Workflow Editor gt Open From Template The Open Processing Workflow Templates dialog box appears as shown in the example in Figure 37 on page 61 listing the available workflow temp
319. ine Save Add Selected to Database Stop Search To refine a filtered protein reference search 1 Select the Custom option from the list in the line below the search operator To make the Custom option available click the down arrow in the line below the operator as shown in Figure 80 Figure 80 Selecting the Custom option Reference mi Does notcontain 4 gt gil1071846 pir A55277 hexokinase EC 2 7 1 1 1 bovine Click this down arrow 110 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 4 Searching for Data Using FASTA Databases The Custom option opens the Custom Filter dialog box shown in Figure 81 so you can add multiple conditions Figure 81 Custom Filter dialog box Custom Filter Fiterbasedon All of the following conditions Reference Does not contain Tel fragment Ix Delete OK _ __ Cancel 2 Click Add A new line appears in the Operator left and Operand right lists 3 Select an operator from the Operator list 4 Type an operand on the line in the Operand column 5 In the Filter Based On list do one of the following Select the All option to indicate whether the search algorithm should search for protein references that meet both conditions or Select the Any option to indicate whether the search algorithm should search for protein references that meet only one of the conditions Figure
320. ines orere prrriii ir tin PEE EEEE E EEA 3 Wizards and Workflow Editor ooun uuaaaaeuuuna anae 5 Quantification seeen na EE E EEEE EA EE ane R 6 The Qual Browser Applicanettosi5 ction nwitnnte aieietandeseemedens 7 Peptides and Fragment Ions cans deo rninteo Keen sah eeenuess 7 Fragmentation Methods uuusssununa aeree 8 M dPIT Experiments rrur cser sk vcnsa tier ee ena E E ee ees 9 World OW dd qt niteen n oir A e EE EEE A 9 lnp ts nd Outputs sese a a bie da thaws hs 12 FASTA Da tabas s ee laceaab ise a a aa E hebben 12 IOPU serne emina EE E NoE E E O EE D Moral at belies 12 ODPUST EREE AE E E T gy E E lores egw 13 Limitations 4 i e cbt so Rape a E a EE ls Re ea Roe ees 14 New Features in This Release 5 icin a oe dua nace een ae ean gase athe daes 14 Sequest HT Search Engine io cedieedemeeeeeenee eereken sass abasaes 14 Spectrum Library Searching och erie where eeL ba wee bbe y ie Sake Wuhs 14 New Workflow Editor Nodes oyeiiue de hiu eh Oradell eeee 34s 15 New Protein Annotations 145 ccc cde iss ase ere oner ee eee ee 16 Mascot Quantification Mode 0 cent ene nes 17 Chapter2 Getting Started ccc cece cece eee eee eee eee 19 Starting the Proteome Discoverer Application 0 0 0 0 eee eee ee 19 Closing the Proteome Discoverer Application 0 0 0 0 0 cece eee 20 Configuring Search Engine Parameters 2 veo ese awk ewer runnerunner 21 Configuring the Sequest HT Search Engine
321. ing On to a Remote Server Figure 50 Job Queue page of the Proteome Discoverer Daemon application for MudPIT processing a Decne scion Configuration Job Queue n Pause Q Resume Abort Remove Refresh Job Queue Message 1 49PM 2 Sequest HT Total search time was 12 7 s 1 49PM 2 Sequest HT Search completed 1 49PM 2 Sequest HT 408 protein s 391 decoy proteins scored and inserted into resultfile in 0 7 s 1 49PM 2 Sequest HT 408 protein s scored 1 49PM 3 TargetDecoyPSM Vali Start calculating relaxed False Discovery Rate 1 49PM 3 TargetDecoyPSM Vali Start calculating strict False Discovery Rate 1 49PM 3 Target DecoyPSM Vali Evaluating peptides of Sequest HT 2 started 1 49PM 2 Sequest HT Reading resultstook 0 6s 1 49PM 2 Sequest HT Storing 996 decoyPSMs for 1620 spectra 1 49PM 2 Sequest HT Start reading results 1 49PM 2 Sequest HT Performing decoy searchcontaining 1620 DTAs took3 5s 1 49PM 2 Sequest HT Start ISE decoy search 1 49PM 2 Sequest HT There is already an adequate decoy FASTA index 1 49PM 2 Sequest HT Looking for existing decoy FASTA index 1 49PM 2 Sequest HT Reading resultstook0 5s 1 49PM 2 Sequest HT Storing 1047 PSMs for 1620 spectra z Logging On to a Remote Server The searches started by the Proteome Discoverer application consume memory and can potentially cause the data acquiring computer to crash and
322. ing new modifications or activating or deactivating existing modifications Note A modification must be active to be usable during a search The Proteome Discoverer application offers two types of modifications dynamic and static Dynamic Modifications Dynamic modifications also known as variable amino acid modifications are modifications that might or might not be present They are mainly used for determining post translational modifications PTMs For example some phosphorylated peptide serines are modified and some are not modified You can set the parameters for a dynamic search on the Select Modifications page of the Mascot and Sequest HT search wizards For instructions on setting these parameters in the wizards see Figure 19 on page 38 and the steps that follow it Static Modifications Thermo Scientific Static modifications apply the same specific mass to all occurrences of that named amino acid as in an exhaustive chemical modification A static modification might result from derivatization or isotopic labeling of an amino acid For example a carboxymethylated cysteine has a delta mass of 58 005479 which is added to each cysteine residue appearing in a protein In static searches the Proteome Discoverer application assumes that every amino acid residue will be modified in that way Constant mass is changed The search wizards perform static modification searches by adding the specified constant value to the ma
323. ing param R Figure 64 Using the Proteome Discoverer Daemon application in the Run Sequence dialog box Windows XP only Run Sequence m Acquisition Options g ii Start Instrument User Instrument LTO Orbitrap xL MS Yes MALDI Source Run Rows fi 3 I Priority Sequence I Start When Ready Change Instruments Processing Action m Instrument Method Start Up Browse T Quan Shut Down Browse Programs Pre Acquisition Create Quan Summary Post Acquisition fessing param R Browse I Pre Acquisition Post Acquisition m After Sequence Set System On Standby C Off Cancel Help C Program Files Thermo Discoverer System Release discovererdaemon exe p C Xcalibur methods BatchProcessing param R 90 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System 3 If the Programs check box in the Processing Actions area on the right is selected clear it The Xcalibur data system sends the acquired raw data files synchronously to the Proteome Discoverer application as shown in Figure 65 Note Only the Xcalibur 2 0 7 data system sends the acquired raw data to the Proteome Discoverer application This functionality is not available in version 2 1 0 Figure 65 Sending the raw data files synchronously to
324. ion The biological process whose specific outcome is the progression of an organism over time from an initial condition for example a zygote or a young adult to a later condition for example a multicellular animal or an aged adult Metabolic process Processes that cause many of the chemical changes in living organisms including anabolism and catabolism Metabolic processes typically transform small molecules but also include macromolecular processes such as DNA repair and replication and protein synthesis and degradation Regulation of biological process Reproduction Any process that modulates the frequency rate or extent of a biological process Biological processes are regulated by many means for example control of gene expression protein modification or interaction with a protein or substrate molecule The production by an organism of new individuals that contain some portion of their genetic material inherited from that organism Response to stimulus A change in state or activity of a cell or an organism in terms of movement secretion enzyme production gene expression and so forth as a result of a stimulus Transport The directed movement of substances such as macromolecules small molecules ions into out of within or between cells Proteome Discoverer User Guide 239 en Quantification This chapter describes how to perform precursor reporter and peak area based quan
325. ion searches for the minimum ion intensity that is detected on all quantification channels and uses it as a best guess for the detection limit It then uses this minimum value instead of the missing quantification values When you specify a value higher than the detected minimum value the application uses the value that you specify instead The Quantification Summary page lists the minimum quantification value detected and the value actually used for the calculations For information on the Quantification Summary page see Summarizing the Quantification on page 292 By default this check box is clear 276 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Setting Up the Quantification Method 5 When you are performing precursor ion quantification and want the Proteome Discoverer application to consider missing quantification channels or quantification channels with just one peak as valid quantification results in the ratio calculation do the following a Select the Use Single Peak Quan Channels check box on the Ratio Calculation page b Set the Single Peak Missing Channels Allowed parameter of the Precursor Ions Quantifier node to 1 For more information on this parameter refer to the Help By default missing quantification channels or quantification channels with just one peak are not used for protein quantification On the Peptides page these peptides are marked Excluded by Method
326. ixed ValuePSM Validator Percolator lih Target Decoy PSM Validator E PTM Analysis Precursor Ions Area Detector fd Precursor Ions Quantifier Reporter Ions Quantifier Workflow Nodes pane Quantification 6 Workflow Editor x 4 gt Workflow Nodes Parameters 9 a Name e Data Input gt 2 m ce Based on template EBH Show Advanced Parameters iu Spectrum Files a er Spectrum amp Feature Retrieval a m m r Proteome Discoverer User Guide Fixed Value PSM Validator ih gt Workspace pane Parameters pane The Proteome Discoverer application offers both isotopically labeled precursor ion quantification and isobarically labeled reporter ion quantification methods which you can also edit SILAC is an isotopically labeled quantification method that uses in vivo metabolic labeling to detect differences in the abundance of proteins in multiple samples SILAC uses the Precursor Ions Quantifier node in the Workflow Editor Thermo Scientific 1 Introduction Features iTRAQ and TMT are very similar isobarically labeled quantification methods that use external reagents or tags to chemically label proteins and peptides to detect differences in abundances TMT quantification offers default 2plex and 6plex quantification methods and iTRAQ offers 4plex and 8plex quantification methods You can use these me
327. izes the time needed for data interrogation or results review increasing the overall throughput of the analysis You can also automatically determine false discovery rates by comparing the results of forward and reversed databases which provides an additional means of increasing confidence in protein identification Sequest HT Search Engine The Sequest HT search engine calculates XCorr scores for peptide matches and provides the peptide matches having the best XCorr score for each spectrum It is similar to the SEQUEST search node which calculates a preliminary SpScore score and uses it to filter peptide candidates It calculates XCorr values for PSMs only if they pass the SpScore filter The Sequest HT node calculates the XCorr value for every peptide candidate It can therefore take longer than the SEQUEST node especially when the number of peptide candidates is large and the processing uses several dynamic modifications In most cases however multiple thread searching is faster with Sequest HT 4 Proteome Discoverer User Guide Thermo Scientific 1 Introduction Features Mascot Search Engine Mascot uses mass spectrometry data to identify proteins from primary sequence databases For more details on the Mascot search engine visit http www matrixscience com Wizards and Workflow Editor Thermo Scientific You can use the Proteome Discoverer application s search wizards or its Workflow Editor to conduct data analysis searches
328. l Returns the filtered search results to the results that were first loaded e Custom Opens the Custom Filter dialog box shown in Figure 110 Figure 110 Custom Filter dialog box Custom Filter x Filterbasedon All of the following conditions Reference m Does not contain zl fragment z Delete For information about using this type of dialog box see Filtering Results with Row Filters on page 167 Blanks Filters out rows that have data filled cells in the column whose funnel icon you clicked e NonBlanks Filters out rows that have empty cells in the column whose funnel icon you clicked 152 Proteome Discoverer User Guide Thermo Scientific ee Filtering Data The single or multiconsensus MSF report displays a list of matching peptides and proteins identified by the search engine that you specify This chapter explains how to sort and filter the data from your Proteome Discoverer results report Contents e Result Filters Page e Filtering the Search Results e Grouping Proteins e Grouping Peptides e Calculating False Discovery Rates Result Filters Page On the Result Filters page shown in Figure 111 you can select the proteins and peptides to filter out of the search results Refining your search results in this way can make your analysis quicker By using filters you can sort and filter your results by charge state modifications or even peptide probability You can also
329. l Acetyl 42 010565 42 0367 H 2 C 2 O Any_N_Te 1 Iv Amidated Amidated 0 984016 0 9848 HN Of 1 Protein_c 2 Vv Amidated Amidated 0 984016 0 9848 HN Of 1 Any_C_Te 2 ie Iv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 N O Any 4 Iv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 NO Any_N_Te 4 ie Vv Carbamyl Carbamyl 43 005814 43 0247 HCNO Any 5 V Carbamyl Carbamyl 43 005814 43 0247 HCNO Any_N_Te 5 144 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Updating Chemical Modifications In the empty row enter the name of the modification the delta masses the chemical substitution the chemical group that is leaving the position and the abbreviations of the modifications If you select Any in the Position column a message box opens to inform you that you must specify which amino acids target amino acids will possibly have the modification For instructions on this procedure see Adding Amino Acids on page 145 To accept the new modifications click the Apply icon Apply Add an amino acid to the modifications See Adding Amino Acids on page 145 To update an existing chemical modification Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 In the Modification column click the cell that you want to update Type your changes for the delta masses t
330. l genes yolk protein genes family 37 69 15 85 1 3 24 30 I mi j m 17533087 Tudor Staphylococcal Nucleasehomolog family member t 36 72 20 68 1 12 13 14 L m I E m 17541098 Heat Shock Proteinfamily member hsp 1 Caenorhabditi 35 21 34 06 3 9 17 18 E m I I bt M 17562024 Heat Shock Proteinfamily member hsp 6 Caenorhabditi 34 91 17 81 2 8 9 14 ii I I i 71994099 ACTin family member act 4 Caenorhabditis elegans 34 83 26 52 12 4 6 17 I E M 17539652 F01G10 1 Caenorhabditis elegans 32 55 28 80 1 7 9 14 L ToolTip In multiconsensus reports the protein information is displayed for the master protein of a protein group 210 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Displaying the Annotated Protein Results To filter the identified proteins by GO Slim categories In the MSF report right click the Proteins page and choose Enable Row Filters Click in the filter row that appears beneath the column headers in one of the GO columns for example Molecular Function Click 7 in this row A dialog box appears that lists the GO Slim categories that you can filter by as shown in Figure 153 Figure 153 Filtering by GO Slim category Peptides PSMs Molecular Function Cellular Component Biological Process TN a
331. l protein sequences from the FASTA file digest them in silico with the specified enzyme calculate the mass of each peptide and compare it to the given precursor mass the Proteome Discoverer application looks for the specific mass in the FASTA index and uses it to find the peptides that have this mass and the associated proteins that contain the peptides For full enzymatic searches the Proteome Discoverer application automatically creates FASTA indexes as they are needed It does not automatically create FASTA indexes during semi enzymatic or no enzyme searches because these searches usually consume a large amount of space on a computer s hard disk However you can manually create FASTA indexes for these types of searches e Specifying the Location and Number of FASTA Indexes Stored Displaying the FASTA Indexes View e Specifying the Columns to Display e Automatically Creating a FASTA Index Manually Creating FASTA Indexes Controlling Automatic FASTA Index Removal Deleting a FASTA Index e Changing Number and Location of Stored FASTA Indexes Removing FASTA Indexes When a FASTA File Is Deleted Specifying the Location and Number of FASTA Indexes Stored If you do not want to store the FASTA indexes in the default directory shown in Figure 85 you can specify an alternate directory in the FASTA Indexes configuration view You can also change the maximum number of FASTA indexes stored Thermo Scientific Proteome Discoverer User Guide
332. lates Click the row displaying the name of the template that you want to delete Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor 3 Click Remove 4 In the Confirm Deletion dialog box click Yes 5 Click Remove again Changing the Name and Description of a Workflow Template You can change the name and the description of a workflow template To change the name and description of a workflow template 1 Choose Workflow Editor gt Open From Template The Open Processing Workflow Templates dialog box appears as shown in the example in Figure 37 on page 61 listing the available workflow templates 2 Click the row displaying the name of the template that you want to change A Pen icon rar now appears to the right of the template name and to the right of the template description as shown in Figure 42 Figure 42 Pen icons in the Open Processing Workflow Templates dialog box Open par Workflow Templates El Time Submitted Name Description z gt o m 10 02 2012 08 46 AM c_elegans_sequest flow c_elegans_100_021411_FWD_combined fasta basic SEQUEST workflow 10 02 2012 06 50 AM WF_Spectrum_Export_MZML 10 02 2012 06 50 AM WF_Re Annotation 10 02 2012 06 50 AM WF_Q_Exactive_SEQUEST_vs_Mascot_Search_Percolator Workflow for processing Q Exactive raw files with Sequest and Mascot gt 10 02 2012 06 50
333. layed side by side In this example the two types of searches are from different samples and the two different types of quantification share no proteins 332 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Identifying Isotope Patterns in Precursor lon Quantification Figure 226 Opened multiconsensus report from three iTRAQ and two SILAC files in replicate mode File Search Report Quantification Processing Workflow Editor Administration Tools Window Help a BS Tl SGOROBH ZEA mm W seuethT g Maso AR LHP AZEBBRIARE Multi Report from 5 Reports x 4 P Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary iabil 2 075 2 118 41 0 0 6 904 2 184 11 0 0 2 249 3 3 3 0 0 11 689 3 3 3 0 000 33 05 28 68 6 826 2 208 1 1 0 0 0 00 4 466 1 882 41 0 0 0 00 4771 2 161 2 2 0 0 0 00 2 345 7 7 7 0 0 12 170 7 7 7 0 0 0 000 81 68 19 28 2 333 2 033 1 1 0 0 0 00 4 454 2 106 1 1 0 0 0 00 9 799 0 00 2 069 3 3 3 0 0 12 289 3 3 3 0 0 0 000 73 95 18 44 2 517 1 1 1 0 0 13 890 1 1 1 0 0 0 000 15 52 17 22 1 225 2 657 3 3 0 0 0 00 1 662 1 666 41 0 0 0 00 6 880 2 366 232 0 0 0 00 3 879 2 409 1 1 0 0 0 00 7 177 2 628 4 1 0 0 0 00 3 152 6 267 232 0 0 0 00 1 587 2 626 2 2 0 0 0 00 2 553 2 013 1 1 0 0 0 00 3 031 2 183 1 1 0 0 0 00 6 175 2 436 1 1 0 0 0 00 2 297 4 4 4 0 0 12 585 4 4 4 0 0
334. lculation quantification 1 In the Workflow Editor set up a quantification workflow For instructions on creating a workflow with the Workflow Editor see Starting a New Search by Using the Workflow Editor on page 42 2 Choose Workflow Editor gt New Workflow 3 In the Workflow Editor drag the Spectrum Files node to the workspace 4 Drag the Spectrum Selector node and the Event Detector node to the workspace 5 Connect the Spectrum Selector node and the Event Detector node to the Spectrum Files node 6 Drag the Precursor Ions Area Detector node to the workspace pane and attach it directly to the Event Detector node Note You cannot use the Precursor Ions Area Detector node in the same workflow with the Precursor Ions Quantifier node or the Reporter Ions Quantifier node 7 Drag the search engine node that you prefer for example SEQUEST to the workspace pane and attach it to the Spectrum Selector node 8 Drag the Fixed Value PSM Validator node or the Percolator node to the workspace pane and attach it to the search engine node Figure 175 illustrates the workflow up to this point 3 Silva J C Gorenstein M V Li G Z Vissers J P C and Geromanos S J Absolute Quantification of Proteins by LCMSE A Virtue of Parallel MS Acquisition Molecular amp Cellular Proteomics 2006 5 144 156 Thermo Scientific Proteome Discoverer User Guide 259 7 Quantification Performing Peak Area Calculation Quantifica
335. le in Figure 31 Is means that the workflow processes data from the CID activation type e Is Not Applies all settings in the list on the right except the selected setting In the example in Figure 31 Is Not means that the workflow processes data from all activation types except CID e Any Applies all settings available for the parameter in the list on the right In the example in Figure 31 Any means that the workflow processes data from any activation type available in the list on the right Any is the default You can filter input data before searching the database to remove lower quality spectral peak lists from your analysis This step might help to decrease search times and false positive identifications The Spectrum Filters area of the Workflow Nodes pane provides three types of spectrum filters to use for your search Use these pre analysis filters to streamline your search results For information about these nodes refer to the Help Use the Scan Event Filter node for high mass accuracy data such as Mascot analysis and Sequest analysis of mixed fragmentation mode type data CID and ETD It can filter information according to fragmentation type mass analyzer identity and other parameters Refer to the Help for information about the Scan Event Filter node To save the workflow as a template 1 Choose Workflow Editor gt Save as Template or click the Save As Template icon Ju To save the workflow in XML
336. le when you open an MSF file generated by a workflow in the Workflow Editor that includes the Reporter Ions Quantifier node the Precursor Ions Quantifier node or the Precursor Ions Area Detector node To activate the Quantification menu e Choose File gt Open Report and follow the procedure in the Help to open an MSF file containing quantification results The commands on the Quantification menu become available If you do not have an MSF file containing quantification results see Performing Precursor Ion Quantification Performing Reporter Ion Quantification on page 249 or Performing Peak Area Calculation Quantification on page 259 for instructions on creating one Proteins Included in the Quantification To determine the proteins to include in the quantification the Proteome Discoverer application first creates protein groups from the identified PSMs When the search results include quantification data it then performs quantification on all protein groups The application calculates the ratio for each of the defined quantification ratios for the protein group as the median of all PSMs belonging to the protein group that are marked as being usable Whether the application considers a PSM usable is determined by the settings of the Quantification Method Editor dialog box including two options on the Protein Quantification page Use Only Unique Peptides and Consider Proteins Groups for Peptide Uniqueness The Use
337. lector node in Figure 29 Note The same options are available in the search wizards Figure 29 Spectrum Selector node parameters in the Parameters pane Parameters an val Show Advanced Parameters 4 1 General Settings Precursor Selection Use MS1 Precursor 4 2 Spectrum Properties Filter Lower RT Limit Upper RT Limit First Scan Last Scan Lowest Charge State 0 Highest Charge State 0 Min Precursor Mass 350 Da Max Precursor Mass 5000 Da Total Intensity Thresho 0 Minimum Peak Count 1 4 3 Scan Event Filters oooco Mass Analyzer Any MS Order Is MS2 Activation Type Any Scan Type Is Full lonization Source Any Polarity Mode Any 4 4 Peak Filters S N Threshold FT onh 1 5 4 5 Replacements for Unrecognized Properties Unrecognized Charge Automatic Unrecognized Mass Ar ITMS Unrecognized MS Orde MS2 Unrecognized Activatic CID Unrecognized Polarity b Set the node s parameters Complete this step for each node that you select Figure 30 shows the parameters set for the SEQUEST node 48 Proteome Discoverer User Guide Thermo Scientific Figure 30 Setting parameters for the workflow File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BHGORH ZB BD KR SeuethT Kms AA LMG EB iva amelet 2 Getting Started BEISA RE Starting a New Search by Using the Workflow Editor K Workflow Editor x amp d 2 k f t
338. les and produce data For more information on these methods see Performing Reporter Ion Quantification on page 249 Specifying the Quantification Channels The first step in setting up the quantification is to specify the quantification channels to use This process includes a validation step For precursor ion quantification the validation step ensures that each peptide is in a valid labeling state according to the labels for the different channels as defined in the quantification method For reporter ion quantification the validation step ensures that only peptides that have one of the specified reporter labels as a modification are considered for protein quantification 266 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Setting Up the Quantification Method The process of specifying label modifications is similar for precursor ion quantification and reporter ion quantification but it also has some differences e For precursor ion quantification you specify the label modifications for each quantification channel For reporter ion quantification you set the label modifications for the whole method e For precursor ion quantification specifying the label modifications for quantification channels other than the unlabeled channel is mandatory For reporter ion quantification specifying the label modifications is optional because the information about the modification of the peptides is not necessary
339. lication in a Window You can start the Proteome Discoverer Daemon application on the command line or in a window To run it on the command line see Running the Proteome Discoverer Daemon Application on the Command Line on page 97 To start the Proteome Discoverer Daemon application in a window 1 Start the Proteome Discoverer Daemon application in Windows by choosing Start gt Programs gt Thermo Proteome Discoverer release_number gt Proteome Discoverer Daemon release_number or by clicking the Daemon icon ee on your desktop 2 After the Proteome Discoverer Daemon application window appears connect to a computer that is running the Proteome Discoverer application Selecting the Server 70 The Proteome Discoverer Daemon application can connect to a remote server so that you can perform searches on multiple raw data files from multiple samples or one sample on a remote computer It can also connect to a local server To specify the server to connect to 1 Click the Configuration tab in the Proteome Discoverer Daemon application window 2 From the Host list select the name of the server that you want to use or type the server name You must connect the Proteome Discoverer Daemon application to a computer running the Magellan server Your local host is the default server that is the computer that you are working on To connect to a remote server see Logging On to a Remote Server on page 76 3 In the User bo
340. ligosaccharides of glycoproteins and the sorting and packaging of proteins for transport to a variety of cellular locations Membrane Double layer of lipid molecules that encloses all cells and in eukaryotic cells many organelles The membrane can be a single or double lipid bilayer It also includes associated proteins Note This term is not restricted to the plasma membrane but applies to all types of membranes present in the cell that is nuclear membranes and mitochondrial membranes Mitochondrion A semiautonomous self replicating organelle that occurs in varying numbers shapes and sizes in the cytoplasm of virtually all eukaryotic cells It is notably the site of tissue respiration Nucleus A membrane bounded organelle of eukaryotic cells in which chromosomes are housed and replicated In most cells the nucleus contains all of the cell s chromosomes except the organellar chromosomes and is the site of RNA synthesis and processing In some species or in specialized cell types RNA metabolism or DNA replication might be absent Spliceosome 236 Proteome Discoverer User Guide A ribonucleoprotein complex containing RNA and small nuclear ribonucleoproteins snRNPs which is assembled during the splicing of messenger RNA primary transcript to excise an intron Thermo Scientific 6 Protein Annotation GO Slim Categories Table 18 GO Slim categories for cellular components Sheet 4 of 4 GO Slim
341. lose the sample in the mass spectrometer To avoid this outcome Thermo Fisher Scientific recommends that you connect the Proteome Discoverer Daemon application to a remote computer running the Magellan server before data acquisition To log on to a remote server 1 Start the Proteome Discoverer application on the remote machine 2 Ifyou want to store the output files in a location other than the default do the following a Choose Administration gt Configuration gt Server Settings gt Discoverer Daemon The PublicFiles folder is the default file displayed in the Current File Directory box as shown in Figure 51 b In the New Directory box browse to the location of the user named folder in the PublicFiles folder on the server where you want to store the output files c Click Apply If the directory already exists it automatically appends the date and an incremental index number to the name 76 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Logging On to a Remote Server If you attempt to create a file other than in the PublicFiles folder in the Current File Directory box Discoverer Daemon issues a message informing you that the Proteome Discoverer application will apply the change the next time that you start it To return to the default directory click Reset Figure 51 Discoverer Daemon area of the Configuration view File Search Report Quantification Processin
342. loss 347 fragmentation methods See activation types 352 Proteome Discoverer User Guide funnel icon 152 G GenBank database 339 342 Gene IDs column 204 206 215 Gene Ontology GO database See GO database General page 221 223 283 GO accessions description 212 displaying 213 GO codes 227 229 230 GO database description 202 displaying annotation results from ProteinCenter in MSF file 208 displaying hierarchical GO terms 16 features 203 GO accessions 212 retrieving information from ProteinCenter 202 206 208 Web site 203 GO Slim categories biological processes 237 cellular components 234 colors 209 definition 228 230 molecular components 233 GO terms 202 228 GO Terms column 212 213 golgi GO Slim category 236 GZ files 132 H HCD activation type analyzed by SEQUEST 3 description 8 fragmenting ions 347 ion factors 37 selecting in search wizards 34 specifying in Reporter Ions Quantifier node 334 Heavy Light Count column 323 Heavy Light Variability column 323 Hidden Markov Model 203 hierarchical GO terms 16 homologous proteins 176 HTML files contents of exported 3 Human Proteome Organization HUPO 12 13 66 Thermo Scientific Import Modifications dialog box 147 Import Workflow dialog box 64 importing chemical modifications 146 incorrect node parameters 62 infrared multi photon dissociation See IRMPD activation type inputs to Proteome Discoverer application 12 International Union of Biochemistry
343. low Editor 42 Rawfile and Scan Range Selection page 32 RDB equivalents 149 reannotation MSF file 216 Proteome Discoverer Daemon 217 Re Annotation node description 216 retrieving protein annotations 216 receptor activity GO Slim category 233 Refresh icon 73 regulation of biological process GO Slim category 239 Remove FASTA indexes confirmation box 127 Renaming Template dialog box 31 replicates calculating protein ratios in multiconsensus reports 326 328 definition 323 in mixed mode 330 ratio counts 324 treating quantification results as 327 variability 324 variability in multiconsensus reports 325 reporter ion quantification calculating peptide ratios 313 316 checking the quantification method 281 co isolation 311 controlling protein and peptide ratios 275 correcting experimental bias 280 correction for isotopic impurities 277 creating a workflow for 253 creating workflow for 258 default methods available in 266 271 description 7 249 displaying quantification channel values 295 displaying Quantification Spectrum chart 298 isotopic distribution values 308 missing reporter ions 300 performing 249 performing on HCD and CID scans 257 setting up protein ratios 278 setting up quantification method 264 specifying label modifications 266 specifying mass tags 272 273 specifying quantification channels 271 summarizing settings for 292 troubleshooting 334 See also iTRAQ quantification Proteome Discoverer User Guide 359 Index S
344. lowing e Choose Quantification gt Edit Quantification Method or click the Edit Quantification Method icon ae either on the toolbar or on the Administration page Note To access the Edit Quantification Method command you must first run a workflow that uses the Reporter Ions Quantifier node or the Precursor Ions Quantifier node The Quantification Method Editor dialog box appears as shown in Figure 181 on page 272 through Figure 186 on page 280 e Choose Administration gt Maintain Quantification Methods or click the Maintain Quantification Methods icon g The Quantification Methods view appears as shown in Figure 177 on page 265 It lists all of the available methods for both precursor ion and reporter ion quantification Then either double click the row for the appropriate method in the Method Name or Description column or click the column to the left of Method Name for the method as shown in Figure 194 and click Edit Figure 194 Selecting the method to edit Gp Add Remove J Edit GI Import A Export Status Method Name Description Is Active vY Dimethylation 3plex C2H6 C2H2D4 13C2D6 Dimethylation 3plex C2H4 C2D4 13C2D4 Method Iv v Full 180 Labeling 02 1802 180 labeling method for fully labeled samples Iv v Incomplete 180 Labeling 02 0180 1802 180 labeling method for incompletely labeled samples Iv v ITRAQ 4plex Method foriTRAQ 4 plex mass tags by Applied Biosystems IV v iTRAQ
345. ls page Quantification Method Editor SILAC 2plex Arg10 Lys6 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Heavy Late Channel Name New 1 Quantification Labels To remove a quantification channel select the quantification channel in the list of q q quantification channels and click beneath the list To specify a name for the new quantification channel backspace over the default name in the Channel Name box and type the new name The example in Figure 180 uses Medium The new name now appears in the quantification channel left box To specify a quantification label to assign to a quantification channel click beneath the Quantification Labels box A default quantification label of New number now appears in the Quantification Labels box and the Label Name box Proteome Discoverer User Guide 269 7 Quantification Setting Up the Quantification Method To remove an existing quantification label select the label in the Quantification Labels box and click beneath the box 6 To change the default quantification channel name backspace over the name in the Label Name box and type the new name The example in Figure 180 uses Arg6 Lys6 7 In the Modification Target area select the location of the label on the peptide e Side Chain Modification Indicates that the label occurs on a side chain
346. lse Discovery Rate area of the Peptide Confidence page shown in Figure 142 on page 195 Proteome Discoverer User Guide 197 5 Filtering Data Calculating False Discovery Rates Figure 144 Results of new relaxed target rate File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BS BOT STDDOBH LOAD Ksqeth g Mot AKRAM RLESTAFAREB Workflow Editor helaRVD1_1_1_1_percolator X Administration x E_coli msf X helaRVD1_1_1_1_percolator msf X Celegans_FT_6ITDDDT_01_decoy msf x pene i A Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Modest Confidence Filter Setti High Confidence Filter Settings XCorr Score Versus Charge a 4 FDR Settings 4 FDR Settings Ga sea F Minimal Score for charge state 1 0 005 Minimal Score for charge state 1 0 005 Sa Type J Minimal Score for charge state 2 2 Minimal Score for charge state 2 2 295 Minimal Score for charge state 3 237 Minimal Score for charge state 3 2 46 rocessiog Docks Neme saan Work Boe Deer za Shoat Deepen ros Minimal Score for charge state 4 3 705 Minimal Score for charge state 4 3 705 gt Bee C FT_6l A2 Minimal Score for charge state 5 371 Minimal Score for charge state 5 371 Minimal Score for charge state 6 3 715 Minimal Score for charge state 6 3 715 Minimal Score for charge state 7 3 72 Minimal Score
347. ly protein quantification ratios from the same type of quantification that is either precursor ion or reporter ion based quantification into replicate ratios The names of the protein ratios must be the same to be combined into replicate ratios For example the ratios to combine into replicates must all be from reporter ion based quantification and they must all be identically named such as 115 114 in the result files to be combined The application reports ratios from different types of quantification or ratios with different names as if they were treatments that is side by side on the protein level of the multiconsensus report Mixed Mode You can also mix replicate and treatment mode For example you can load three result files from an iTRAQ 4plex experiment and two files from a SILAC experiment and specify treating the quantification results as replicates In this case the Proteome Discoverer application tries to treat all defined protein quantification ratios as replicates if possible It reports everything else side by side at the protein level of the multiconsensus report In this example it calculates the combined averaged ratios from the three iTRAQ 4plex files and the two SILAC 2plex files and reports the TRAQ and SILAC ratios side by side as shown in Figure 225 and Figure 226 on page 332 In this way the application can mimic complex experimental setups Thermo Scientific Proteome Discoverer User Guide 331 7 Quantifica
348. lysine because these are the cleavage sites for the generally used trypsin protease Both populations incorporate these amino acids into proteins through natural cellular protein synthesis The cells growing in the medium with the heavy isotopes incorporate these isotopes into all of their proteins After altering the proteome in one sample through chemical treatment or genetic manipulation you then combine equal amounts of protein from both cell populations and digest with trypsin before MS analysis Because peptides labeled with heavy and light amino acids are chemically identical they co elute during reverse phase chromatographic separation This means they are detected simultaneously during MS analysis To determine the average change in protein abundance in the treated sample you use the relative peak intensities of multiple isotopically distinct peptides from each protein as shown in Figure 171 244 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Performing Precursor lon Quantification Figure 171 Schematic workflow for SILAC based peptide and protein quantification Relative intensity Quantification Sample A Sample B E Toes G in light DMEM in heavy DMEM 12c6 14N2 Lys and 12Ce 14N4 Arg E Mix heavy and light lysates in 1 1 Digest with Trypsin Heavy and light peptides XXxxxpTQxxxxR XXXXXP TOxxxxR XXXXXPSOQXxxxKXXK XXXXXSOXXXXxxXK XXXXPSXxxxR
349. m TMT6plex PQD 1 0000 0 0000 3365 Unique Used 0 904 16 7 QAAQNIIPASTGAAk Unambiguous G3P_PIG N Term TMT plex K15 T PQD 0 8868 0 0000 2968 Redundant Not Used 0 988 17 nTDGSTDYGILQINSR Unambiguous LYSC_CHICK N Term TMT plex PQD 1 0000 0 0000 3331 Unique Used 1 029 18 I iSQAVHAAHAEINEAGR Unambiguous OVAL_CHICK N Term TMT plex CID 0 8482 0 0000 3224 Unique Used 1 687 19 eDPQTFYYAVAVWk Unambiguous TRFE_HUMAN N Term TMT plex K14 T PQD 0 7486 0 0000 4347 Unique Used ae 4 3 F Discoveren Other Dsta PQD_test_dats 10mix_TMT_1to1_32CE_repeat4_Ross RAW 7639 RT 121 44 mn ITMS PQD Precursor 2 0 Monom z 557 47467 Da MH 558 48195 Da Integration MostConfident Centroid Integefion totrance 0 2 Da 500 400 300 4 Intensity counts Quan Channels Ready 10 10 Protein Group s 10 10 Protein s 525 525 Peptide s 6470 6470 PSM s 14702 14702 Search Input s Displaying Quantification Channel Values for Precursor lon Quantification For precursor ion quantification you can generate a Quan Channel Values chart that displays the area of the isotopes detected for the available quantification channels Heavy isotopes are incorporated into proteins in precursor ion quantification You can use the quantification value area to calculate the relative ratio of a peptide You might also want to view the quantification value area to verify that the peptide ratio calculation is correct
350. matically calculates confidence levels according to the outcome score distribution of the target decoy search that preceded it The phosphoRS 3 0 node updates the preliminary version of the phospho site localization algorithm that was distributed with the 1 3 Proteome Discoverer application The new features of this update are the following e Improved performance The updated phospho site localization algorithm performs parallel calculations using multiple processor cores if available Proteome Discoverer User Guide 15 1 Introduction New Features in This Release e Individual peak depth approach The algorithm determines the optimal number of peaks that is the best peak depth considered for localization of phosphorylation sites for each m z window individually which increases the sensitivity of site localization for CID data e Optimized scoring parameters Depending on the applied fragmentation technique the algorithm uses different fragment ion types for scoring to provide the highest possible sensitivity For CID data it scores only singly and doubly charged b and y ions For analysis of HCD spectra the algorithm also considers neutral loss ions In contrast when localizing phosphorylation sites in ETD spectra the algorithm considers only singly charged c z and y H ions e Additional node parameters The phosphoRS 3 0 node adds new parameters For example you can specify whether the Proteome Discoverer application should con
351. mation that includes this string e Does Not Contain Excludes information that does not include this string c Click the first line in the Condition column and type the condition that the information must meet in order to be excluded d Repeat step a through step c to add more sets of conditions for the information that you want to exclude e To delete a set of conditions in the Active column select the line that you want to delete and click a Click Compile Database Click Start Search on the Find Protein References page to view the results of the extraction as shown in the example in Figure 84 on page 115 You do not have to enter information into the Search For box Excluding Individual Protein References and Sequences from a FASTA Database You can exclude individual entries from a FASTA file To exclude individual protein references and sequences from a FASTA file Choose Tools gt FASTA Database Utilities In the FASTA Database Utilities dialog box click the Compile FASTA Database tab In the Original box browse for the FASTA database that contains the protein that you want to remove or type its path and name In the Please Select a FASTA Database dialog box click Open In the Target box browse for the output FASTA file or type its path and name In the Save Add to FASTA File dialog box select the file verify that the file extension is fasta and click Save Select the Ignore Case of Referenc
352. me that the samples were prepared with the iTRAQ 4plex quantification method giving quantifiable reporter peaks at 114 115 116 and 117 m z When you open these three MSF files the Proteome Discoverer application adds the files to the Input Files page as shown in Figure 221 Thermo Scientific Proteome Discoverer User Guide 327 7 Quantification Calculating and Displaying Protein Ratios for Multiconsensus Reports Figure 221 Loading three single result files containing quantification data File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BAB OTSBDO OSH LORD g sqetht Kut ARMA AEBRRBARE Administration Xx myo_8plex_hcd_pqd 02 msf X Celegans_FT_6ITDDDT_01_decoy msf xX Workflow Editor 10mix_TMT_45CE_1is x Loading x Fab Input Files Result Filters Eon Selected Files Filename Status Loading Progress Task Progress Current Task R C Program Files Proteome Discoverer source files Smoke_Test_Files result_1 msf w C Program Files Proteome Discoverer source files Smoke_Test_Files result_2 msf C Program Files Proteome Discoverer source files Smoke_Test_Files result_3 msf m Treat quan results as replicates E Show quan spectra on separate tab _ Result Items Per File Filename Spectra Processing Nodes Proteins Peptides Unfiltered Peptides Filtered a result_1 15153 SEQUEST 2 90 200 200 E result
353. meters page Sequest HT Search Wizard 2 a Scan Extraction Parameters Define the parameters used for the scan extraction process f d Limits and Thresholds First Mass 350 0 Last Mass 5000 0 Activation Type Any X Unrecognized Charge Replacements Automatic v Intensity Threshold 0 0 Minimum lon Count if S N Threshold 15 Grouping Parameters E Group Spectra Precursor Mass Criterion Precursor Tolerance ppm Max RT Difference min 7 Set the scan extraction parameters a In the First Mass box type the mass of the first precursor ion in daltons In the Last Mass box type the mass of the last precursor ion in daltons These two parameters define the range of ion fragments to search for in the database From the Activation Type list select the fragmentation method to use to activate the scan e CID Collision Induced Dissociation e MPD Multi Photon Dissociation e ECD Electron Capture Dissociation e PQD Pulsed Q Collision Induced Dissociation ETD Electron Transfer Dissociation Proteome Discoverer User Guide 33 2 Getting Started Starting a New Search by Using the Search Wizards 34 Proteome Discoverer User Guide e HCD High Energy Collision Dissociation e Any Activation Type See Fragmentation Methods on page 8 for descriptions of these methods The default is Any Activation Type In the Unrecognized Charge Replacements list sele
354. methods batch BSA2 2 C Projects Daemon __C caliburmethods D aemon C calibur methods batch_export BSA3 a C Projects Daemon 9C Xcalibur methods D aemon C gt lt caliburmethods batch 4 Click OK In this example the Xcalibur data system starts two different workflows performing a Sequest search and exporting a raw file for the recorded raw data files in the Proteome Discoverer application as shown in Figure 62 88 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 62 Two workflows in the job queue started by two different processing methods Start Jobs Configuration Job Queue Gi Pause Gp Resume gg Abort l Remove amp Refresh oa Job Queue Execution State Progress Spectrum Source Submitted at Completed 100 BSA3 BSA3 raw 10 25 2012 4 06 PM Time Processing Node Message 4 06 PM 2 Spectrum Exporter 429 spectra exported in 0 47 s 4 06PM 2 Spectrum Exporter Start exporting of spectra to BSA3 4 06PM 2 Spectrum Exporter 2 Received 429spectrs 4 06PM 2 Spectrum Exporter 1000 spectra exported in 0 96 s 4 06PM 2 Spectrum Exporter Start exporting of spectra to BSA3 4 06PM 2 Spectrum Exporter 2 Received 1000 spectra 4 06PM 1 Spectrum Selector Reading from File 1 of 1 C Projects Daemon BSA3 raw 4684 spectra to
355. might have a file size limitation A search that generates large amounts of data for example a search with multiple raw files could create an MGF file that exceeds this limitation The Max MGF File Size parameter avoids this limitation by performing several separate Mascot searches and merging the results 2 To split the MGF file and avoid any potential file size limitations on the Web server enter the maximum size in megabytes that the MGF file can be in the Max MGF File Size MB box as shown in Figure 11 This size should be less than the file size permitted by the Web server The minimum file size is 20 and there is no maximum The default file size is 500 megabytes 26 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Configuring Search Engine Parameters Figure 11 Maximum MGF file size on the Mascot server 4 1 Mascot Server 500 Mascot Server URL Number of attempts to submit the search Time interval between attempts to submit a search sec 4 2 Mascot Server Authentication User Name Password 4 3 Web Server Authentication User Name Password 4 4 Default Confidence Thresholds Significance High Significance Middle 20 90 For information on these parameters refer to the Help 3 In the Number of Attempts to Submit the Search box specify the number of times that the Proteome Discoverer application tries to submit the search when the Mascot server is busy The minimum value is
356. mo Scientific 7 Quantification Using Reporter lon Isotopic Distribution Values To Correct for Impurities Table 22 The meaning of colors in the Quantification Spectrum chart Sheet 2 of 2 Color Meaning Yellow box Indicates that the pattern includes peaks from only one channel This ion pattern peak is not used in calculating the quantification values for the different quantification channels Pink bar Indicates that a quantification pattern peak is expected but is missing This ion pattern peak is not used in calculating the quantification values for the different quantification channels Blue bar Indicates that a quantification pattern peak is present but is unsuitable because of errors in peptide labeling or because of the wrong centroid retention time a range out of the delta mass the wrong intensity or a peak that has been used by another isotopic pattern This ion pattern peak is not used in calculating the quantification values for the different quantification channels Using Reporter lon Isotopic Distribution Values To Correct for Impurities Thermo Scientific iTRAQ and TMT kits consist of labels that contain different numbers of 1 C atoms 15N atoms or both For simplicity assume that a 4plex kit yields peaks at 114 115 116 and 117 m z which correspond to 1 C1 C2 C3 and C4 respectively Because the label substances are not 100 percent isotopically pure each label contains a certain number of ot
357. mples see Using the Proteome Discoverer Daemon Utility on page 69 Through settings that you specify in the Proteome Discoverer application you can search filter and sort raw files with the Sequest and Mascot algorithms In addition to creating reports from the analyzed data the application extracts relevant MS MS spectra from the raw file and determines the precursor charge state Filters in the application remove false positives and other irrelevant information with a variety of user specified methods Note You can filter data according to false discovery rates that you define through the use of decoy databases that you specify in the workflow Using the standard Proteome Discoverer workflow involves the following steps when you 8 8 step y process analyze and interpret mass spectrometry data These steps are shown graphically in Figure 3 1 Upload a FASTA database if necessary to use Sequest 2 Choose a search wizard or create a workflow in Workflow Editor Identify the raw file 3 Select parameter settings in the search wizard or the nodes of the Workflow Editor Proteome Discoverer User Guide 9 1 Introduction Workflow 4 Begin a search of the raw data The Proteome Discoverer application initiates a search against a FASTA database 5 Sort and filter the search report generate graphs and views and interpret the search results 6 Optional Review the quantification results and change parameters 7 R
358. mport Modifications Import From Unimod v http Awww unimod orgAanl unimod_tables xml E Overwrite Existing Close In the Import From list select Unimod The UNIMOD URL appears in the adjacent box To overwrite an existing upload select the Overwrite Existing check box Click Import A status message appears When the upload is complete click Close Proteome Discoverer User Guide 147 4 Searching for Data Updating Chemical Modifications For chemical modifications imported from unimod org you can only edit the Is Active Modification and Abbreviation columns You do not have access to the Delta Mass Delta Average Mass Substitution Leaving Group Position and UNIMOD Accession No columns Chemical modifications imported from unimod org have a number greater than zero in the Unimod Access No column If you select the Overwrite Existing check box the Proteome Discoverer application does the following when it imports chemical modifications from unimod org e Updates the columns that are inaccessible to you e Updates the names and the abbreviations of the modifications e Adds any new amino acids found in unimod org Adds any amino acids that you removed if they are defined in unimod org e Removes any amino acids that you added if they are defined in unimod org If you do not select the Overwrite Existing check box the Proteome Discoverer application performs the same tasks as it does during installa
359. n E The Structure Of Bovine F1 Atpase Covalently Inhibited With 4 Chloro 7 Nitrobenzofurazan gt gil2624889 pdb 1AG8 D Chain D Aldehyde Dehydrogenase From BovineMitochondna gi 2624888 pdb 1AG8 C Chain C Aldehyde Dehydrogenase From 4 gt gil3401963 pdb 1AQL B Chain B Crystal Structure Of BovineBile Salt Activated Lipase Complexed With Taurocholate gi 3401962 pdb 1AQLJA Chain 6 gt gi 1127257 pdb 1LCP B Chain B Bovine Lens Leucine Aminopeptidase Complexed With L Leucine PhosphonicAdd giJ1127256 pdb 1iLCP A Chain A Bov al a ake a 11 21 31 4 51 61 7 81 91 KETAAAKFER QHMDSSTSAA SSSNYCNQMM KSRNLTKDRC KPVNIFVHES LADVQAVCSQ KNVACKNGQT NCYQSYSTMS ITDCRETGSS KYANCAYKIT 101 QANKHIIVAC EGNPYVPVHF DASV Start Search Save Add Selected to Database Stop Sear 4 Searching for Data Using FASTA Databases Boolean search operators Protein references Amino acid sequence of selected protein 10 Optional To save a protein result row in another FASTA database select the protein row click Save Add Selected to Database select the database in the Save Add to FASTA File dialog box and click Save To filter a protein reference search 1 On the Find Protein References page of the FASTA Database Utilities dialog box click the line below Reference in the middle of the page to access a list of operators that you can use to filter the references The default operator is Starts wit
360. n at physiological temperatures In biologically catalyzed reactions the reactants are known as substrates and the catalysts are naturally occurring macromolecular substances known as enzymes Enzymes possess specific binding sites for substrates and are usually composed wholly or largely of protein DNA binding Selective interaction with DNA deoxyribonucleic acid Enzyme regulator activity Modulation of an enzyme Metal ion binding Selective interaction with any metal ion Motor activity Catalysis of movement along a polymeric molecule such as a microfilament or microtubule coupled to the hydrolysis of a nucleoside triphosphate Nucleotide binding Selective interaction with a nucleotide which is any compound consisting of a nucleoside that is esterified with ortho phosphate or an oligophosphate at any hydroxyl group on the ribose or deoxyribose moiety Protein binding Selective interaction with any protein or protein complex a complex of two or more proteins that may include other nonprotein molecules Receptor activity The mediation by protein or gene products of a signal from the extracellular environment to a intracellular messenger Proteome Discoverer User Guide 233 Table 17 GO Slim categories for molecular functions Sheet 2 of 2 GO Slim molecular function RNA binding Description Selective interaction with an RNA molecule or a portion of it Signal transduc
361. n page 43 b Click the Browse button in that row The Select Analysis File s dialog box appears as shown in Figure 25 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 25 Select Analysis File s dialog box Ey Select Analysis File s Selected Files i F Folder i Remove ok Cancel c Click Add Files to open the Add Analysis File s dialog box d Browse to the location of the data input file select the file and click Open e Click OK to close the Select Analysis File dialog box 8 Ifyou selected the Spectrum Files node in step 5 drag the Spectrum Selector node to the workspace and place it beneath the Spectrum Files node Figure 26 shows the addition of the Spectrum Files and Spectrum Selector nodes to the workspace Selecting the Spectrum Selector node in the workspace pane displays the available parameters for that node in the right pane The numbers that appear on each workflow node indicate the order in which the Proteome Discoverer application processes the nodes Note You can set the Spectrum Selector node to select which precursor mass to use for a given MS scan such as choosing the precursor from the parent scan Thermo Scientific Proteome Discoverer User Guide 45 2 Getting Started Starting a New Search by Using the Workflow Editor Figure 26 Spectrum Files and Spe
362. nce XCorr 07 z 2 High Confidence XCorr 19 z 2 Medium Confidence XCorr 08 z 3 High Confidence XCor 23 z 3 Medium Confidence XCor 1 z gt 4 High Confidence XCorr 26 z gt 4 Medium Confidence XCorr 12 For information on these parameters refer to the Help 3 If you are using the SEQUEST search engine to search high resolution data set the XCorr confidence thresholds under the XCorr Confidence Thresholds high resolution data parameter The default values appear in Figure 8 4 If you changed any settings click Apply 24 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Configuring Search Engine Parameters The message box shown in Figure 9 appears Figure 9 Administration message box Administration Changes were saved They will have no effect for currently open workflows 5 Click OK Note Click A Reset to return to the default values Configuring the Mascot Search Engine Before using the Mascot search engine you must direct the Proteome Discoverer application to the location of the Mascot server and configure the parameters that control access to the Mascot server If your Mascot search fails the following procedure can help you check for server problems e Directing the Proteome Discoverer Application to the Mascot Server Location Configuring Mascot Parameters e Troubleshooting Failed Mascot Searches Directing the Proteome Discoverer Application to the Mascot Ser
363. nclude in quantification 242 diseases associated with 231 displaying filtered out 170 general information about 223 group members 177 filtering applying filters 155 deactivating filters 167 removing filters 166 result filters 154 155 row filters 167 with protein filters 155 function of 227 grouping algorithm used 179 by algorithm in previous releases 184 displaying other proteins belonging to same group L77 on Proteins or Peptides page shortcut menu 174 175 on Result Filters page 174 176 peptides with sequences not belonging to master protein 183 PSMs identified by multiple workflow nodes 184 ranking 174 sequence redundancy 174 turning off 178 groups in status bar 184 homologous 176 master 174 178 183 members of groups 174 number of 174 PSM Ambiguity column 182 ranking 174 retrieving information from ProteinCenter 201 scoring 153 sequence features 225 Web links to information 231 Proteins page displaying filtered out rows 170 GO database information from ProteinCenter 202 master proteins 174 Pfam annotations 203 Ratio columns 273 275 Proteome Discoverer User Guide 357 Index Q ratio count and variability 323 Ratio Count columns 323 row filters 167 Proteome Discoverer application closing 20 features 2 filtering data 154 155 inputs 12 limitations 14 main window 19 new features in this release 14 opening 19 outputs 13 search wizards 29 system requirements xii workflow 9 Proteome Discoverer application icon
364. ne 33 0 Yes No 100 0 300 100 33 300 mussing fora 33 75 No No 100 0 300 100 0 300 quan 33 75 Yes No 100 0 300 100 75 300 channel 33 0 No Yes 100 0 300 0 0 0 33 0 Yes Yes 100 0 300 100 33 300 33 75 No Yes 100 0 300 0 0 0 33 75 Yes Yes 100 0 300 100 75 300 Quan 33 0 No Irrelevant 0 0 0 0 0 0 as 33 0 Yes Irrelevant 0 0 0 0 0 0 mussing for all 33 75 No Irrelevant 0 0 0 0 0 0 quan 33 75 Yes Irrelevant 0 0 0 0 0 0 channels Table 24 Calculating peptide ratios when values are very high or low Sheet 1 of 2 Use Ratios Calculated ratios Displayed ratios Above Maximum Maximum Case Allowed Fold Allowed Fold Change setting Chango for 115 114 116 114 115 114 116 114 Quantification setting Ratio is within 100 Irrelevant 2 000 3 000 2 000 3 000 the limits 100 Irrelevant 0 500 0 250 0 500 0 250 Ratio isO oreo 100 No 0 3 000 0 000 3 000 because one 700 Yes o0 0 250 100 000 0 250 quan channel value is missing 320 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Calculating Protein Ratios from Peptide Ratios Table 24 Calculating peptide ratios when values are very high or low Sheet 2 of 2 Case A ratio exceeds the limits All ratios exceed the limits Use Ratios Calculated ratios Displayed ratios Above Maximum Maximum Allowed Fold Allowed Fold Change setting Change for 115 114 116 114 115 114 116 114 Quantification setting 100 No 2000 000 3 000 100 000 3 000 100 No 0 300 0
365. nfiguration gt Server Settings gt Discoverer Daemon command on the remote server If you attempt to create a file other than in the PublicFiles folder in the Current File Directory box Discoverer Daemon issues a message informing you that the Proteome Discoverer application will apply the change the next time that you start it This option performs the same function as the f foldername option except that you can use the name of the folder more than once When you use the name more than once the Proteome Discoverer application appends the date and an incremental index number to the name Local server Does nothing a foldername SpectrumF7 e Remote server Uploads the spectrum file to the location specified on the configured server SpectrumF 1e is the name of the spectrum file Local server Does nothing e h Lists the options available with the Thermo Magellan DiscovererDaemon command e 1 serverName userName Connects Discover Daemon to the specified local or remote host machine servername Specifies the name of the local or remote host userName Specifies the name to log on e r outputFi7ename Specifies the name of the output file You must use this option with the e option as in this example DiscovererDaemon e sfcid any mascot3 xml r silaclnoMT_AS4DE msf 98 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteom
366. nfiguration a 5 9 Workflow Nodes amp Annotation Mascot 3 MSPepSearch amp SEQUEST amp Sequest HT amp SpectraST B4 Server Settings amp Discoverer Daemon amp FASTA Indexes Ready Now you are ready to search the spectrum library To search with the MSPepSearch node see Searching Spectrum Libraries with the MSPepSearch Node on page 139 For more information on the MSPepSearch node refer to the Help Deleting a Spectrum Library You can delete a spectrum library from the application To delete a spectrum library 1 Choose Administration gt Maintain Spectrum Libraries The Administration page appears with the Spectrum Libraries view 2 Click at the beginning of a row to select the row 3 Click E Remove 4 In the Remove Spectrum Libraries Databases dialog box click OK 136 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Searching Spectrum Libraries The Spectrum Libraries file that you selected appears as a job in the job queue After you start the deletion of the file you cannot cancel the deletion You can remove the completed job from the job queue by clicking Remove and then clicking OK in the Delete Jobs dialog box Searching Spectrum Libraries with the SpectraST Node Figure 100 shows the basic workflow for searching spectrum libraries with the SpectraST node You can use this node as an alternative to a search node such as SEQUEST Figure 100
367. ng iTRAQ or TMT tags from HCD scans mS Reporter lons 1 Se Quantifier Parameters 4 Show Advanced Parameters 4 4 1 Quantification Method Quantification Method TMT 6plex 2 Peak Integration Integration Window Tolerar 20 ppm Integration Method Most Confident Centroid 3 Scan Event Filters Mass Analyzer FTMS MS Order MS2 Activation Type HCD Proteome Discoverer User Guide 335 7 Quantification Troubleshooting Quantification To quantify PQD scans from an ion trap use the typical settings shown in Figure 229 Figure 229 Typical settings for quantifying iTRAO or TMT tags from the ion trap POD scans ee Spectrum 2 AS Reporter lons 1 mi Selector lt Quantifier Fixed Value PSM Validator Parameters Sar 2 Show Advanced Parameters 4 1 Quantification Method Quantification Method TMT 6plex 4 2 Peak Integration Integration Window Tolerar 20 ppm Integration Method Most Confident Centroid 4 3 Scan Event Filters Mass Analyzer FTMS MS Order MS2 Activation Type PQD Make sure that you have used the correct set of static and dynamic modifications for the search engine For example if you are searching TMT 6plex data with SEQUEST check that your settings resemble those in Figure 230 336 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Troubleshooting Quantification Figure 230 Modifications required for searching TMT 6plex samples
368. ng rule B if the accession number and the description are separated by a space A typical FASTA title line that matches this parsing rule would look like this one gt HP0001 hypothetical protein Helicobacter pylori 26695 FASTA ID e Accession HPOOOL e Description hypothetical protein Helicobacter pylori 26695 Custom Parsing Rule C The application uses custom parsing rule C if the FASTA title line only contains the accession number A typical FASTA title line that matches this parsing rule would look like this one gt 143B_HUMAN FASTA ID e Accession 143B_HUMAN e Description 143B_HUMAN Thermo Scientific Proteome Discoverer User Guide 343 Ooo i Chemistry References The tables in this appendix list amino acid symbols and mass values enzyme cleavage properties and the fragment ions used in the Proteome Discoverer application Contents e Amino Acid Mass Values e Enzyme Cleavage Properties e Fragment Ions Amino Acid Mass Values The Proteome Discoverer application uses the amino acid symbols and mass values listed in Table 25 and Table 26 Table 25 Amino acid mass values Sheet 1 of 2 Anno acid One letter Three letter Monoisotopic Average Sum code code mass mass formula Glycine G Gly 57 02147 57 0517 C H3NO Alanine A Ala 71 03712 71 0787 C3H NO Serine S Ser 87 03203 87 078 C H NO Proline P Pro 97 05277 97 1168 C H NO Valine V Val 99 06842 99 1328 C5 H NO Thr
369. ng s to Include box A line enabling you to specify the first set of conditions appears in the box Click the first line in the Select Operator column and select the operator to apply to the information to be extracted You can select from the following e Starts With Extracts information that begins with this string e Does Not Start With Extracts information that does not begin with this string Ends With Extracts information that ends with this string Does Not End With Extracts information that does not end with this string e Contains Extracts information that includes this string e Does Not Contain Extracts information that does not includes this string Click the first line in the Condition column and type the condition that the information must meet in order to be extracted Repeat step a through step c to add more sets of conditions for the information to be extracted To delete a set of conditions in the Active column select the line that you want to delete and click x The Compile FASTA Database page should now resemble the example in Figure 83 Proteome Discoverer User Guide 113 4 Searching for Data Using FASTA Databases Figure 83 Compile FASTA Database page of the FASTA Database Utilities dialog box FASTA Databases Original C Program Files Proteome Discoverer source files FASTA_Files H influenzae fasta Target C Program Files Proteome Discoverer source files FASTA_Files uniprot_sprot_20
370. nsus reports the Quantification Method Editor dialog box features a Common Quan Parameters box shown in Figure 187 on page 282 so that you can set common quantification parameters for all contained result files at once On the General page of the dialog box shown in Figure 223 you can switch between treatment and replicate mode Proteome Discoverer User Guide 329 7 Quantification Calculating and Displaying Protein Ratios for Multiconsensus Reports Figure 223 Switching between treatment and replicate mode by editing the common quantification parameters Quantification Method Editor ea Common Quan Parameters x General Ratio Calculation Protein Quantification Experimental Bias As in treatment mode multiconsensus reports are no different from single reports for replicates at the peptide level At the protein level the Proteome Discoverer application combines the protein ratios of the single result files into averaged protein ratios as shown in Figure 224 It calculates the combined protein ratio as the arithmetic mean of the protein ratios of the single reports and calculates the protein ratios as the median of the used peptide ratios of the particular result file See Calculating Ratio Count and Variability on page 323 for information on how the application calculates and displays the values in the Ratio Count and Variability columns for multiconsensus reports Figure 224 shows
371. nt is the number of peptides marked Used for this protein If more than one ratio was used for example the ratios in iTRAQ or TMT data the count for a particular protein ratio might be smaller than the number of peptides marked Used The count could be lower than the number of peptides marked Used in cases where some of the ratios are excluded by the different settings or thresholds defined by the quantification method For replicates the Ratio Count columns display a list of the separate counts for each replicate If a protein was not identified in one of the replicates or no peptide usable for calculating the protein was identified for this replicate a appears in the Ratio Count cell If none of the replicates provide a usable peptide the Ratio Count cell is empty The Ratio Variability columns show the variability of the peptide ratios that are used to calculate a particular protein ratio They are similar to a coefficient of variation for the calculated protein ratios as a normalized measure of the peptide ratio spread used for calculating the protein ratio The Proteome Discoverer application calculates the displayed variability differently for single search reports multiconsensus reports that are treated as treatments and multiconsensus reports that are treated as replicates Single Search Reports Thermo Scientific For single search reports the protein ratio variability is calculated as a coefficient
372. ntents Searching for Data ce wad che eae cal uie cx earner een wanes 101 Using FASTA atabases ii a eta ac abut yen ska ioiai Lente hod si eT Eai EE 101 Displaying FASTA Files og icc i Balin Ws Baler Wie al we We ding aa lly 101 Adding FASTA Piles ius aaah sate tae hate Bale Aik s eet we 104 Deleting FASTA Files oprasrssei eee ed seek as week ae eee ee 104 Compressing a Protein Database o50 baci eee bee abe cise eas 105 Displaying Temporary PAs VA Piles to cisco segs wale asda elt 105 Adding a Protein Sequence and Reference to a FASTA Database File 106 Finding Protein Sequences and References 0 000 cece eee eee 107 Compiling a FASTA Databases cdc ca nu ti tam nauar 112 Excluding Individual Protein References and Sequences from a FASTA D t bdse vey ot oye hapten tebe ered re nae ghost ie driers a a tong bees 116 Managing FASTA Indexes nie cana dese epaaes aaa ales 117 Searching Spectrum Libraries ac nd cla eiad a Dainese Ae tes 129 Displaying Spectrum Libraries onunu naear tied oe were deere 130 Adding a Spectrum Library ii i 0 5 tales as teas Rea rnaar eer 131 Deleting a Spectrum Library esses ie to s eatin ebhe lace gta pia stole a ale 136 Searching Spectrum Libraries with the SpectraST Node 137 Searching Spectrum Libraries with the MSPepSearch Node 139 Visually Verifying Spectrum Library Matches 000048 140 Updating Chemical Modifications 00 c cee eee e
373. ntification you would select a dynamic modification of TMT6plex 229 163 Da K If you do not find this label you can enable it by following the instructions in Updating Chemical Modifications on page 141 c In the Static Modifications area select the static modifications For example for TMTe 6plex quantification you would select TMT6plex 229 163 Da K in the Peptide N Terminus box d Set any other parameters that you prefer 11 Set the parameters for all other nodes in the Parameters pane For information about all the parameters that you can set for each node refer to the Help For information on the parameters that you can set for the Reporter Ions Quantifier node see step 12 of this procedure 12 Click the Reporter Ions Quantifier node and set the parameters for it in the Parameters pane a Set up the quantification method Click the Quantification Method parameter and follow the procedure in Setting Up the Quantification Method on page 264 to specify the quantification method b Set the parameters that specify the peak integration i In the Integration Tolerance box specify the mass to charge m z window that enables you to look for the reporter peaks The default is 20 ppm ii In the Integration Method box select which peak to choose when more than one peak is found inside the integration window Thermo Scientific Proteome Discoverer User Guide 255 7 Quantification Performing Reporter lon
374. ntification Modifications with Mascot In the editor in the Mascot server window you can specify that these groups be variable fixed or exclusive You can also define them directly for the method in report ion quantification or for each component in precursor ion quantification R kod l To specify the quantification modifications to search for Choose Administration gt Configuration gt Mascot and configure the Mascot search engine by following the instructions in Configuring the Mascot Search Engine on page 25 Be sure that in the Mascot Server URL box you enter the URL of the Mascot server to be used for Mascot searches Set up a workflow that includes at a minimum the nodes shown in Figure 172 on page 247 for precursor ion quantification Figure 174 on page 254 for reporter ion quantification or Figure 175 on page 260 for Precursor Ions Area Detector quantification Click the Mascot node Select the dynamic modifications to search for e Select a dynamic modification from the list in each Dynamic Modification parameter You can select up to nine modifications e Click the From Quan Method parameter in the Parameters pane under Modification Groups and from the list see Figure 176 for an example select the modifications that you want to search for You can select more than nine modifications Note Do not use the modifications that you specify as part of the modification groups in the selected
375. ntification method by following the instructions in the next section Correcting Experimental Bias Correcting Experimental Bias The purpose of the Experimental Bias page of the Quantification Method Editor dialog box is to correct experimental bias which is the difference in the total observed protein abundance between two or more samples Assuming that in real samples most of the proteins are not regulated the intensity of the median protein in sample x should be the same as the intensity of the median protein in sample y If it is not it may indicate experimental bias caused by for example errors in pipetting or the determination of protein concentration in the mixed samples You must correct for the difference For best results always enter a small normalization factor To correct experimental bias 1 Click the Experimental Bias tab shown in Figure 186 This page is the same for both precursor ion and reporter ion quantification Figure 186 Experimental Bias page of the Quantification Method Editor dialog box G Quantification Method Editor SILAC 2plex Arg10 Lys6 x Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Normalize On Protein Median Manual Normalization 2 Select the normalization factor to apply from the list at the top of the page e Default None Performs no normalization e Normalize on Protein Median Normalizes all pept
376. ntifier node of 0 3 Da for extracting the reporter peaks from the quantification spectrum 298 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart Figure 200 Quantification Spectrum chart for an iTRAO 8plex sample using Proteome Discoverer scans File Search Report Quantification Processing Workflow Editor Administration Tools Window Help Ou aoao on OMe a g a nm K sesti Kut AR AAE GCEBTAPARE Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x Workflow Editor myo_Splex_hcd_pad x Administration xX myo_8plex_hcd_pqd 02 msf x v4 gt Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Sequence PSM Ambiguity Proteins ProteinGroups Protein Group Accessions Modifications Activation Type AScore ACn Rank Search Engine Rank Qua 1 TIVTALKVSADEIAAASRR Unambiguous 2 1 P59401 PQD 0 1698 0 0000 1 1 2 J7 IGGRVQLVGDDLFVINKK Unambiguous 1 1 Q8KG25 PQD 0 3175 0 0000 1 Y 3 J7 LAGYKDALAQHGIALNEK Unambiguous 1 1 P24242 PQD 0 2517 0 0000 1 4 I LLHEEPMTKLDLSTIR Unambiguous 1 1 013682 PQD 0 3474 0 0000 k 1 sr ronment Unambiguous 1 1 p24242 PQD 0 2435 0 0000 1 1 6 P EFMPMPIALAHRLSRR Unambiguous 1 1 Q97F85 PQD 0 1933 0 0000 1 7 I LTTKFQQALADAQSLAAR Unconsidered 3 1 Q7vwe PQD 0 1698 2 2 8 I LGIPVMVYGLRTDFQGK Selected 1 1 Q2KSN6 PQD 0 0923 0 0000 1 1 9 J7
377. o Spectra One Peptide and One Protein cine dine eumantes ee eats pe ete 321 Case 3 Quantification Result Associated with Two Spectra Two Peptides and One Protein hose tacos ae yaaa taeda Gia Be 321 Case 4 Quantification Result Associated with One Spectrum Two Peptides and One Protein wd ae bo Veda seas ethos Raed woe de 322 Case 5 Quantification Result Associated with One Spectrum One Peptide Two Proteins wv wae Suid oot cet oy iia te Wate a 322 Case 6 Quantification Result Associated with One Spectrum Two Peptides and Two Proteins juice s 0 u dtetadaseancthcte unner 322 Case 7 Quantification Result Associated with Two Spectra Two Peptides and Two Proteins siiiieas cesta eae ares 323 Calculating Ratio Count and Variability cis pctopacediwpiucd swt ns 323 Replicates bap ccctsts niota a a AE E E E E a rei E 323 Treatrnents os mi Ua aie EE E AEE E E AE A A ace 324 HAO COUR oe x dy ee a ee EA O AE ATE RA E Aah 324 PATO AEE IES co Beane tae eros a hae aoe get ahaa a ae ae aed Aa 324 Calculating and Displaying Protein Ratios for Multiconsensus Reports 326 Calculating Protein Ratios in Multiconsensus Reports Treated as Treatments iieaoe aea a aa Ahh Pucci nit E a AT ieni 328 Calculating Protein Ratios in Multiconsensus Reports Treated as Replicatesaio ei siis ert Ue a e eae a ins lata She A ar 328 Mixed Modes korren srine i seat ENSE et Math ER ae 330 Identifying Isotope Patterns in Precursor Ion Quantification
378. o reannotate an MSF file in the Workflow Editor e To reannotate an MSF file in Proteome Discoverer Daemon To reannotate an MSF file in the Workflow Editor 1 Choose Workflow Editor gt New Workflow 2 In the Annotation area of the Workflow Nodes pane select only the Re Annotation node and drag it to the Workspace pane 3 Select the Re Annotation node 4 Click the MSF File Path box and then click the Browse button to open the Select Analysis File dialog box 5 Browse to the MSF file to save the new annotations in or type the path and name of the file in the File Name box and click Open The name of the MSF file appears in the Name box in the Workflow Editor 6 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon e The Proteome Discoverer application submits the workflow to standard workflow processing and displays the reannotation progress in the job queue Note If you created the MSF file that you want to reannotate with a previous version of the Proteome Discoverer application the application updates the file first to comply with the current result file schema 216 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Reannotating MSF Files To reannotate an MSF file in Proteome Discoverer Daemon 1 Create a reannotation workflow in the Workflow Editor according to the instructions in To reannotate an MSF file in the Workflow Editor on page 216 2
379. o return an included or excluded peptide to its default status On the Proteins page click the plus sign next to the protein of interest to display its constituent peptides Right click the peptide of interest and choose Include Exclude Peptide s from Protein Quantification gt Default from the shortcut menu The Quan Usage column now displays the peptide s usage status when the MSF file was first opened Excluding Peptides with High Levels of Co lsolation Thermo Scientific To create a fragment spectrum you select a precursor mass for isolation isolate and fragment the ions within a mass window that you define and record the product ion masses created Ideally you would isolate and fragment only the precursor ions of a single selected component However in practice you isolate the precursor ions within a user specified window typically 1 or 2 daltons around the isolation mass Co eluting components with a mass falling into this isolation window are also isolated and fragmented This process is called co isolation The co isolating components are likely to be peptides whose fragments are observed in the created fragment spectra The co isolation can exacerbate the identification of the selected peptide and lower the identification confidence Proteome Discoverer User Guide 311 7 Quantification Classifying Peptides Co isolation is an issue in reporter ion quantification In this type of quantification
380. o1 Se Construct spectrum file collection WF_Re Annotation For more information about processing files with Discoverer Daemon see Using the Proteome Discoverer Daemon Utility on page 69 Uploading Results to ProteinCenter If you have a user account on a ProteinCenter server you can upload search results directly from the Proteome Discoverer application to ProteinCenter To upload search results to ProteinCenter 1 Open an MSF file and be sure that it is selected 2 Choose Tools gt Options 3 In the Options dialog box click ProteinCenter The ProteinCenter page opens as shown in Figure 159 218 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Uploading Results to ProteinCenter Figure 159 ProteinCenter page of the Options dialog box On es Fragment Match Options Upload ur http webservice proteincenter proxeon com ProXweb Fragment Match Colors and Fonts ProteinCenter User name a In the URL box type the URL of the ProteinCenter server to use b In the User Name box type the user name of your ProteinCenter user account c In the Password box type the password of your ProteinCenter user account d Click OK A message box appears with the following message Settings of Protein Center changed Do you want to save your changes 4 Click Yes 5 Open an MSF file in the Proteome Discoverer application Refer to the Help 6 Choose Tools gt Ex
381. oBGRIARE Parameters Show Advanced Parameters 4 1 input Data Protein Database Enzyme Name Trypsin Maximum Missed Cleav 1 Instrument Default Taxonomy All entries 4 2 Tolerances Precursor Mass Toleran 10 ppm Fragment Mass Toleran 0 8 Da Use Average Precursor False 4 3 Modification Groups From Quan Method a4 15N 13C Metabolic MD 4 Static Modification 5 Static Modification 6 Static Modification From Quan Method The modifications specified within the modification group of the selected quan method are used in addition to the set dynamic and static modifiations Oo Modifications specified within the selected quan method Data Export must not be set additionally as dynamic or static u Spectrum Exporter modifications Annotation Ready Thermo Scientific 5 Optional If you want to group these modifications go to the editor in the Mascot server window and choose Configuration Editor gt Quantitation Once you group the modifications you can define them as fixed variable or exclusive You can also define them directly for the method in reporter ion quantification or for each component in precursor ion quantification Refer to the Mascot documentation for information on grouping modifications and defining the groups For the final search results it does not matter whether you explicitly specified a modification as either a dynamic or a static
382. oRS node in a workflow Connect it to all search nodes whose results you want to submit to phosphorylation site localization scoring Figure 34 gives an example of a workflow with two different search nodes attached to the phosphoRS node Figure 34 Workflow with two different search nodes attached to the phosphoRS node File Search Report Quantification Processing Workflow Editor Administration Tools Window Help SE BB BSOTSHODOSH LOAD K seth g Mot i A RARE AQAEBSRRFARB Workflow Editor x gt 4 Workflow Nodes 4a F Parameters 4 a A i TRA Data Input amp os pore l Show Advanced Parameters Qi Spectrum Files Spectrum amp Feature Retrieval Event Detector Spectrum Selector ig Noise Peak Filter i Non Fragment Filter ig Spectrum Grouper i Spectrum Normalizer iu Top N Peaks Filter Spectrum Filters Ty Scan Event Filter TW Spectrum Confidence Filter Ti Spectrum Properties Filter E Sequence Database Search ge Mascot Q SEQUEST W Sequest HT Spectral Library Search Q MsPepSearch Q SpectrasT E PSM Validation jih Fixed ValuePSM Validator Percolator lih Target Decoy PSM Validator E PTM Analysis if phosphoRS3 0 E Quantification 2 Precursor Ions Area Detector 2 Precursor Ions Quantifier fd Reporter Ions Quantifier E Data Export i Spectrum Exporter S Annotation Annotation 7j Ready m
383. obability for a target amino acid of at least the minimum value pass the filter If you do not select any target acids all rows containing a site probability of at least the defined minimum probability pass the filter 5 Click OK Grouping Proteins Although MS MS based proteomics studies are centered around peptides you can also explore what proteins are present in a sample and their associations through related peptides Deducing protein identities from a set of identified peptides becomes difficult because of sequence redundancy such as the presence of proteins that have shared peptides These redundant proteins are automatically grouped and are not initially displayed in the search results report In the results report you can turn protein grouping on or off with the Enable Protein Grouping command on the shortcut menu or with the settings in the Protein Grouping Enabled area on the Result Filters page The latter method enables you to select more options in grouping Grouping is turned on by default For information about the grouping mechanism that the Proteome Discoverer application uses to group proteins see Protein Grouping Algorithm on page 179 The proteins within a group are ranked according to the number of peptide sequences the number of PSMs their protein scores and the sequence coverage The top ranking protein of a group becomes the master protein of that group By default the Proteins page displays only the mast
384. ocabularies ontologies that describe gene products in a species independent manner biological processes cellular components molecular functions Each gene ontology is divided into categories and subcategories called GO terms which define the protein in more specific terms For example chloroplast a term in the cellular component ontology is subdivided as follows 202 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Pfam Annotation chloroplast p chloroplast envelope p chloroplast membrane i chloroplast inner membrane i chloroplast outer membrane You can obtain more information on the GO Ontology Web site at www geneontology org Pfam Annotation In addition to GO annotations you can also retrieve from ProteinCenter Pfam annotations from the Pfam database at the Wellcome Trust Sanger Institute pfam sanger ac uk These are annotations of protein families which are proteins with similar sequences and similar biological functions A special sequence comparison algorithm called the Hidden Markov Model groups proteins into the families by comparing the sequences Each family has its own ID number that starts with Pf The Proteins page of the MSF file displays this number in the Pfam IDs column You can use the Pfam identification number to go to the Pfam database to obtain more details about the protein family You can also activate the ProteinCard for each protein by double clicking the Pfam
385. ocessing Node 5 28PM 0 Spectrum Library Creator Total processing time was 38 min 31s 5 28PM 0 Spectrum Library Creator Finished extraction of reference spectra from 2011_05_24 c_elegans_nist tar gzin 38min 10s 4 50PM 0 Spectrum Library Creator Extracting spectra for vizualization 4 50PM 0 Spectrum Library Creator Preparing Spectrum Library 2011_05_24_c_elegans_nist tar gz for usage Message B 43 Workflow Nodes amp Annotation a Mascot E MSPepSearch 6 SEQUEST amp Sequest HT amp SpectraST E Server Settings amp Discoverer Daemon amp FASTA Indexes When the Proteome Discoverer application finishes adding the spectrum library the spectrum library file appears in the Spectrum Libraries view as shown in Figure 99 Thermo Scientific Proteome Discoverer User Guide 135 4 Searching for Data Searching Spectrum Libraries Figure 99 Added NIST spectrum library in the Spectrum Libraries view File Search Report Report File Quantification Processing Workflow Editor Administration Tools Window Help 2B OT AOD FSR BT HT Zom Rs KMart BPARKRAATAOBSSAAB l Administration X Workflow Editor C elegan SpectraST Benchmark 2 0 x 4 WR Add Remove Process Management R Y S4 Job Queue Content Management a a FASTA Files is FASTA Indexes m Spectrum Libraries t a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management a R Licenses Co
386. of the reporter ions in the MS MS spectra to assess the relative abundance of the peptides and therefore the proteins that they are derived from 252 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Reporter lon Quantification iTRAQ includes two default mass tags available from Applied Biosystems ABI that you can use to label all peptides e iTRAQ 4plex which is standard e iTRAQ 8plex The Proteome Discoverer application includes default quantification methods for processing data from iTRAQ 4plex and iTRAQ 8plex labeled samples You can use these methods to create your own workflow templates For information on adding quantification methods see Changing a Quantification Method on page 288 iTRAQ quantification works exactly the same as TMT quantification except that TMT quantification offers 2plex 6plex and 10plex quantification methods and iTRAQ offers 4plex and 8plex quantification methods Creating a Workflow for Reporter lon Quantification Thermo Scientific To use an isobarically labeled reporter ion quantification method you must open an MSF file generated from a workflow that includes the Reporter Ions Quantifier node Setting up the workflow for TMT and iTRAQ quantification is basically the same To create a workflow for reporter ion quantification 1 Choose Workflow Editor gt New Workflow For instructions on creating a workflow with the Workflow Editor see Starting a New
387. of variation for log normal distributed data CVpg normal In this case the protein ratio variability is calculated from the used peptide ratios 7 7 as follows CV og normal exp StdDev log r log r 1 1 where StdDev log 7 log r 1 483 x MAD log r log 7 Proteome Discoverer User Guide 325 7 Quantification Calculating Ratio Count and Variability 2 CF ise normal fexp 1 483 x MAD log r log 7 1 variability r r 100 x CV igg normal where MAD 7 is the median absolute deviation MAD of the peptide ratios r 7 In statistics the median absolute deviation is a robust measure of the variability of a univariate sample of quantitative data MAD log r 7 median log r median log r r Starting with the residuals deviations from the data s median the median absolute deviation is the median of their absolute values The 1 483 constant ensures consistency for the 7 distributed normally as N 02 and large N E 1 483 x MAD r r 6 The Proteome Discoverer application uses these statistics because they are more robust in the presence of outliers as a classical coefficient of variation CV It also uses them to calculate the protein ratio as the median of the used peptide ratios Calculating Variability in Multiconsensus Reports Treated as Treatments For multiconsensus reports that treat quantification data as different treatments the r
388. ome Discoverer User Guide 161 5 Filtering Data Filtering the Search Results The A Cn peptide filters out all PSMs with a A Cn score larger than the specified value On the Peptides page or the peptides sections of the Proteins and Search Input pages the A Cn column displays the A Cn values For example Figure 115 shows how the score of a peptide ranked 2 compares to other multiple high confidence peptides from the same spectrum Figure 115 A Cn scores for multiple high confidence peptides from the same spectrum File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OF BB B OTF SBRBROORH LB WD KR seueth KmMst A AAEL ABCEBERBPARG Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x v dp MassAnalyzer Activation Type MS2 55 103 658 86029 1316 71330 PQD MS2 15 73 704 36084 2111 06797 PSM Ambiguity Proteins ProteinGroups Protein Group Accessions Modifications Activation Type AScore ACn Rank hPGDFGADAQGAMTk Unambiguous 5 2 494711 1942750 N Term iTRAQ8plex K15 PQD 0 1599 0 0000 hPGDFGADAQAAMSk Unconsidered 1 2 494711 1942750 N Term iTRAQSplex K15 PQD hPGNFGADAQGAMTk Unconsidered E 1 1942750 N Term iTRAQ8plex K15 PQD 0 376 3 GGSVTLPESGTDLLTHRLEK Unconsidered t 0 PQD 0 3941 4 PSM Ambiguity Mass Analyzer Activation Type MS Order Identified Peptides Isolation Interference Ion Inject Time ms Precursor m z Da Precursor MH D
389. ometry analyses Contents e Related Documentation e System Requirements e Special Notices e Contacting Us To provide us with comments about this document click the link below Thank you in advance for your help SURVEY Related Documentation The Proteome Discoverer application includes Help and these manuals as PDF files e Proteome Discoverer User Guide Proteome Discoverer Installation Guide To view product manuals Proteome Discoverer User Guide Go to Start gt Programs gt Thermo Proteome Discoverer 1 4 gt Proteome Discoverer 1 4 User Guide Proteome Discoverer Installation Guide Go to Start gt Programs gt Thermo Proteome Discoverer 1 4 gt Proteome Discoverer 1 4 Installation Guide Thermo Scientific Proteome Discoverer User Guide xi Preface To open Help e From the main Proteome Discoverer window choose Help gt Help Contents e Ifavailable for a specific window or view click Help or press F1 for information about setting parameters For more information visit www thermo com You can find application notes at www thermo com appnotes System Requirements The Proteome Discoverer application requires a license In addition your system must meet the following minimum requirements System Requirements Hardware e 2 GHz processor with 2 GB RAM e DVD R ROM drive e Video card and monitor capable of 1280 x 1024 resolution XGA e Screen resolution of 96 dpi e 75 GB available on the
390. on Processing Workflow Editor Administration Tools Window Help jeu aalsler Celegans_FT_6ITDDDT_01_01 msf_annotation msf x BOOO88 4088 GD CK SequestHT GK Mascot A R Le ASB Sadar Bl B A oj Thermo Scientific Description Search Input Result Filters Peptide Confidence Search Summary qgogggaqgaggaqgg aa aa aaa UNCoordinated family member unc 54 Caenorhabditis x F07A5 7 Caenorhabditis elegans ATPsynthase subunitfamily member atp 2 Caenorhab VITellogenin structural genes yolk protein genes family MYOsin heavy chain structural genes family member myo VITellogenin structural genes yolk protein genes family VITellogenin structural genes yolk protein genes family ENOLase family member enol 1 Caenorhabditis elegans Protein Disulfide Isomerasefamily member pdi 1 Caen Malate DeHydrogenase family member mdh 1 Caenom VITellogenin structural genes yolk protein genes family Tudor Staphylococcal Nucleasehomolog family member t Heat Shock Proteinfamily member hsp 1 Caenorhabdit Heat Shock Proteinfamily member hsp 6 Caenorhabditi ACTin family member act 4 Caenorhabditis elegans F01G10 1 Caenorhabditis elegans Ribosomal Protein Small subunitfamily member rps 3 R05G6 7 Caenorhabditis elegans Temporarily Assigned Genename family member tag 6l Peptides Molecular Function
391. on application is running click the Refresh icon 2 to display the workflow 7 Connect to the server If you have a local connection the Proteome Discoverer application disables the Server Output Directory box and displays local connection Then it places the output files are placed beneath the input files If you connect to a remote server in the Server Output Directory box type the name of the directory where you want the original output files placed on the server By default the Proteome Discoverer Daemon application places this directory under the following directories e Windows 7 c ProgramData Thermo Discsoverer lt release_number gt PublicFiles e Windows XP c Documents and Settings All Users DiscovererDaemon Spectrum Files If you choose this directory you must type a file folder name in the Server Output Directory box You can specify a different directory by choosing Administration gt Configuration in the Proteome Discoverer application clicking Discoverer Daemon in the Server Settings section and browsing for the location in the New Directory box If you selected the MudPIT option in the Spectrum Files area in the Output Filename box type the name of the output file that you want to store the results of the search in The Start Jobs page should now resemble Figure 47 for batch processing or Figure 48 for MudPIT processing Proteome Discoverer User Guide 73 3 Using the Proteome Discoverer Daemon
392. on of the tags in the different TMT kits Sheet 2 of 2 TMT 2plex Tag Mass TMT 6plex Original TMTe 6plex Current TMT 10plex iodo TMT 6plex Tag Mass Tag Mass Tag Mass Tag Mass 128 128 134433 128 128 134433 128_N 128 128115 128 128 134433 128_C 128 134433 129 129 13779 129 129 131468 129 N 129 131468 129 129 1311468 129_C 129 13779 130 130 141141 130 130 141141 130_N 130 134825 130 130 141141 130_C 130 141141 Thermo Scientific 131 131 138176 131 131 138176 131 131 138176 131 131 138176 The iodo TMT 6plex includes cysteine reactive TMT reagents The TMT 10plex leverages the high resolution of recent mass spectrometers to routinely differentiate the 3C isotopes from the N isotopes For the 127 128 129 and 130 tags the TMT 10plex contains two reagents the C and the N reagent For the monoisotopic masses of the different reporter ions after CID or HCD fragmentation see Table 20 Figure 173 shows the position of the C and N atoms in the different reagents In this illustration the stars indicate the positions of the 3C and the N substitutions the red lines indicate the position of the ETD fragmentation sites and the blue lines indicate the position of the CID fragmentation sites McAlister G C Huttlin E L Haas W Ting L Jedrychowski M P Rogers J C Kuhn K Pike I Grothe R A Blethrow J D and Blethrow G S P Increasing the Multiplexing Capacity of TMTs Using Report
393. only PSMs that meet the criteria set in the Protein Grouping Enabled area of the Results Filters page a protein group might contain proteins that have identified peptides whose sequences are not all contained in the master protein of the protein group For example if you specify that the protein grouping inference process consider only PSMs that have at least medium confidence a protein group might include a protein with a low confidence peptide that does not belong to a master protein Proteome Discoverer User Guide 183 5 Filtering Data Grouping Proteins Protein Groups in the Status Bar The status bar shows the actual number of protein groups versus the total number of protein groups refer to the Help The difference is the number of protein groups that the application removed to comply with the selection of the Apply Strict Maximum Parsimony Principle option on the Results Filters page By enabling the display of filtered out protein groups you can investigate the protein groups that were removed during this process Proteins Grouped by the Grouping Algorithm in Previous Releases The Proteome Discoverer application removes some protein groups that the protein grouping mechanism created in previous versions of the application The previous algorithm might have created these groups from only low confidence peptides or the application removed them to comply with the selection of the Apply Strict Maximum Parsimony Principle option on the
394. ontology terms are related in hierarchical graphs called GO accessions The GO term annotated to a special protein is always part of a complex directed graph All ancestor elements that is the elements between the annotated GO term and one of the three top level terms molecular functions cellular components and biological processes are additional less specific descriptions of the annotated value For example the iron ion binding GO 0005506 term contains in its graph the metal ion binding GO 0046872 value which is less specific All GO terms contained in the graph of the annotated GO term of the protein are represented in the GO Terms column on the Proteins page 212 Proteome Discoverer User Guide Thermo Scientific KD Ww 6 Protein Annotation Displaying the Annotated Protein Results To display GO accessions 1 Open the generated MSF file by following the instructions in Opening the Results Report on page 195 2 In the Column Chooser dialog box of the Proteins page select the GO Terms column For information on the Column Chooser dialog box see Selecting the Columns to Display on page 197 The Proteome Discoverer application displays the protein s GO terms contained in the graph of the annotated GO term on the Proteins page of the MSF report in the GO Terms column as shown in Figure 155 Figure 155 GO Terms column in results report GO Terms column File Search Report Quantificati
395. or ion or Excluded by Method both Are the peptide quan results compatible with the quan method Does the peptide have protein references Mark peptide as No Proteins Is the peptide ranked the best in the spectrum Mark peptide as Redundant Is the number o protein links or groups gt 1 Mark all peptides not yet classified as Not Unique Is the peptide the most confident Mark peptide as Redundant Mark peptide as Unique Mark all peptides not yet classified as Redundant Proteome Discoverer User Guide 313 7 Quantification Calculating Peptide Ratios Calculating Peptide Ratios For both precursor ion and reporter ion quantification the Proteome Discoverer application calculates protein ratios as the median not the mean of all peptide hits belonging to a protein that is marked Used in the Quan Usage column of the report It chooses the median to calculate the protein ratios because it is relatively robust in the presence of outliers In principle the Proteome Discoverer application uses only the peptides in the filtered results for protein ratio calculation when the result filters are applied to the search result These result filters are what you want to apply to quantification For example protein ratios that change because you filter peptides having a specific sequence tag will skew the results Protein ratios are the median of the peptid
396. ost important databases 339 parsing rules 342 FASTA files view Residues column 103 Sequences column 103 Cancel icon 103 Compact icon 103 105 Display Temporary icon 103 Last Modified column 103 Name column 103 Proteome Discoverer User Guide 351 Index G opening 101 parameters in 103 Size column 103 Status column 103 FASTA Index Creator dialog box 121 FASTA indexes automatic removal 126 automatically creating 121 changing maximum number stored 128 changing storage location 128 deactivating automatic removal 126 definition 117 deleting 126 deleting from deleted FASTA files 129 discarding changes from previous session 129 displaying 119 manually creating 125 restoring deleted 127 FASTA Indexes Options dialog box 128 FASTA Indexes view after adding FASTA index 124 126 opening 119 FDR See false discovery rate Features page 221 225 filter sets copying from one installation of Proteome Discoverer to another 163 creating 164 deleting 165 loading 163 164 saving 164 using 163 filtered out rows 170 filters deactivating 167 false discovery rates 186 protein reference searches 109 removing 166 Find Protein References page 107 Fixed Value PSM Validator node attaching to search engine nodes 46 description 15 Fourier Transform mass spectrometer 256 fragment ions activation types producing 347 ammonia loss 347 charged on C terminal side 347 charged on N terminal side 347 factors dependent on 7 types 7 347 water
397. ot score 138 F value 137 138 scores reported 137 Spectrum Exporter node exporting spectra 66 Spectrum Files node creating a search workflow 44 peak area calculation quantification 259 260 Thermo Scientific Index T precursor ion quantification 246 248 reporter ion quantification 253 254 spectrum libraries adding 131 to search with MSPepSearch node 134 to search with SpectraST node 131 deleting 131 136 displaying date last modified 131 name 131 number of spectra found 131 size 131 displaying downloaded 130 generating mirror plots 15 140 searching with MSPepSearch node 14 129 139 searching with SpectraST node 14 129 137 Spectrum Libraries view Spectra column 131 displaying 15 130 Last Modified column 131 Name column 131 parameters in 131 Size column 131 Type column 131 Spectrum Selector node creating a search workflow 45 peak area calculation quantification 259 260 precursor ion quantification 247 248 reporter ion quantification 253 254 selecting precursor mass to use 45 spliceosome GO Slim category 236 Start Jobs page 71 73 217 Start Workflow icon 207 static modifications definition 141 selecting in Sequest HT wizard 38 setting for FASTA indexes 123 status bar 184 185 Status column 103 structural molecule activity GO Slim category 234 survey link xiv SwissProt database 339 342 system requirements xii T tab delimited TXT files 13 tandem mass tag quantification See TMT quantification Target Decoy PS
398. ot server In this case file an error report e Ifyou can perform simple Mascot searches investigate your failing searches more closely Does the search finish successfully on the Mascot server according to the Mascot search log Do the process messages sent to the job queue during the search indicate the problem 4 If the search problems persist after you take these measures file an error report 28 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards Starting a New Search by Using the Search Wizards As mentioned earlier the quickest way to begin using the Proteome Discoverer application is to define your search parameters using the search wizards You can access the Sequest HT and Mascot search wizards from the Proteome Discoverer application interface Use these search wizards to perform basic functions such as setting the search parameters selecting a database and a search engine and selecting the chemical modifications that you will use to conduct your search To perform the application s more sophisticated operations such as quantification or using decoy searches to estimate the number of incorrect PSMs that exceed a given threshold you must use the nodes available in the Workflow Editor The SEQUEST search engine is only available as a node in the Workflow Editor You can also access the Sequest HT and Mascot search engines through nodes in the Workflow
399. otation msf x o B S O A AA cocoa E s Accession Description Score F Coverage Proteins UniquePeptides Peptides PSMs M jar Function lar Component Biological Process d id lolecul Cellul logical im mm a 17509401 17509391 25144756 193209657 32566139 17570201 71991083 17536383 17554386 17554310 17570197 17533087 17541098 17562024 71994099 17539652 17554770 17541790 71991728 71988080 17555558 17553980 32566204 71989658 17555492 17540874 42538971 71983985 17508669 25150292 17507559 71997105 71996641 17552884 17506493 17506425 17551718 25144271 17563146 UNCoordinated family member unc 54 Caenorhabditi F07A5 7 Caenorhabditis elegans ATP synthasesubunit family member atp 2 Caenorha VITellogenin structural genes yolk protein genes famil MYOsin heavy chain structural genes family member m VITellogenin structural genes yolk protein genes famil VITellogenin structural genes yolk protein genes famil ENOLase family member enol 1 Caenorhabditis eega Protein Disulfide Isomerasefamily member pdi 1 Cae Malate DeHydrogenase family member mdh 1 Caeno VITellogenin structural genes yolk protein genes famil Tudor Staphylococcal Nucleasehomolog family member Heat ShockProtein family member hsp 1 Caenorhab Heat Shock Protein family member hsp 6 C
400. otein s 26480 26480 Peptide s 28142 28142 PSM s 13472 13472 Search Input s In the box in the upper left of the Peptide confidence page you can switch between validation based on Percolator and validation based on the calculation of target and decoy estimated FDRs from the search engine scores This choice is always available even if Percolator refused to process the data because it did not meet one of the requirements for the number of target and decoy matches Use the Peptide Confidence page to do the following e Set new filters and recalculate new FDRs based on these new filter criteria e Set new target FDRs and then recalculate new filter settings that when applied lead to FDRs no higher than the new target Note If you filter on peptide confidence during the loading of the report all of the options on the Peptide Confidence page are unavailable because you can no longer adjust the settings 196 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Calculating False Discovery Rates Recalculating the FDRs You can recalculate the false discovery rate on the Peptide Confidence page To recalculate the FDRs 1 Open an MSF file and click the Peptide Confidence tab 2 In the filter list select the filter for determining the peptide confidence The available options are different for each search engine e Sequest Default XCorr Score Versus Charge Uses this filter to calculate the F
401. otopic label Label for quantification Non standard residue Amino acid derivative like selenomethionine Multiple More than one classification possible AA substitution Amino acid replaced by another amino acid mutation Other Modification not fitting into another category The Proteome Discoverer application automatically imports the classifications from unimod org the protein modifications online database for mass spectrometry applications You can also manually define your own classifications Adding Chemical Modifications You can create new chemical modifications and add them to the Chemical Modifications view For example you might have a new or experimental label that you want to add to the list of chemical modifications e To add a new chemical modification e To update an existing chemical modification To add a new chemical modification 1 Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 2 Click the Add a Modification heading An empty row appears as shown in Figure 105 Figure 105 Adding a row in the Chemical Modifications view Is Active Modification Abbreviation DeltaMass Delta Average Mass Substitution Leaving Group Position Unimod Accession No y A E wl A E O E E iy Vv Acetyl Acetyl 42 010565 42 0367 H 2 C 2 O Any 1 aA Iv Acetyl Acetyl 42 010565 42 0367 H 2 C 2 O Protein_N 1 Vv Acety
402. ous experiments Using a library of already well identified peptides avoids identifying already known peptides over and over again by a time consuming database search Restricting the library to previously identified peptides also drastically reduces the search space and therefore the search time In addition comparisons that use consensus spectra consider the measured peak intensities increasing the selectivity and making the identification more accurate You can use the SpectraST and the MSPepSearch nodes to search large spectrum libraries downloaded from the NIST or the PeptideAtlas home page All currently available libraries are for collision induced dissociation CID or quadrupole time of flight QTOF data The QTOF libraries also work for high energy collision induced dissociation HCD data Proteome Discoverer User Guide 129 4 Searching for Data Searching Spectrum Libraries Displaying Spectrum Libraries You can display a list of all the spectrum libraries that you registered in the Proteome Discoverer application 1 To list the available spectrum libraries e Choose Administration gt Maintain Spectrum Libraries or on the Administration page click the Maintain Spectrum Libraries icon Ti on the toolbar or in the Content Management area The Spectrum Libraries view shown in Figure 95 appears It lists all the spectrum libraries that you downloaded from NIST or the Peptide Atlas home page and registered It displa
403. out rows 170 row filters 167 search wizards FASTA files used 29 spectrum source files used 29 starting searches 30 workflow involved in using 30 Seattle Proteome Center 12 sector field mass spectrometer specifying in Reporter Ions Quantifier node 256 Select Analysis File s dialog box 44 54 Select Modifications page 38 141 Select Processing Method dialog box 86 Sequence Setup icon 85 Sequence View icon 85 Sequences column 103 Sequest adding FASTA files 104 availability of FASTA files for searches 103 105 calculating peptide rank 160 creating FASTA index 121 description 4 options for calculating FDR 197 workflow 9 See also Sequest HT search engine See also SEQUEST search engine Sequest HT search engine configuring parameters for 21 22 data types analyzed 3 description 4 14 options for calculating FDR 197 wizard 2 5 29 Completing the Wizard_name Search Wizard page 40 Rawfile and Scan Range Selection page 32 Scan Extraction Parameters page 33 Search Description page 40 Select Modifications page 38 141 Sequest ST Search Parameters page 35 starting 31 Welcome to the Search Wizard page 31 See also Sequest SEQUEST search engine configuring 24 data types analyzed 3 description 3 4 output 13 See also Sequest Sequest ST Search Parameters page 35 Show Peptide Groups command grouping peptides 185 Thermo Scientific Show Peptide Ratios command icon 313 Show Protein Group Members command 174 175 Show Protein Peptide ID De
404. overer application recognizes the following file formats for searching spectrum libraries with the SpectraST node e msp files which you can find in the _consensus_final_true_lib tar gz file on the library download site at NIST or on the PeptideAtlas home page You will need an unpacking tool such as 7 Zip or WinRAR to unpack the downloaded gz file before you can add the msp file to the Proteome Discoverer application Thermo Scientific Proteome Discoverer User Guide 131 4 Searching for Data Searching Spectrum Libraries e zip gz files from the NIST or PeptideAtlas You can find these files named _spectrast tar gz or _splib zip on the library download site at NIST or on the PeptideAtlas home page The zip file must contain four files with suffixes splib sptxt pepidx and spidx If one of these files is missing the file is not added to the Proteome Discoverer application 2 In the Proteome Discoverer application choose Administration gt Maintain Spectrum Libraries or click the Maintain Spectrum Libraries icon TA on the toolbar Click Add In the Select a Spectrum Library dialog box do the following If you want to add an msp file to the Proteome Discoverer application a In the list box in the lower right corner of the Select a Spectrum Library dialog box select All Spectrum Library Files gz msp zip or msp files msp b Browse to the location of the spectrum library wh
405. owing information Gene Ontology GO annotations which are displayed in the GO Accessions column of the Proteins page of the MSF file GO Slim annotations which are displayed in the Molecular Function Cellular Component and Biological Process columns of the Proteins page of the MSF file In addition you can define your own categories of GO Slim annotations Gene identifications from the Entrez gene database which are displayed in the Gene IDs column of the Proteins page of the MSF file Protein family Pfam annotations which are displayed in the Pfam IDs column of the Proteins page of the MSF file UniProt PTM modifications documented in the UniProt database which are displayed on the Proteins Identification Details view in the Proteins page of the MSF file 206 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Creating a Protein Annotation Workflow The Proteome Discoverer application retrieves the annotation data after all the search nodes have finished processing To create an annotation workflow 1 Choose Workflow Editor gt New Workflow 2 Set up your workflow by following the instructions in Starting a New Search by Using the Workflow Editor on page 42 3 In the Annotation area of the Workflow Nodes pane select the Annotation node and drag it to the Workspace pane The Annotation node automatically connects to the other nodes in the workflow 4 Optional After you join all you
406. ows Connect the nodes a Click the top node so that a blue handle is activated at the bottom center of the node as shown in Figure 27 Figure 27 Activated node example SpectrumFiles 0 b R Joining the nodes together creates a sequence of steps for the Proteome Discoverer Blue handle application to follow b Drag the blue handle down to the top center of the node below it as shown in Figure 28 Figure 28 Joining two nodes me SpectrumFiles 0 Drag arrow from top node to bottom node IMPORTANT Ifthe next node appears with a red edge at this point you cannot connect to the previous node Proteome Discoverer User Guide 47 2 Getting Started Starting a New Search by Using the Workflow Editor If the Workflow Editor prevents you from connecting two nodes the workflow is erroneous c Link all the nodes to develop a workflow 12 After you join all your chosen nodes align them by choosing Workflow Editor gt Auto Layout or clicking the Auto Layout icon or right clicking a node and choosing Auto Layout from the shortcut menu 13 Optional You can renumber the workflow nodes in the workflow in consecutive order by choosing Workflow Editor gt Auto Number 14 Set the parameters for each node in the workspace pane a Click the node to activate its functions The available parameters for the node appear in the Parameters pane as shown in the example for the Spectrum Se
407. ows the names of the quantification channels and the y axis shows the intensity of the reporter ions in counts The 4plex quantification method in iTRAQ has four reporter ions Suppose that they are used to label four biological samples 114 115 116 and 117 Figure 198 shows the Quan Channel Values chart created by the Show Quan Channel Values command for these samples It shows the relative intensities of the samples labeled with the 114 115 116 and 117 reporter ions Clearly the sample labeled 115 is the sample with the greatest reporter ion intensity Proteome Discoverer User Guide 295 7 Quantification Displaying the Quantification Channel Values Chart Figure 198 Quan Channel Values chart for reporter ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE BB BOT STB O SH g amm K suet g Mascot AAAS AQOESBTABAREB Sequest SILAC Dimethylation Search 1 3 0 297 msf X Multi Reportfrom2Reports x PQD with TMT 6 plex 10 175 min msf x _ waa Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Quan Spectra PSM Ambiguity Protein Group Accessions Modifications Activation Type AScore I eVVGSAEAGVDAASVS FR Unambiguous OVAL_CHI K N Term TMT plex CID 1 0000 0
408. ox Quantification Method Editor SILAC 2plex Argi0 Lys6 Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Show the Raw Quan Values Minimum Quan Value Threshold 0 0 E Replace Missing Quan Values With Minimum Intensity E Use Single Peak Quan Channels Apply Quan Value Corrections Reject All Quan Values If Not All Quan Channels Are Present Fold Change Threshold for Up Down Regulation 2 0 Maximum Allowed Fold Change 100 E Use Ratios Above Maximum Allowed Fold Change for Quantification Percent Co lsolation Excluding Peptides from Quantification 2 To create additional columns in the results report that display the reporter ion intensities or the corrected reporter ion intensities when you selected Apply Quan Value Corrections for every peptide select the Show the Raw Quan Values check box By default this option is clear 3 To set all quantification values whose intensity falls below a specified threshold to zero type the threshold in the Minimum Quan Value Threshold box The default threshold value is 0 0 4 When the ratio of the ion intensity of the peptide in a sample to the ion intensity of the peptide in the control sample is missing or is 0 and you want to replace it with the minimum ion intensity detected select the Replace Missing Quan Values With Minimum Intensity check box The Proteome Discoverer applicat
409. pane and place it directly under the Spectrum Files node Set the parameters 3 Drag two Scan Event Filter nodes to the workspace pane and place them side by side beneath the Spectrum Selector node In the Parameters pane set the Activation Type parameter to CID for one node and to ETD for the other node 4 Drag the SEQUEST node to the workspace pane and place it beneath the Scan Event Filter node set to the CID activation type 5 Drag the Mascot node to the workspace pane and place it beneath the Scan Event Filter node set to the ETD activation type 6 Drag two Fixed Value PSM Validator nodes to the workspace pane and place one beneath the SEQUEST node and one beneath the Mascot node 7 Connect the nodes as shown in Figure 35 8 Choose Workflow Editor gt Start Workflow to start the parallel workflow Adding a Non Fragment Filter Node for High Resolution Data The main purpose of the Non Fragment Filter node is to remove precursor peaks from the spectra that are not related to peptide fragments and could therefore increase the risk of the search engines making false positive matches If you add a Non Fragment Filter node to the workflow for processing data taken from Orbitrap instruments Thermo Fisher Scientific recommends that you remove most of the precursor peaks Setting the window to a smaller width increases the risk of leaving some of the precursor peaks or their side bands in the spectrum Figure 36 shows the recommended se
410. pen Report Display Row Filter Job Queue Execution State Progress Name Spectrum Source Description Submitted E Completed Import of 2011_05_26_human_spectrast tar gz Import of Spear 11 9 2012 3 Time Processing Node Message 4 04PM_ 0 Spectrum Library Creator Total processing timewas 1h 3min amp 04PM_ 0 Spectrum Library Creator Finished extraction of reference spectra from2011_05_26_human_spectrast tar gzin 36 min 20 s 3 27PM_ 0 Spectrum LibraryCreator Extracting spectra for vizualization 3 27PM 0 Spectrum Library Creator Finished creationof decoy spectrallibrary from 2011_05_26_human_specrast tar gzin 23 min45 s 3 27PM 0 Spectrum Library Creator NREPS 20 77542 10 19 49702 4 9 95862 2 3 87582 1 0 3 27PM 0 Spectrum LibraryCreator PROBABILITY gt 0 9999 194054 0 999 0 9999 73846 0 99 0 999 33589 0 9 0 99 9199 lt 0 9 0 3 27PM 0 Spectrum LibraryCreator TERMINI Tryptic 288561 Semi tryptic 22127 Non tryptic 0 3 27PM 0 Spectrum Library Creator CHARGE 1 23452 2 169509 3 97513 4 19214 5 899 gt 5 101 3 27PM 0 Spectrum Library Creator Total number of distinct stripped peptides inlibrary 190539 3 27PM 0 Spectrum Library Creator Total numberof distinctpeptideionsin library 310688 3 27PM 0 Spectrum Library Creator Total number of spectra in library 310688 3 27PM 0 Spectrum Library Creator Peptide Indexfile C PROGRA 2 Thermo DISCOV 2 4 SPECTR 1 2011_0 1 TAR hum
411. peptides and proteins versus the total number of peptides and proteins displayed in the Result Items Per File area at the bottom of the Input Files Result Filters page Affects the quantification results of proteins 154 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results For information about filtering with the Result Filters page see Filtering Results with the Filters on the Result Filters Page on page 155 Row filters on the shortcut menu of the Proteins Peptides and Search Input pages display filters only Use these filters with the filters on the Result Filters page to narrow your search results even further When you display the filtered out rows the affected lines for both filters are seen as unavailable rows Excluding peptides by setting row filters does not change the number of identified peptides and the percentage coverage values of the proteins For information about filtering with row filters see Filtering Results with Row Filters on page 167 If you save your report you can save the filters that you set on the Result Filters page with your results report You cannot save the filters that you set with the row filters with your results report The row filters only work on the visible rows in the report However you can save the row filters in a saved layout For information about saving layouts refer to the Help Filtering Results with the Filters on the Resul
412. ph 0 0000 S 12 100 0 S 13 100 3 ic a 7 AAAAGLGHPAsPGGsEDGPP 1 2 1 1P100552870 2 S11 Phospho S15 Phosph 0 0000 S 11 100 0 S 15 10C nen 3 E er I7 GPPDFssDEEREPTPVLGSG 42 4 1 1P100216230 3 S6 Phospho S7 Phospho 0 0000 S 6 99 8 S 7 99 8 T 14 8 49 0 00 3 amp Ej 7 SEAAAPHTDAGGGLssDEE 10 4 1 1P100156793 5 S15 Phospho S16 Phosph 0 0000 S 1 0 0 T 8 0 4 S 15 8 39 0 00 3 E ve I7 GLRDsHssEEDEASSOTDLS 10 5 1 1P100012280 3 5 Phospho S7 Phospho 0 0000 5 100 0 S 7 100 0 S 8 38 0 00 3 amp an I7 KEDsDEEEDDDsEEDEEDDE 1 4 1 1P100183526 5 SA Phospho 12 Phospho 0 0000 S 4 100 0 S 12 100 0 8 30 0 00 3 10 gt GPPDFssDEEREPtPVLGSGA 32 4 1 1P100216230 3 S6 Phospho S7 Phospho 0 0000 6 100 0 S 7 100 0 T 8 22 0 00 3 H 11 7 SLEETLHTVDLssDDDLPHD 10 1 1 1P100005809 7 12 Phospho 13 Phosph 0 0000 S 1 0 0 T 5 0 0 T 8 6 8 14 0 00 3 H 12 gt SQSDLDDQHDyDSVASDED 10 4 1 1P100789714 1 Y11 Phospho S16 Phosp 0 1242 S 1 0 0 S 3 0 0 Y 11 7 97 0 00 3 H 13 7 KSLDsDEsEDEEDDYQQK 22 1 1 1P100013297 1 5 Phospho S8 Phospho 0 0000 2 0 0 S 5 100 0 S 8 7 96 0 00 2 H 14 gt AAAAGLGHPAsPGGsEDGPP 3 2 1 1P100552870 2 11 Phospho S15 Phosph 0 0000 S 11 99 2 S 15 99 2 S 7 81 0 00 3 H 15 gt FTDKDQQPsGsEGEDDDAE 12 2 1 1P100550243 2 9 Phospho Si1 Phospho 0 0000 T 2 0 0 S 9
413. plication on the Command Line Syntax Thermo Scientific You can run the Proteome Discoverer Daemon application on the command line or in an interface window Torun the Proteome Discoverer Daemon application on the command line 1 Open a command shell and use the cd command to move to Program Files gt Thermo gt Discoverer gt System gt Release 2 Type DiscovererDaemon and any of the following options on the command line DiscovererDaemon e foldername Filecount workflow ParameterAssignment c foldername a foldername SpectrumF7 e h 1 serverName userName r outputFi ename p parameterFile rawFi e f foldername The Discoverer Daemon command line syntax includes the following parameters e e foldername Filecount workflow ParameterAssignment Executes the workflow on the server using these specified parameters foldername Specifies the location where the raw files are stored You can give it any name for example RawFiles or Fractions Fi Tecount Specifies the number of spectrum files that must be included before the workflow is executed This parameter is intended to be used with MudPIT experiments and acquisition on several machines If the workflow should be executed regardless of the number of files contained in the file collection use ANY instead of a number workflow Specifies the name of the template file containing the workflow in xml format You must have created thi
414. port to ProteinCenter The Export to ProteinCenter dialog box opens 7 In the Destination box specify the name of the data set to upload to ProteinCenter as shown in Figure 160 Figure 160 Export to ProteinCenter dialog box Celegans_FT_6ITDDDT_01_01 msf_annotation Criteria Checked protein groups 8 If you want to export only the result data from selected protein groups select the Checked Protein Groups check box Thermo Scientific Proteome Discoverer User Guide 219 6 Protein Annotation Uploading Results to ProteinCenter If you do not select Checked Protein Groups the Proteome Discoverer application exports the result data of all protein groups 9 Click Export After the Proteome Discoverer application exports the data set to ProteinCenter you can log in to your ProteinCenter account The uploaded data set appears under the Incoming node in the ProteinCenter window as shown in Figure 161 Figure 161 Uploaded data set in the ProteinCenter window O lel http o0000q proteincenter proxeon com ProXweb contentview WORKSPACE INFO e3e3f787 8842 4fl a 9ec6 64393031a2b6 File Edit View Favorites Tools Help x Google oly Favorites ProteinCenter ProteinCenter E Free Hotmail a Suggested Sites v g Web Slice Gallery v Tele E 24 Search qj Share More gt gt 6 090412_Hela_test_2hr_01 prot 3065 20120724_TEAB_Hyb_1ug_NoTrap_25cm_2hr_03 prot 3157 ABRF_4
415. porter Intensities with Minimum Intensity option In the example the 126 reporter ion has been replaced with a minimum intensity value This is not exactly the true value but it is better than having no estimates for the ratios of this protein Whether this option gives valuable results for you depends on your experimental design and quantification strategy Proteome Discoverer User Guide 303 7 Quantification Displaying the Quantification Spectrum Chart Figure 203 Quantification results after applying the Replace Missing Reporter Intensities with the Minimum Intensity option File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OU BBBOTSATO SH g mT K suet Kmart AAA Me ROBTABARE Sequest TMT 6 plex Benchmark 1 4 0 175 msf x 4d Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary o Accession Description Score V Coverage Proteins UniquePeptides Peptides PSMs 127 126 127 126 Count io 127 126 Vari_ Sequence PSM Ambiguity Proteins Protein Groups Protein Group Accessions Modifications Activation Type AScore Rank Search Engine Rank p irs 8 I7 iSQAVHAAHAEINEAGR Unambiguous 1 1 129293 N Term TMT plex CID 0 6588 0 0000 1 1 9 I eVVGsAEAGVDAASVSEFR Unambiguous 1 1 129293 N Term TMT6plex S5 Ph CID 0 4219
416. precursor ion and reporter ion quantification The Status column indicates whether the quantification method is valid for use in quantification e A green check mark means that the quantification method is valid and can be used for quantification e An exclamation point in a yellow triangle means that the quantification method is provides information on how to fix it Figure 191 provides examples of these symbols in the Status column Figure 191 Method validity symbols in the Quantification Methods view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 7 6 a aajao oono m 48 Gi m QR SequestHT Q Mascot ei ARAZA LPPE JSAR B not valid Double click this mark to view a message that describes the error and Administration x Sequest TMT 6 plex Benchmark 1 4 0 175 msf my 4a a 286 x nae Shel a FASTA Files is FASTA Indexes Spectral Libraries Chemical Modifications Cleavage Reagents Quantification Methods amp Sequest HT E SpectraST B Server Settings amp Discoverer Daemon amp FASTA Indexes Proteome Discoverer User Guide m a idm Add 3 Remove Edit J Import E Export gt Is Active LJ Status Method Name d Description v Dimethylation 3plex C2H6 C2H2D4 13C2D6 Dimethylation 3plex C2H4 C2D4 13C2D4 Method Ww v Full 180 Labeling 02 1
417. precursor isolation window This percentage is the relative amount of ion current within the isolation window that is not attributed to the precursor itself lass recursor_intensity_in_isolation_window _isolation_interference 100 x j ESA ena da a nindo total_intensity_in_isolation_window The application displays the calculated interference value in the Isolation Interference column on the Peptides and Search Input pages For reporter ion quantification a high isolation interference value could indicate that a calculated peptide ratio is skewed by the presence of co isolated peptide species Note The Proteome Discoverer application only calculates the Isolation Interference value if the precursor scans are high resolution high mass accuracy scans You can use the Percentage Co Isolation Excluding Peptides from Quantification parameter on the Ratio Calculation page of the Quantification Method dialog box shown in Figure 202 on page 302 to specify a threshold of between 0 and 100 percent for the allowed co isolation interference The default value is 100 percent which means that no PSM is excluded This parameter is only available for reporter ion quantification Classifying Peptides The flowchart in Figure 209 shows how the Proteome Discoverer application classifies peptides for protein quantification It displays this classification in the Quan Info column of the results report Refer to the Help for descriptions of these
418. psin Full a queur aan E File Size kB 1278 E Last Access Time 10 4 2012 11 51 06 AM License Management a Max Missed Cleavage Sit 2 Precursor Mass Range Da 350 5000 R Licenses Use Average Precursor M False Auto Remove Indexed FASTA File Enzyme Index Size kB Last Access Time Vv yeast5protmix fasta Trypsin Full 56 10 04 2012 01 37 PM Configuration a E g j Name 5 4 Workflow Nodes FASTA Database _yeast5protmix fasta amp Annotation Enzyme Trypsin Full E Mascot File Size kB 56 MSPepSearch Last Access Time 10 4 2012 1 37 08 PM E SEQUEST Max Missed Cleavage Sit 2 amp Sequest HT Precursor Mass Range Da 350 5000 i SpectraST Use Average Precursor M False B4 Server Settings amp Discoverer Daemon E FASTA Indexes Manually Creating FASTA Indexes As noted earlier you can manually create FASTA indexes for semi enzymatic or no enzyme searches To manually create a FASTA index 1 Follow the procedure in Automatically Creating a FASTA Index on page 121 Also set the Create Additional Decoy Database Index parameter in the FASTA Index Creator dialog box to True as shown in Figure 90 on page 122 The Proteome Discoverer application starts creating the FASTA index and the job queue appears 2 When the job finishes choose Administration gt Maintain FASTA Indexes or click the Maintain FASTA Indexes icon f to display the FASTA Indexes view 3 In the FASTA Indexes view click
419. ptide 4 1 lonScore 33 Rank 2 If you did not select the Merge Results of Equal Search Nodes option the Proteome Discoverer application ranks the peptides shown in Table 8 on page 159 as follows Sequest 2 Peptide 2 1 XCorr 20 Rank 1 Peptide 2 2 XCorr 8 Rank 2 Sequest 3 Peptide 3 1 XCorr 12 Rank 1 Peptide 3 2 XCorr n12 Rank 1 Mascot 4 e Peptide 4 1 IonScore 33 Rank 1 Mascot 5 e Peptide 5 1 lonScore 34 Rank 1 160 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Filtering the Search Results Recalculating Peptide Rank The Proteome Discoverer application does not consider filtered out peptides in calculating peptide ranks Filtered out peptides have a rank of infinite If you apply filters to an open MSF report the application recalculates the peptide ranks It also recalculates the delta score values each time that the peptide ranks change Using the Peptide Rank Filter If you use the Peptide Rank filter when you open a report the Proteome Discoverer application reads the peptides twice In the first step it collects identifications and the main scores of all peptides passing the peptide filters except the Peptide Rank filter Then it calculates the ranks for these peptides and loads all peptides having a higher rank than the maximum allowed rank It loads the remaining peptides in the second step If you apply the Peptide Rank filter to an open repor
420. quantification method as additional dynamic or static modifications 262 Proteome Discoverer User Guide Thermo Scientific Figure 176 From Quan Method list File Search Report Quantification Processing Workflow Editor Administration Tools Window Help x Workflow Editor x POA SBB SBSTISBaDRBDOBH amp B BD R SequsthT RK Mascot A dh ok B Sequest SILAC Dimethylation Search 1 3 0 297 msf x Multi Report from 2 Reports X PQD with TMT 6 plex 10 175 min msf rat A Name E Data Input A i Cc Based on template i Spectrum Files Spectrum amp Feature Retrieval Event Detector Spectrum Selector Spectrum Processing ig Noise Peak Filter i Non Fragment Filter ud Spectrum Grouper iu Spectrum Normalizer ig Top N Peaks Filter Spectrum Filters Ty Scan Event Filter Tif spectrum Confidence Filter Ti Spectrum Properties Filter Sequence Database Search Q Mascot W SEQUEST W Sequest HT Spectral Library Search W MSPepSearch W SpectrasT PSM Validation jih Fixed ValuePSM Validator m Percolator Target Decoy PSM Validator 2 PTM Analysis i phosphoRS Quantification 2 Precursor Ions Area Detector 4 Precursor Ions Quantifier 4 Reporter Ions Quantifier Description V Merge results of equal search nodes 7 Quantification Searching for Quantification Modifications with Mascot B
421. r 2 Spectrum Properties Filter Lower RT Limit 0 00730166666666667 Upper RT Limit 119 980918333333 First Scan 0 Last Scan 0 Lowest Charge State 0 12 Optional Save the search parameters as a template that you can use in the future a Click Save as Template The Save Processing Workflow Template dialog box appears as shown in Figure 23 Figure 23 Save Processing Workflow Template dialog box Template Name Template Description c_elegans_100_021411_FWD_combined fasta basic SEQUEST workflow a __seve__ cancel b In the Template Name box give the search workflow a name The Template Description box reflects the description that you entered on the Search Description page shown in Figure 21 on page 40 c Click Save Thermo Scientific Proteome Discoverer User Guide y 2 Getting Started Starting a New Search by Using the Workflow Editor 13 Click Finish on the Completing the Wizard_name Search Wizard page to start the search You can monitor the progress of the search in the job queue Refer to the Help 14 Choose File gt Open Report to display your search results Refer to the Help a Filter and sort your results See Filtering the Search Results on page 154 b Use different views to aid in your analysis Refer to the Help Starting a New Search by Using the Workflow Editor You can create a customized search by using the Proteome Discoverer Workflow Editor instead of the sea
422. r components for GO annotation Evidence codes describe how the GO information was proven for example by computer prediction or by experiment PMIDs Lists the cellular component codes in the PubMed database which is maintained by the NLM and the NIH Each code is linked to the PubMed browser Go Slim Specifies the basic GO Slim category for the GO term GO Slim categories are reduced versions of the GO ontologies containing a subset of the terms in the entire GO database They give a broad overview of the ontology content without the detail of the specific fine grained terms able 18 provides the Go Slim categories for cellular components Name Describes the cellular component for a GO term This description is created by the GO consortium Enzymes with an EC number for IUBMB Enzyme Nomenclature are displayed with links to detailed information at the International Union of Biochemistry and Molecular Biology Biological Processes Page The Biological Processes page of the ProteinCard page shown in Figure 168 summarizes information about the biological processes that the protein is a part of Thermo Scientific Proteome Discoverer User Guide 229 6 Protein Annotation ProteinCard Parameters Figure 168 Biological Processes page of the ProteinCard page Protein Identification Details toles Coverage ProteinCard E P General PP keys P Features J Molecular Functions P Cellular Components ME Ext
423. r Guide 1 1 Introduction Features The Proteome Discoverer application includes the following features e Support for the Sequest HT SEQUEST and Mascot search engines The Sequest HT and Mascot search engines are available as wizards or as nodes in the Workflow Editor Search Engines on page 3 describes these search engines Note This document refers to the algorithm and general capabilities of SEQUEST and Sequest HT collectively as Sequest It refers to the nodes implementing Sequest s features as SEQUEST or Sequest HT e The Workflow Editor for searching with multiple algorithms and merging results from multiple dissociation techniques See Starting a New Search by Using the Workflow Editor on page 42 e Support for both precursor ion quantification for example SILAC reporter ion quantification for example iTRAQ and Tandem Mass Tag TMT and peak area calculation quantification For details see Performing Precursor Jon Quantification on page 243 Performing Reporter Ion Quantification on page 249 and Performing Peak Area Calculation Quantification on page 259 respectively e Access to annotation information from ProteinCenter including information from the Gene Ontology GO database Protein Family Pfam database from the Wellcome Trust Sanger Institute and gene identifications from the Entrez gene database maintained by the National Center for Biotechnology Information NCBI Yo
424. r chosen nodes align them by choosing Workflow Editor gt Auto Layout or clicking the Auto Layout icon 8 or right clicking a node and choosing Auto Layout from the shortcut menu 5 Optional Renumber the workflow nodes in the workflow in consecutive order by choosing Workflow Editor gt Auto Number Figure 149 shows the basic protein annotation workflow Figure 149 Protein Annotation workflow x spectrum Fies 0 w Spectrum 1 d Selector Fixed Value PSM Validator 3 j Annotation 4 6 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon Thermo Scientific Proteome Discoverer User Guide 207 6 Protein Annotation Displaying the Annotated Protein Results Displaying the Annotated Protein Results The Proteome Discoverer application retrieves GO Pfam Entrez gene and UniProt PTM annotation data from ProteinCenter when it finishes processing all search nodes You can display the annotated protein results in the MSF file For GO annotations the application can filter the list of identified proteins by selected Go Slim categories Note The Proteome Discoverer application cannot retrieve annotations from searches conducted in the UniRef FASTA database because of the prefix appended to the accession number e Displaying GO Protein Annotation Results e Displaying GO Accessions e Displaying Protein Family Pfam Annotation Results e Displaying Entrez Gene Identifications e Displa
425. r use 3 In the sequence table select the row or rows that you want to run 4 Choose Actions gt Run Sequence or click the Run Sequence icon i If you have changed the instrument configuration in Foundation platform after the previous sequence run the Change Instruments In Use dialog box opens Otherwise the Run Sequence dialog box opens as shown in Figure 59 For an LC MS system the autosampler or device with an autosampler is specified as the start instrument When the autosampler makes an injection it triggers the mass spectrometer to begin data acquisition Figure 59 Run Sequence dialog box Run Sequence x Acquisition Options User Chemist Instrument Start Instrument Thermo EASY nLC Yes LTG Orbitrap XL MS Run Rows 1 3 ty WI Start When Ready Change Instruments Processing Actions Instrument Method Quan Start Up Browse Qual Shut Down Browse Reports Programs ee Programs Pre Acguisiti Browse ine Create Quan Summary Post Acquisition Browse Run Synchronously 7 Pre Acquisition V Post Acquisition After Sequence Set System On D Standby D Off 86 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System 5 Click OK If you have not already saved the sequence the File Summary Information dialog box
426. ragment ions have a charge on the N terminal side and x y and z fragment ions have a charge on the C terminal side Fragment ions a b and y are ions that have lost ammonia 17 Da and fragment ions a b and c are ions that have lost water 18 Da The subscript next to the letter indicates the number of residues in the fragment ion Table 28 summarizes the fragment ions used in the Proteome Discoverer application 1 For more information about fragment ions and nomenclature see Roepstorff P and Fohlman J Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides Biomed Mass Spectrum 1984 11 11 601 Proteome Discoverer User Guide 347 B Chemistry References Fragment lons Table 28 Fragment ions lons Description a A ion with charge on the N terminal side b B ion with charge on the N terminal side c C ion with charge on the N terminal side y Y ion with charge on the C terminal side Z Z ion with charge on the C terminal side a A ion that has lost ammonia 17 Da b B ion that has lost ammonia 17 Da y Y ion that has lost ammonia 17 Da a A ion that has lost water 18 Da b B ion that has lost water 18 Da c C ion that has lost water 18 Da 348 Proteome Discoverer User Guide Thermo Scientific ee i Index A a fragment ions 347 348 a fragment ions 347 348 a fragment ions definition 7 348 accession keys 224
427. rch wizards The Workflow Editor is a flexible and complex tool that you can use to create customized data processing workflows Instead of using the standard wizards available through the Processing menu you can develop a workflow specific to your needs The Workflow Editor searches with multiple algorithms and merges results from multiple fragmentation methods It also provides great flexibility in creating custom search results Unlike the search wizards the Workflow Editor can accept multiple input raw files You can create a reusable processing workflow template by saving your design to load and use at another time A unique workflow gives you the ability to set parameters that are normally static settings in the wizard or use a function that would not normally be available such as deconvoluting the precursor ions for all high mass accuracy data or exporting a spectrum The workflow is the layout of processing nodes or workflow steps which you then submit to process your data The nodes are like building blocks that you can use to create a unique search sequence You can use them to define your own search parameter tolerances and criteria WARNING As a prerequisite to using the Proteome Discoverer Workflow Editor you must know how each workflow node functions If you do not understand the function or interconnectivity of these nodes you can potentially build a sequence that creates bad results and makes no analytical sense For a deta
428. re produced by a collision induced dissociation CID process in which a peptide ion is fragmented in a collision cell Low energy CID spectra are generated by MS MS and ESI and are sequence specific The fragment ion spectra contain peaks of the fragment ions formed by cleavage of the peptide bond and are used to determine the amino acid sequence A fragment must have at least one charge for it to be detected If this charge is retained on the N terminal fragment the ion is classed as a b or c If the charge is retained on the C terminal fragment the ion type is x y or z A subscript indicates the number of residues in the fragment In addition to the proton carrying the charge c ions and y ions abstract an additional proton from the precursor peptide as shown in Figure 2 Proteome Discoverer User Guide 7 1 Introduction Features Figure 2 Structures of six singly charged sequence ions RI O R2 R3 O H N C CN C o c N 7 l i COOH H H H H a X gt RI R2 R4 HN C C N C C 0 H N i L N C COOH H H H A H b 2 Li TI aaa HN C CN C O NH G G N E COOH H H H H H H C2 Z2 Fragmentation Methods The Proteome Discoverer application supports the following fragmentation types e CID Uses the collision induced dissociation CID method of fragmentation where molecular ions are accelerated to high kinetic energy in the vacuum of a mass spectrometer and then allowed to collide with neutral
429. reak bonds resulting in fragmentation e PQD Uses the pulsed Q collision induced dissociation PQD method of fragmentation where precursor ions are activated at a high value a parameter that determines the stability of an ion s trajectory in an ion trap mass analyzer Then a time delay occurs to allow the precursor to fragment and then a rapid pulse is applied to a low value where all fragment ions are trapped The product ions can then be scanned out of the ion trap and detected PQD fragmentation produces precise reproducible fragmentation and has been used for iTRAQ peptide quantification on the LTQ mass spectrometer using both electrospray and MALDI source ionization MudPIT Experiments Workflow Thermo Scientific Multidimensional Protein Identification Technology MudPIT experiments investigate complex proteomes by applying multidimensional chromatography to the samples before acquisition in the mass spectrometer Typically this process results in several dozen or even a few hundred fractions that are separately analyzed by LC MS resulting in one raw file per sample fraction Analyzing gel slices or performing in depth follow up acquisitions also results in multiple fractions Because all these fractions belong to the same sample the Proteome Discoverer application can process all raw files from these fractions as one contiguous input file and generate a single result file For detailed information about processing MudPIT sa
430. refer to the Help Displaying the Quantification Channel Values Chart You can generate a chart that displays the absolute intensity for reporter ion quantification or the area for precursor ion quantification of the quantification values detected for the available quantification channels To display the quantification channel values chart 1 Click the row of the peptide that interests you To obtain meaningful results Used must appear in the Quan Info column of the report 2 Choose Quantification gt Show Quan Channel Values or click the Show Quan Channel Values icon To see the results see the following sections e Displaying Quantification Channel Values for Reporter Ion Quantification e Displaying Quantification Channel Values for Precursor Ion Quantification Displaying Quantification Channel Values for Reporter lon Quantification Thermo Scientific For reporter ion quantification you can generate a Quan Channel Values chart that displays the absolute intensity of the reporter ions detected for the available quantification channels Reporter ions or reporters are the labels affixed to peptide samples in reporter ion quantification They fragment in the MS MS process You can use the quantification value intensity to calculate the relative ratio of a peptide You might also want to view the absolute quantification value intensity to verify that the peptide ratio calculation is correct The x axis of the chart sh
431. refore there can be multiple peptides having rank 1 for each spectrum For example consider the workflow with two SEQUEST nodes and two MASCOT nodes shown in Figure 114 158 Proteome Discoverer User Guide Thermo Scientific Figure 114 Workflow with two Mascot nodes and two SEQUEST nodes File Search Report Quantification Processing Workflow Editor Administration Tools Window Help 5 Filtering Data Filtering the Search Results OM SE BOT BROOD AH omm K swath KMart AMHR AGOBTRIAREB _ Workflow Editor x Workflow Nodes ideal a Name P o Based on template became P Description Spectrum amp Feature Retrieval A Event Detector Spectrum Selector Spectrum Processing ij Noise Peak Filter i j Non Fragment Filter Merge results of equal search nodes i Spectrum Grouper ij Spectrum Normalizer ug Top N Peaks Filter E Spectrum Filters Ty Scan Event Filter Spectrum tL Selector i im Spectrum Confidence Filter TW Spectrum Properties Filter Loe Yy R lih Fixed ValuePSM Validator Percolator Target Decoy PSM Validator 5 PTM Analysis Cid phosphoRS3 0 Quantification Precursor Ions Area Detector Precursor Ions Quantifier Reporter Ions Quantifier Data Export z iu Spectrum Exporter LJ Annotation Annotation EEI m Ready The search engines find the peptides shown in Table 8 for spectrum 10
432. rer Daemon Application from the Xcalibur Data System 2 In the Std QC Unk and Other columns do the following e To senda sample to the Daemon application make sure that Yes appears in the column for its sample type e To avoid processing a sample with the Discoverer Daemon application clear the column for its sample type Tip Use the Other column for the Blank sample type For example if you do not want to send blank samples to the Discoverer Daemon application for further processing clear the Other column 3 Save the processing method Batch Processing with a Processing Method That Calls the Discoverer Daemon Application To inject samples and to acquire and process data files with the Xcalibur data system you must create one or more instrument methods one or more processing methods and a sequence that defines the sample injection set For information about creating an instrument method for your LC MS system refer to the Help for the LC devices and the Help for the mass spectrometer For information about creating processing methods and sequences refer to the Xcalibur Help Tip For a typical LC MS experiment an autosampler automates the sample injection process and the position nomenclature depends on the autosampler tray type For information about specifying the autosampler tray type and the position nomenclature for the specified tray type refer to the Help for the autosampler For some autosampler
433. res page of the ProteinCard page Coverage ProteinCard General Keys Molecular Functions Cellular Components Biological Processes Features Source Category From To Acc Description PrediSi Signal 1 18 Interpro SSF56574 1 409 IPR023796 Serpin domain Interpro PTHR11461 1 413 IPR000215 Serpin family Interpro PF00079 48 409 IPR023796 Serpin domain PFAM PFAM 50 409 PF00079 Interpro SM00093 52 409 IPR023796 Serpin domain tmap TRANSMEM 65 84 UNIPROT CARBOHYD 120 120 UNIPROT CARBOHYD 125 125 Interpro TransMembrane domain N linked GIcNAc N linked GIcNAc Diseases External Links Protein contains a signal peptide at the beginning of the sequence Serpin serine protease inhibitor Structure is a multi domain fold containing a bundle of helices and a beta sandwich PS00284 382 392 IPR023795 Protease inhibitor 14 serpin conserved site Table 13 lists the parameters on the Features page of the ProteinCard page Table 13 Parameters on the Features page of the ProteinCard page Command Source Description Specifies the name of the database that the information about the feature was taken from e InterPro Tmap computational enrichment e PrediSi computational enrichment e Pfam computational enrichment e UniProt Category Displays the type of information that UniProt InterPro and Tmap include for each row For example UniProt might include CARBOHYD as one of its type
434. rn excluded or included peptides to their default status You cannot include peptides if No Quan Values Inconsistently Labeled or Excluded by Method appears in the Quan Info column 1M To exclude a peptide from the quantification results 1 On the Proteins page click the plus sign next to the protein of interest to display its constituent peptides 310 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Excluding Peptides with High Levels of Co Isolation Right click the peptide of interest which must display Used in the Quan Usage column and choose Include Exclude Peptide s from Protein Quantification gt Exclude from the shortcut menu Not Used Excluded now appears in the Quan Usage column To save the information in the MSF file resulting from this setting choose File gt Save Report To include an excluded peptide in the quantification results On the Proteins page click the plus sign next to the protein of interest to display its constituent peptides Right click the peptide of interest which must display Not Used Excluded Redundant or Not Unique in the Quan Info column and choose Include Exclude Peptide s from Protein Quantification gt Include from the shortcut menu Used Included now appears in the Quan Usage column To save the information in the MSF file resulting from this setting choose File gt Save Report T
435. robability value to identifiers and the percentage of false discoveries that you can expect typically 1 percent Note You must select the Search Against Decoy Database check box to see peptide confidence determined by FDR e To specify a strict target false discovery rate for peptide matches with high confidence type a value of 0 0 through 1 0 in the Target FDR Strict box The default is 0 01 1 percent FDR e To specify a relaxed target false discovery rate for peptide matches with moderate confidence type a value of 0 0 through 1 0 in the Target FDR Relaxed box The default is 0 05 5 percent FDR j Click Next The Select Modifications page appears as shown in Figure 19 Figure 19 Select Modifications page Select Modifications Select the dynamic and static modifications to be used during the search Dynamic Side Chain Modifications Static Side Chain Modifications None Ea X None A None X y None X None i x None X None X x None None M4 X None MA None Z X None Z Dynamic Peptide Modifications Static Peptide Modifications N Terminus None X N Terminus None C Terminus None X C Terminus None 10 Specify which modifications you want the search algorithm to include during its in silico digestion of the protein
436. rom several searches the Proteome Discoverer application does not calculate a combined protein score and it orders the proteins by their coverage e Search the fractions all at one time in MudPIT To search the fractions of only one sample use MudPIT In this mode the Proteome Discoverer application searches all fractions as one logical sample and creates a single MSF result file It automatically merges all identified peptides and proteins from all fractions and creates a single combined score for every protein that includes all peptides identified from the different fractions Opening a MudPIT report is faster and consumes less memory than combining separate reports into a multiconsensus report For example if the Proteome Discoverer application identifies a protein in every fraction and opens all fractions into a multiconsensus report a copy of the same protein resides in memory for every fraction that the protein was identified in It must merge the proteins into an additional protein instance that it displays in the multiconsensus report slowing performance and consuming memory unnecessarily However if you searched the fractions in MudPIT mode the proteins are already merged from the different fractions and the Proteome Discoverer application only needs to load the identified merged proteins The following procedure describes how to create a workflow for multiple raw files from the same sample This workflow is basically the same as t
437. roteins Figure 134 shows the steps involved in this process Thermo Scientific Proteome Discoverer User Guide 179 5 Filtering Data Grouping Proteins Figure 134 Protein grouping inference process in the Proteome Discoverer application All PSMs j Step 1 Collect PSMs meeting criteria specified for protein grouping PSMs relevant to protein grouping Step 2 Group all proteins that share the same set or subset of identified peptides Preliminary protein groups Step 3 Filter out protein groups that have no unique peptides among the considered peptides Step 4 Iterate through all spectra and select which PSM to use in ambiguous cases Step 5 Resolve cases where protein groups form circular rings of identified peptides Final protein groups 180 Proteome Discoverer User Guide Steps 3 5 are performed only if you select the Apply Strict Maximum Parsimony Principle option in the Protein Grouping area of the Result Filters page Thermo Scientific Thermo Scientific 5 Filtering Data Grouping Proteins 1 In the first step the application collects all peptide spectrum matches PSMs that meet the selection criteria that you specified through the settings of the parameters in the Protein Grouping Enabled area on the Result Filters page see Figure 131 on page 176 The Help explains these parameters You can
438. rough or granular with ribosomes adhering to the outer surface and smooth with no ribosomes attached Extracellular The space external to the outermost structure of a cell For cells without external protective or external encapsulating structures this term refers to the space outside of the plasma membrane It only applies to proteins that are not attached to the cell surface It covers the host cell environment outside an intracellular parasite Proteome Discoverer User Guide 235 6 Protein Annotation GO Slim Categories Table 18 GO Slim categories for cellular components Sheet 3 of 4 GO Slim cellular component Golgi Description A compound membranous cytoplasmic organelle of eukaryotic cells consisting of flattened ribosome free vesicles arranged in a more or less regular stack The Golgi apparatus differs from the endoplasmic reticulum in often having slightly thicker membranes appearing in sections as a characteristic shallow semicircle so that the convex side cis or entry face abuts the endoplasmic reticulum secretory vesicles emerging from the concave side trans or exit face In vertebrate cells there is usually one such organelle but in invertebrates and plants where they are known usually as dictyosomes there may be several scattered in the cytoplasm The Golgi apparatus processes proteins produced on the ribosomes of the rough endoplasmic reticulum Such processing includes modification of the core o
439. rs for it in the Parameters pane a In the Mass Precision box specify the expected standard deviation of the mass P p precision Three times the standard deviation is used to create extracted ion chromatograms The minimum value is 1 ppm The maximum value is 4 ppm The default is 2 ppm In the S N Threshold box specify a threshold signal to noise value that determines whether the Proteome Discoverer application removes peaks from the spectrum It removes peaks with a signal to noise value below this threshold The minimum value is 1 0 and there is no maximum value The default is 1 11 Click the Spectrum Selector node and set the parameters for it in the Parameters pane 12 13 248 Proteome Discoverer User Guide a Change the setting in the Max Precursor Mass box to an appropriate setting For example for SILAC 2plex Arg10 Lys6 quantification set this option to 6500 Change the setting in the S N Threshold box to an appropriate setting For example for SILAC 2plex Arg10 Lys6 quantification set this option to 1 5 For other parameters that you can optionally set for the Spectrum Selector node refer to the Help Click the search engine node for example SEQUEST and set the parameters for it in the Parameters pane a b d In the Protein Database box select the FASTA database In the Dynamic Modifications area select the dynamic modifications For example for SILAC 2plex Arg10 Lys6
440. rum Library dialog box so you can ip Add choose the spectrum library to import a Remove Deletes a spectrum library Name Displays the name of the spectrum library File Size kB Displays the current size of the spectrum library Spectra Displays the number of spectra found in the spectrum library during processing Type Displays the type of spectrum library downloaded either SpectraST which are spectrum libraries that you can use with the SpectraST node or NIST which are spectrum libraries that you can use with the MSPepSearch node Last Modified Displays the date when the spectrum library was last modified or created Adding a Spectrum Library You must add a spectrum library to the Proteome Discoverer application before you can conduct a search with the SpectraST or MSPepSearch node In the registration process the Proteome Discoverer application automatically recognizes the type of the spectral library The type determines the search node that you can use the library with Adding the spectrum libraries is similar to the procedure for adding FASTA files e To add a spectrum library for searching with the SpectraST node e To add a spectrum library for searching with the MSPepSearch node To add a spectrum library for searching with the SpectraST node 1 Download the appropriate spectrum libraries from the NIST at http peptide nist gov or from Peptide Atlas at http www peptideatlas org speclib The Proteome Disc
441. s Thermo Scientific Proteome Discoverer User Guide 27 2 Getting Started Configuring Search Engine Parameters Figure 12 Administration message box Administration x Changes were saved LD They will have no effect for currently open workflows 9 Click OK Note Click A Reset to return to the default values Troubleshooting Failed Mascot Searches If all your searches with Mascot fail follow these instructions to locate the problem To troubleshoot failed Mascot searches 1 Verify that the Mascot server is running and accessible from the computer that is running the Proteome Discoverer application For details on how to do this see Directing the Proteome Discoverer Application to the Mascot Server Location on page 25 2 With the Mascot server is running verify that it is operating properly by submitting a simple search from the Mascot Web interface Do one of the following e Ifthe search from the Mascot Web interface is successful go to step 3 e Ifthe search fails contact your system administrator There might be a problem with the Mascot server itself 3 If your Mascot server is operating properly and you can access it from the Proteome Discoverer application try to perform a very simple search using the Mascot wizard Do one of the following as applicable e Ifsimple searching fails there might be a general problem in the interaction between the Proteome Discoverer application and the Masc
442. s i Spectrum Files i Description Spectrum amp Feature Retrieval Event Detector m lt Spectrum Selector Spectrum Processing ig Noise Peak Filter E i Non Fragment Filter gt spectrum Files o i Spectrum Grouper i Spectrum Normalizer 7 Merge results of equal search nodes ig Top N Peaks Filter Spectrum Filters Ti Scan Event Filter i Seco w Spectrum Confidence Filter i m E Sequence Database Search Mascot W SEQUEST W SequestHT Cl Spectral Library Search W MSPepSearch W Spectrast Fixed Value A E PSM Validation lih Fixed ValuePSM Validator Percolator Target Decoy PSM Validator El PTM Analysis i phosphoRS3 0 E Quantification Precursor Ions Area Detector m 2 Precursor Ions Quantifier Reporter Ions Quantifier El Data Export us Spectrum Exporter 5 Miimi Ready EMM After you install the Proteome Discoverer Daemon application the Proteome Discoverer application places the directory where it saves the raw files and stores the results in the following files Thermo Scientific Proteome Discoverer User Guide 79 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System e Windows 7 c ProgramData Thermo Discsoverer lt release_number gt PublicFiles e Windows XP c Documents and Settings All Users Application data
443. s you can change the tray type from the Sequence Setup view by choosing Change gt Tray Name and then selecting a different tray type To start the Discoverer Daemon application from the Xcalibur data system version 2 10 or later you must add a processing method that calls the Discover Daemon application to the sequence 1M To set up and run an injection sequence with a processing method that starts the Discoverer Daemon application 1 From the Home Page window of the Xcalibur data system do one of the following e Click the Sequence View icon on the Home Page window toolbar or nnn e Click the Sequence Setup icon lll on the Roadmap view Thermo Scientific Proteome Discoverer User Guide 85 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System The Sequence Setup view opens with an empty sequence table Refer to the Xcalibur Sequence Setup view Help for information about filling out the sequence table 2 In the Proc Meth column select a processing method with a parameter file that calls the Daemon application as follows e Type the file location and name of the processing method or e Double click the column to open the Select Processing Method dialog box where you can browse to and select the processing method You can now start the sequence without first saving it or you can save the sequence for late
444. s Filter Spectrum Filters T Scan Event Filter Ti Spectrum Confidence Filter ml Spectrum Properties Filter Sequence Database Search Q Mascot W SEQUEST W SequestHT Spectral Library Search W MsPepSearch 5 PSM tion jh Fixed ValuePSM Validator Percolator lah Target Decoy PSM Validator E PTM Analysis iff phosphoRS3 0 Quantification Precursor Ions Area Detector 2 Precursor Ions Quantifier 2 Reporter Ions Quantifier Parameters i Fide Advanced Parameters H Target FDR Relaxed 0 05 3 In the Target FDR Strict box set the target FDR for high confidence peptide hits 4 In the Target FDR Relaxed box set the target FDR for Peptides medium confidence peptide hits 5 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon Thermo Scientific Proteome Discoverer User Guide 191 5 Filtering Data Calculating False Discovery Rates To set up FDRs by using the Percolator node 1 Create a search workflow that includes at least one of the search engine nodes SEQUEST Mascot or Sequest HT and the Percolator node 2 For information about creating a workflow see Creating a Search Workflow on page 44 3 Connect all search nodes whose results you want to submit for validation to the Percolator node Figure 140 gives an example of such a workflow No
445. s of information and InterPro might include SSF57184 as one of its types of information From Specifies the start position of the amino acid To Specifies the end position of the amino acid Acc Specifies the accession identifier for the domain linked to InterPro or Pfam Description 226 Proteome Discoverer User Guide Describes the feature Thermo Scientific 6 Protein Annotation ProteinCard Parameters Molecular Functions Page The Molecular Functions page of the ProteinCard page shown in Figure 166 summarizes information about the function of the protein It consolidates GO data and Enzyme Category EC information The EC designation indicates whether a protein has been categorized with a certain enzyme function Figure 166 Molecular Functions page of the ProteinCard page P Protein Identification Details seca Coverage ProteinCard General Keys Features Cellular Components Biological Processes Diseases External Links Molecular Functions GO Id Evidence codes PMIDs Go Slim Name GO 0004867 IEA ISS TAS IBA 1309665 enzyme regulator activity serine type endopeptidase inhibitor activity GO 0005518 NAS 7656593 protein binding collagen binding GO 0051082 IEA protein binding unfolded protein binding Table 14 lists the parameters on the Molecular Functions page of the ProteinCard page Table 14 Parameters on the Molecular Functions page of the ProteinCard pa
446. s the workflow in c Workflows MascotEcoli xml DiscovererDaemon 1 protlab2 leo_davinci c sfcid a sfcid iTRA_BSA_3ITMS2_3HCD raw e sfcid any c workflows MascotEcoli xml The following sequence of commands submits multiple raw files for processing on a remote server DiscovererDaemon exe c AllTrypMyo DiscovererDaemon DiscovererDaemon DiscovererDaemon DiscovererDaemon exe exe exe exe a AllTrypMyo_020110303 C DaemonTest mudpit4 Tryp_Myo raw a AllTrypMyo_020110303 C DaemonTest mudpit4 Tryp_Myo_1 raw a AllTrypMyo_020110303 C DaemonTest mudpit4 Tryp_Myo_2 raw e AllTrypMyo_020110303 3 C DaemonTest mudpit4 wf_sequest xm The next sequence of commands submits multiple raw files for processing on a local server DiscovererDaemon DiscovererDaemon DiscovererDaemon DiscovererDaemon exe exe exe exe a AllTrypMyo C DaemonTest mudpit4 Tryp_Myo raw a AllTrypMyo C DaemonTest mudpit4 Tryp_Myo_1 raw a AllTrypMyo C DaemonTest mudpit4 Tryp_Myo_2 raw e AllTrypMyo 3 C DaemonTest mudpit4 wf_sequest xml The Discoverer Daemon appends a time stamp to each file when it processes the files on a remote server Proteome Discoverer User Guide Thermo Scientific ee Searching for Data This chapter describes the features that you can use when searching for and analyzing data in the Proteome Discoverer application Contents e Using FASTA Databases e Searching Spectrum Libraries
447. s workflow template file in the Proteome Discoverer application by choosing Workflow Editor gt Export Workflow to XML ParameterAssignment Specifies the name and value of a parameter in the format of parameter value Some examples follow This example sets the FASTA database for any node to equine fasta FastaDatabase equine fasta Proteome Discoverer User Guide 97 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application on the Command Line The next example sets the FASTA database for all Mascot nodes to equine fasta Mascot FastaDatabase equine fasta The last example sets the FASTA database for Mascot nodes having 4 as the processing node number to equine fasta It is equivalent to 4 FastaDatabase equine fasta because the processing node numbers are unique Mascot 4 FastaDatabase equine fasta c foldername Remote server Creates a user named folder in the PublicFiles folder on the server where you store output files The PublicFiles folder is the default file in the Current File Directory box in the view displayed in the Proteome Discoverer application when you select Administration gt Configuration gt Server Settings gt Discoverer Daemon The c option automatically appends the date and if the directory already exists an incremental index number to the name You can only create a folder in the directory configured in the view opened by the Administration gt Co
448. scoverer Daemon Utility 055 Starting the Proteome Discoverer Daemon Application in a Window Selecting the Servers Mi Ml ditt dain Wadena le hails winded ia ale Mle tent a aed Starting a Workflow vocal sada a dab ed wena o eed wks athe Ae eee Monitoring Job Execution in the Proteome Discoverer Daemon Applicaton naie ah ease wwe S she adh a pies dole ees Logging On to a Remote Server 0 cee eee eee Running the Proteome Discoverer Daemon Application from the Xcalibur Data System eroii Sear deg Piety She coe Bar testy E trig eet oes Before You Statt ese en ee Meee eee pee eats oun shud Creating a Parameter File That the Discoverer Daemon Application Creating a Processing Method That Calls the Discoverer Daemon Application Vee vs ovis ieee AREER ew BOE EEE Raya Wee Ree tes Batch Processing with a Processing Method That Calls the Discoverer Daemon Application seco ea ccah ees Wl sndra od sap leaebet man apc hake ed Batch Processing with Multiple Processing Methods Batch Processing by Using a Post Acquisition Method Xcalibur Data Systemi 20 7 Only oie eiga ie antaa fice a i iaa ia aE anaes Processing MudPIT Samples by Using a Processing Method MudPIT Processing Using the Run Sequence Dialog Box Running the Proteome Discoverer Daemon Application on the Command SYNTAX o winds E N ta agr A E E a E eee N Thermo Scientific Thermo Scientific Chapter 4 Chapter 5 Co
449. se the name in quotation marks as in this example p C Xcalibur methods batch processing param R 7 In the Std QC Unk Other and Sync columns accept the default settings or modify them according to your requirements For information about setting the sample types to be sent to the Discoverer Daemon application see To specify the sample types to be sent to the Discoverer Daemon application To send all sample types to the Discoverer Daemon application make sure that all of the sample type columns are set to Yes as shown in Figure 58 Figure 58 Program table with a call to the Daemon application Programs Jove Sample type Enable Std QC Unk Other Action Program or Macro Name Sync Parameters 1 Yes Yes Yes Yes Yes Run Program C Program Files T hermo Discoyvere Yes X Yes Yes Yes Yes Run Program Yes p C Daemon data daemon param R Click OK to save the changes to the processing method Choose File gt Save To specify the sample types to be sent to the Discoverer Daemon application If the processing method that you want to modify is not open open it and make sure that the parameter file and its location are specified as described in To add a processing method that calls the Discoverer Daemon application to a processing method on page 82 84 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discove
450. se the parameter file created in the Proteome Discoverer Daemon application to call the application from the Xcalibur data system For the Xcalibur 2 0 7 data system you can start the Discoverer Daemon application in two ways e You can add a parameter file that calls the Discoverer Daemon application to the processing method specified in the Xcalibur injection sequence e You can select a parameter file for post acquisition processing in the Programs area of the Run Sequence dialog when you start a sequence run For the Xcalibur 2 1 0 or later data system you can start the Discoverer Daemon application only by adding a parameter file to the processing method specified in the Xcalibur injection sequence These topics describe how to run the Discoverer Daemon application from the Xcalibur data system e Before You Start e Creating a Parameter File That the Discoverer Daemon Application Uses e Creating a Processing Method That Calls the Discoverer Daemon Application e Batch Processing with a Processing Method That Calls the Discoverer Daemon Application e Batch Processing with Multiple Processing Methods e Batch Processing by Using a Post Acquisition Method Xcalibur Data System 2 0 7 Only e Processing MudPIT Samples by Using a Processing Method e MudPIT Processing Using the Run Sequence Dialog Box Before You Start Before you start running the Proteome Discoverer Daemon application from the Xcalibur data system perform the follo
451. sed The Quantification Spectrum chart always compares the exact same isotopic pattern peaks for each label For example the chart in Figure 205 compares the first three isotopic pattern peaks among all three types Light Medium and Heavy But the chart also contains an additional Light isotopic pattern peak and an additional Heavy isotopic pattern peak that are not used so these two peaks are represented by unfilled circles Thermo Scientific Proteome Discoverer User Guide 305 7 Quantification Displaying the Quantification Spectrum Chart Figure 205 Extra isotopic pattern peaks represented by unfilled circles in the Quantification Spectrum chart File Search Report Quantification Processing Workflow Editor Administration Tools Window Help iea ap OT BODOSH g a BD K seuethT g Mascot i A R aE AZSEBTRBIARGE C Documents and Settings ussjo prodeval2 My Documents Smoke_Test_Files SILAC Smoke Tests 081023_RR_BSA_1_1_1_2plus RAW RT 28 82 29 85 min Event Spectrum FTMS Quantified lon z 2 Mono m z 714 80090 Da MH 1428 59453 Da 717 93505 716 30107 ORT 29 36 i z2 Area counts sec 10 3 Light PoRT 29 33 1716 42507 i i gRT 28 87 BoP JerT 29 330 ere Ready 5 6 Protein Group s 2911 2911 Merged Protein s 7353 7353 Peptide s 11823 11823 PSM s 1564 1564 Search Input s The Quantification spectrum chart can also indicate whether an expected quantification pattern peak is absent Regions in pink
452. semi enzymatic digestion Unspecific Specifies a non specific digestion No Cleavages Specifies that no cleavages occur 122 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases e Maximum Missed Cleavage Sites Specifies the maximum number of internal cleavage sites per peptide fragment that is acceptable for an enzyme to miss when cleaving peptides during digestion Normally the digestion time is too short to enable the enzyme to cleave the peptide at all positions so you must specify the number of missed positions in one resulting peptide fragment where the enzyme could cleave but did not The minimum value is 0 and the maximum value is 12 The default is 2 5 In the Mass Range Settings section set the limits of the mass range of the singly charged precursor ion to be processed e Minimum Precursor Mass Specifies the minimum mass of the precursor ion The minimum value is 0 0 Da and the maximum value is 10000 0 Da The default is 350 Da e Maximum Precursor Mass Specifies the maximum mass of the precursor ion The minimum value is 0 0 Da and the maximum value is 10000 0 Da The default is 5000 Da e Use Average Precursor Mass Determines whether the average mass is used to match the precursor ion e True Uses the average mass to match the precursor ion e False Default Uses the monoisotopic mass to match the precursor ion which is the mass of the most abundant isotope of t
453. sh Job Queue Spectrum Source Submitted at Completed Files BSA3 raw BSA4 raw 10 26 2012 10 30 AM Completed 100 mudPIT Files BSA1 raw BSA2 raw 10 26 2012 10 28 AM The Proteome Discoverer application saves the data in the two MudPIT samples in two directories each one containing the raw data files of one MudPIT sample in this example two raw data files as shown in Figure 73 Thermo Scientific Proteome Discoverer User Guide 95 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Figure 73 Saving the raw data files of each MudPIT group in two directories ole es Ou Search export 20121026 P Organize v Include in library v Share with Burn a fil 5 Name Date modified Type Size a F Favorites BE Desktop BSA1 raw 10 26 2012 10 28 Xcalibur Raw File 220 006 KB B Downloads 2 BSALxml 10 26 2012 10 28 XML Document 1 KB T Recent Places BSA2 raw 10 26 2012 10 28 Xcalibur Raw File 220 006 KB BSA2 xml 10 26 2012 10 28 XML Document 1KB E Libraries g mudPIT msf 10 26 2012 10 29 Thermo s Mass Sp 1 000 KB E Documents L mudPIT mzML 10 26 2012 10 29 MZML File 16 008 KB a Music Pictures a 6 items jaje OW M Thermo gt Discoverer1 4 PublicFiles DiscovererDaemon SpectrumFiles export_20121026 1 4 Organize v Include in library Share with Burn
454. sider neutral loss peaks for scoring Moreover you can set the maximum number of phospho isoforms and PTMs per peptide that the application considers If a certain peptide exceeds this cutoff the application does not analyze it e Changed output column headings The phosphoRS 3 0 output appears in three columns in the MSF file phosphoRS Site Probabilities Binomial Peptide Score and Isoform Confidence Probability The Site Probabilities column appears by default but you must choose the other two columns with the Column Chooser New Protein Annotations Entrez Gene IDs The Proteome Discoverer application has added new features to its retrieval of protein annotations The Proteome Discoverer application can retrieve Entrez gene identifications from ProteinCenter The Entrez gene identification is a unique identification assigned to the genes in the Entrez database maintained by the National Center for Biotechnology Information NCBI The database assigns an identifier to all proteins transcribed from the corresponding gene The Proteins page of the results report displays these identifications in the Gene IDs column You can use this information to group or cluster together biologically meaningful proteins Hierarchical GO Terms Gene ontology GO terms are related in hierarchical graphs These graphs contain all the ancestor terms of the term associated with a protein You can display the annotated GO term and all its hierarchical terms
455. space pane beneath the Spectrum Files node and continue with the process of creating a workflow as described in Creating a Search Workflow on page 44 7 Choose Workflow Editor gt Start Workflow to start the workflow You can use the Proteome Discoverer Daemon utility to monitor multiple searches on multiple raw data files For information about this tool see Using the Proteome Discoverer Daemon Utility on page 69 54 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Starting a New Search by Using the Workflow Editor Creating a Quantification Workflow To perform quantification you must run a quantification workflow A quantification workflow is a search workflow that includes one of three quantification nodes found in the quantification section of the Workflow Nodes pane of the Workflow Editor Table 1 lists these nodes and where you can obtain information about creating a quantification workflow for each Table 1 Quantification nodes Quantification node Use For more information Precursor Ions Quantifier For precursor ion See Performing Precursor Ion node quantification for Quantification on page 243 example SILAC Reporter Ions Quantifier For reporter ion See Performing Reporter Ion node quantification for Quantification on page 249 example iTRAQ and TMT Precursor Ions Area For peak area calculation See Performing Peak Area Detector node quantification Calculation Qu
456. splay Temporary Displays FASTA files that contain the proteins found by a Mascot search The Proteome Discoverer application temporarily imports these FASTA files which are not available for Sequest searches Name Displays the name of the FASTA file Size kB Displays the current size of the FASTA file Sequences Residues Displays the number of sequences found in the FASTA file during processing Displays the number of amino acids found in the FASTA file during processing Status Displays the current status of the FASTA file Imported Indicates that the FASTA file has been downloaded from a source and registered e Available Indicates that the FASTA file is available for Sequest searches e Processing Indicates that the FASTA file is in the process of being registered Last Modified Thermo Scientific Displays the date when the FASTA file was last modified or created Proteome Discoverer User Guide 103 4 Searching for Data Using FASTA Databases Adding FASTA Files You must add a FASTA file to the Proteome Discoverer application before you can conduct a search with Sequest 1 bd 1 To add a FASTA file Choose Administration gt Maintain FASTA Files or click the Maintain FASTA Files icon amp The Administration page appears with the FASTA files view shown in Figure 74 on page 102 Click f Add In the Open dialog box that appears browse for and sel
457. ss of the specified amino acid You can set the parameters for a static search on the Select Modifications page of the Mascot and Sequest HT search wizards For instructions on setting these parameters in the search wizards see Figure 19 on page 38 and the steps that follow it Proteome Discoverer User Guide 141 4 Searching for Data Updating Chemical Modifications Opening the Chemical Modifications View The Chemical Modifications view is an advanced feature of the Proteome Discoverer application You use it to build and maintain the static and dynamic modifications data that is available when you define your search settings In the Chemical Modifications view you can explore the default types of modifications and their corresponding amino acids It contains the modification s delta mass amino acids and substitutions By using the Chemical Modifications view you can add amino acids to existing modifications and create new modifications To open the Chemical Modifications view 1 Choose Administration gt Maintain Chemical Modifications or click the Maintain Chemical Modifications icon a either on the toolbar or on the Administration page The Chemical Modifications view appears on the Administration page as shown in Figure 103 The amino acids listed are those where the modifications can appear Figure 103 Chemical Modifications view File Search Report Quantification Processing Workflow Editor Administration Tools Windo
458. ss the Quantification Method Editor dialog box to set up a quantification method the default methods available in the top list of the Quan Channels page are as follows e SILAC 2plex Arg10 Lys6 e SILAC 2plex Arg10 Lys8 e SILAC 2plex Ile6 e SILAC 3plex Arg6 Lys4 Arg10 Lys8 e SILAC 3plex Arg6 Lys6 Arg10 Lys8 Dimethylation 3plex C2H6 C2D4H2 13C2D6 e 180 labeling For a description of these methods see Performing Precursor lon Quantification on page 243 However after you have chosen a method or set up your own method only that method appears in the top list of the Quan Channels page after you execute the workflow 268 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Setting Up the Quantification Method The left box of the Quan Channels page displays two types of default isotopic labels e Heavy Refers to amino acid labels that use heavy isotopes for example Arg10 and Lys8 e Medium 3plex methods only Refers to amino acid labels that use less massive isotopes for example Arg6 and Lys4 e Light Refers to amino acid labels that use normal isotopes 3 To add a quantification channel click beneath the list of quantification channels in the box on the left The default name of New number now appears in the list of quantification channels and in the Channel Name box as shown in Figure 179 Figure 179 New quantification channel on the Quan Channe
459. st specify the fragmentation method used to activate the scan Note You cannot perform TMT quantification on both PQD and HCD scans You can choose only one activation type CID Collision Induced Dissociation ECD Electron Capture Dissociation ETD Electron Transfer Dissociation Default HCD High Energy Collision Dissociation MPD Multi Photon Dissociation PQD Pulsed Q Collision Induced Dissociation Thermo Scientific 7 Quantification Performing Reporter lon Quantification For a description of these fragmentation types see Fragmentation Methods on page 8 13 Choose Workflow Editor gt Start Workflow or click the Start Workflow icon E Performing TMT Quantification on HCD and CID Scans Ifa raw file contains both CID scans for identification and HCD scans for quantification you can use the following workflow to both quantify the HCD scans and identify peptides in the CID scans the HCD scans or both To perform TMT Quantification on HCD and CID scans 1 Drag the Reporter Ions Quantifier node to the workspace pane and connect it to the workflow 2 Set the Activation Type parameter for the Reporter Ions Quantifier node to HCD 3 Set the Activation Type parameter for the Spectrum Selector node to Any Is CID HCD or Is CID depending on your method setup and identification strategy 4 Set all other parameters modifications tolerances FASTA files and so forth and choose Workflo
460. stration X Celegans_FT_6ITDDDT_01_01 msf_annotation msf x wade Accession Description Coverage Proteins UniquePeptides Peptides PSMs Molecular Function Cellular Component Biological Process 17509401 UNCoordinated family member unc 54 Caenorhabditis 139 42 25 17 4 27 40 61 TM i Wii TT a 17509391 F07A5 7 Caenorhabditis elegans 67 19 31 19 3 14 20 28 25144756 ATPsynthase subunitfamily member atp 2 Caenorhab 59 45 38 64 10 10 13 2 E a i 193209657 VITellogenin structural genes yolk protein genes family 59 36 21 57 3 9 23 34 I 1 m 17570201 VITellogenin structural genes yolk protein genes family 45 81 16 34 3 5 24 30 1 I im m 71991083 VTTellogenin structural genes yolk protein genes family 44 29 22 42 4 13 27 34 nucleotide binding 1 M 17536383 ENOLase family member enol 1 Caenorhabditis elegans 43 66 43 55 3 9 11 19 Lii Mii ml p 17554386 Protein Disulfide Isomerasefamily member pdi 1 Caen 43 16 39 18 2 13 15 25 li Or I I E M 17554310 Malate DeHydrogenase family member mdh 1 Caenor 39 95 58 65 1 8 13 24 ME nt DOO m 17570197 VITellogenin structura
461. t select the label modification that would be found on the target amino acid residue From the adjacent list select the appropriate letter to indicate that the modification should occur on the indicated residue and will have an increased mass From the N Terminal Modification list select the label modification that would be found on the N terminus of each peptide The left box of the Quan Channels page displays a list of mass tags which are the fragmented labels To add a mass tag click beneath the list of mass tags in the box on the left To remove a mass tag select the mass tag you want to remove and click beneath the list of mass tags When you add a mass tag or change the settings of an existing mass tag do the following a In the Tag Name box enter the name of the new mass tag if you do not want to use the default name b In the Monoisotopic m z box enter the monoisotopic mass to charge ratio of the new mass tag c Inthe Average m z box enter the average mass to charge ratio of the new mass tag d In the Reporter Ion Isotopic Distribution area select the correction factor for the mass tags Click and to add and delete correction factors Thermo Scientific 7 Quantification Setting Up the Quantification Method For information on these correction factors see Using Reporter Ion Isotopic Distribution Values To Correct for Impurities on page 308 You must correct the purity of mass ta
462. t the application filters out those peptides that do not pass the peptide filters except the Peptide Rank filter It calculates the ranks for the remaining peptides Finally it applies the Peptide Rank filter To filter peptides by rank 1 Open the MSF file Refer to the Help 2 Click the Result Filters tab 3 Select Peptide Rank in the Peptide Filters area of the Result Filters page if it is not already selected The Maximum Peptide Rank option appears in the middle of the Result Filters page 4 Optional In the Maximum Peptide Rank box set the maximum rank that a peptide must have to avoid being filtered out The minimum value is 1 and there is no maximum value The default value is 1 Filtering Peptides by the Delta Cn Value Thermo Scientific Search engines often provide multiple possible matching peptides as explanations for the same spectrum Most of the time you can clearly distinguish the top scoring match from the other PSMs but sometimes especially in the presence of dynamic modifications the best scoring matches of the same spectrum have very similar scores In this case you can filter the results to select the best scoring PSMs and the matches that have very similar scores by using the A Cn peptide filter The A Cn value displays the normalized score difference between the currently selected PSM and the highest scoring PSM for that spectrum score ACn rank i ranki score SCOPE sankl rankl Prote
463. t Filters Page The following procedures describe how to filter your results using the result filters on the Result Filters page Filtering Search Results with Protein Filters Filtering Search Results with Peptide Filters Filtering Peptides by Rank Filtering Peptides by the Delta Cn Value Filtering Results by the Original Rank Assigned by the Search Engine Filtering Search Results with Protein Filters Thermo Scientific Follow this procedure to apply protein filters to your search results 1 bd I 2 To filter your search results with protein filters Open your search results Refer to the Help Click the Result Filters tab which is shown in Figure 111 on page 154 Click Add a Filter in the Protein Filters area A list of filters appears For a description of the available filters refer to the Help Select the filter to apply from the list of filters Settings pertaining to the selected filter appear in the Filter or Grouping Settings area on the right as shown in Figure 112 For a description of the available settings refer to the Help Proteome Discoverer User Guide 155 5 Filtering Data Filtering the Search Results Figure 112 Protein filter options File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OU BB BIOTI BODO SA amp SB GB g SequethHT Q Mco RA L MET AZEBTRRBBPRAARB Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x 4 Proteins P
464. t from 2 Reports x 41 gt ZCoverage 114 113 114 113 Count 114 113 Variability Score A2 Coverage A2 Peptides A2 PSM A Score B2 Coverage B2 Peptides B2 PSM B2 a a 7 TYPE I RESTRICTION ENZYME BA 10 80 1466 zo o i ZC Ta 5 0 00 188 8 hypothetical yok protein Badllus 13 33 1 422 1 0 00 5 45 Z 6 0 00 7 88 1 1 9 hypothetical protein H_GS541B18 1 6 82 1 389 1 1 65 2 03 4 5 220 479 9 10 101 7 DYSTROPHIN 7 35 1 371 1 0 00 142 6 13 545 5 93 24 30 11 gt GLUCOSAMINE FRUCTOSE6FHO 9 00 1 352 1 3 07 4 09 2 z 0 00 491 2 E ETI X58534 crystal protein Bacillus t 8 25 1 350 1 0 00 2 00 2 0 00 6 26 13 PECTIN LYASEA PRECURSOR PLA 3 17 1 348 1 2 04 3 17 1 2 0 00 14 DNA methyltransferase cytosines 375 1 346 2 553 3 75 2 4 0 00 G Til gt HYPOTHETICAL 12 3 KD PROTEIN 10 48 1 343 1 0 00 8 57 1 2 0 00 10 48 1 1 16 7 PHOSPHORIBOSYLFORMIMINO 5 29 80 1 322 1 0 00 16 33 3 5 171 19 59 3 3 17 7 NIPP 1 nuclearinhibitorof protein 8 55 1 317 1 183 7 41 2 5 0 00 1 14 1 1 18 7 conserved hypothetical proteinyoq 8 77 1 286 1 2 09 8 77 1 2 0 00 19 PUTATIVEALIPHATIC SULFONATE 4 52 1 282 1 0 00 452 1 3 0 00 20 7 CARBONICANHYDRASE III CARB 18 15 1 270 1 1 78 463 1 1 0 00 13 51 3 4 H 21 _ transposon related protein homolo 14 58 1 256 1 535 11 46 5 12 177 3 13 2 z 22
465. t is 3 0 Optional Select the Group Spectra check box The rest of the boxes in the Grouping Parameters area become available In the Grouping Parameters area you can set grouping parameters to group similar spectra in the raw data file into a single spectrum Thermo Scientific Thermo Scientific 2 Getting Started Starting a New Search by Using the Search Wizards Grouping spectra speeds up the analysis The application evaluates an ion only once rather than every time it is observed within the given retention time limits In the Precursor Mass Criterion list select the criteria for grouping You can select either of these settings e Same Measured Mass to Charge Groups spectra according to the mass to charge ratio m z of the precursor ion e Same Singly Charged Mass Groups all charge states with the same singly charged precursor mass For example this option groups 2 and 3 ions for the same peptide because they have the same singly charged parent In the Precursor Tolerance box type the range of the precursor tolerance in daltons Da milli mass units mmu or parts per million ppm For example if the mass to charge ratio of a spectrum is 100 0001 Da and the tolerance is 2 Da all the spectra with masses in the range of 100 0001 plus or minus 2 Da are valid mass candidates In the Max RT Difference min box enter the maximum retention time in minutes Retention time is the time in the mass chromatogram when an
466. t s Displaying the Quantification Spectrum Chart You can generate a chart showing the spectrum used for quantification This chart is available for every peptide with an associated quantification result To display the Quantification Spectrum chart 1 Select the peptide of interest If Show Peptide Groups is already selected you might need to ungroup the peptides first by right clicking and choosing Show Peptide Groups The peptide must be labeled Used in the Quan Usage column of the Peptides page Thermo Scientific Proteome Discoverer User Guide 297 7 Quantification Displaying the Quantification Spectrum Chart 2 Choose Quantification gt Show Quantification Spectrum or click the Show Quantification Spectrum icon F To see the results see the following sections e Displaying the Quantification Spectrum Chart for Reporter Ion Quantification isplayin e Quantification Spectrum art for Precursor lon Quantification e Displaying th tification Spectrum Chart for P I tificat Displaying the Quantification Spectrum Chart for Reporter lon Quantification For reporter ion quantification the Quantification Spectrum chart displays the intensity of the reporter ions in counts It shows a spectrum for each peptide except for those peptides labeled No Quan Values Figure 200 shows an example of a quantification spectrum from an iTRAQ 8plex sample quantified with an Integration Tolerance setting in the Reporter Ions Qua
467. t the Proteome Discoverer application should try to obtain the requested annotations if the ProteinCenter Web service issues an error The default is 3 5 In the Time Interval Between Attempts to Submit the Annotation Request sec box specify the amount of time in seconds that the Proteome Discoverer application should Thermo Scientific Proteome Discoverer User Guide 205 6 Protein Annotation Creating a Protein Annotation Workflow 8 wait between tries to obtain the requested annotations if the ProteinCenter Web service issues an error The default is 90 seconds In the Timeout of the Annotation Request min box specify the amount of time in minutes that the Proteome Discoverer application should continue to try to access the ProteinCenter Web service The default is 15 minutes If you changed any settings click Apply The message box shown in Figure 148 appears Figure 148 Administration message box Administration x Changes were saved They will have no effect for currently open workflows Click OK Tip Click A Reset to return to the previous values Click Factory Defaults to return to the values set when you first installed the Proteome Discoverer application 9 Restart your machine Creating a Protein Annotation Workflow You can retrieve annotations of all identified proteins from ProteinCenter by using the Annotation node in a workflow This node can retrieve the foll
468. tags by Applied Biosystems Vv FASTA Files v iTRAQ 8plex Thermo Scientific Instruments Method for iTRAQ 8 plex mass tags by Applied Biosystems optimize Vv v SILAC 2plex Arg10 Lys6 SILAC 2plex Arg10 Lys6 Method Vv FASTA Indexes v SILAC 2plex Arg10 Lys8 SILAC 2plex Arg 10 Lys8 Method Vv Y SILAC 2plex Ile6 SILAC 2plex Ile6 Method Vv Pee v SILAC 3plex Arg6 Lys4 Arg10 Lys8 SILAC 3plex Arg6 Lys4 Arg10 Lys8 Method Vv Pm sesso W SILAC3plex Arg6 Lys6 Arg10 Lys8 SILAC 3plex Arg6 Lys6 Argi0 Lys8 Method Vv v TMT 2plex Method for 2 plexTandem Mass Tag ofProteome Sciences plc Vv a Chemical Modifications v TMT 6plex Method for 6 plexTandem Mass Tag of Proteome Sciences plc Vv 3 Cleavage Reagents 7 a Quantification Methods License Management ri R Licenses Configuration R B4 Workflow Nodes amp Annotation E Mascot iG MSPepSearch E SEQUEST E Sequest HT i SpectraST L B4 Server Settings E Discoverer Daemon 9 FASTA Indexes Ready 4 From the list at the top of the Quantification Method Editor dialog box select the quantification method For precursor ion quantification you can choose from the following methods when you initially set up a workflow and first access the Quantification Method Editor dialog box e SILAC 2plex Arg10 Lys6 Uses arginine 10 and lysine 6 e SILAC 2plex Arg10 Lys8 Uses arginine 10 and lysine 8 e SILAC 2plex Ile6 Uses isoleucine 6 e SILAC 3plex Arg6 L
469. tails command icon 221 Show Proteins Covered by This Set of Peptides command icon 177 Show Quan Channel Values command icon 295 Show Quantification Spectrum command icon 298 signal transducer activity GO Slim category 234 SILAC 2plex Arg10 Lys6 quantification method description 243 precursor ion quantification 243 selecting in Quantification Editor dialog box 265 268 troubleshooting precursor ion quantification 336 SILAC 2plex Arg10 Lys8 quantification method description 243 precursor ion quantification 243 selecting in Quantification Editor dialog box 265 268 troubleshooting precursor ion quantification 336 SILAC 2plex Ile6 quantification method description 243 precursor ion quantification 243 selecting in Quantification Editor dialog box 265 268 troubleshooting precursor ion quantification 336 SILAC 3plex Arg6 Lys4 Arg10 Lys8 quantification method description 246 precursor ion quantification 243 selecting in Quantification Editor dialog box 265 268 troubleshooting precursor ion quantification 336 SILAC 3plex Arg6 Lys6 Arg10 Lys8 quantification method description 246 precursor ion quantification 243 selecting in Quantification Method Editor dialog box 266 268 troubleshooting precursor ion quantification 337 SILAC See precursor ion quantification single quadrupole mass spectrometer 256 single search reports 324 Size column 103 131 Spectra column 131 SpectraST node description 15 129 137 dot bias score 138 d
470. tal Execution State Progress Name Spectrum Source Description Submitted at E Completed 100 BSA2 BSA2 raw 10 25 2012 4 05 PM H Time Processing Node Message 4 05PM 2 Spectrum Exporter 429 spectra exported in 0 43 s 4 05PM 2 Spectrum Exporter Start exporting of spectra to BSA2 4 05PM 2 Spectrum Exporter 2 Received 429 spectra 4 05PM 2 Spectrum Exporter 1000 spectra exported in 1 05 s 4 05PM 2 Spectrum Exporter Start exporting of spectra to BSA2 4 05PM 2 Spectrum Exporter 2 Received 1000 spectra 4 05PM 1 Spectrum Selector Reading from File 1 of 1 C Projects Daemon BSA2 raw 4684 spectra total Execution State Progress Name Spectrum Source Description Submitted at 4 05PM_ 2 Spectrum Exporter 429 spectra exported in 0 46 s 4 05PM 2 Spectrum Exporter Start exporting of spectra to BSA2 4 05PM 2 Spectrum Exporter 2 Received 429 spectra 4 05PM 2 Spectrum Exporter 1000 spectra exported in 2 42 s 4 05PM 2 Spectrum Exporter Start exporting of spectra to BSA2 4 05PM 2 Spectrum Exporter 2 Received 1000 spectra 4 05PM 1 Spectrum Selector Reading from File 1 of 1 C Projects Daemon BSA2 raw 4684 spectra total Batch Processing by Using a Post Acquisition Method Xcalibur Data System 2 0 7 Only You can perform batch processing by using different processing methods for different samples However editing the processing method is complicated For qu
471. tarting a New Search by Using the Search Wizards e Starting a New Search by Using the Workflow Editor Starting the Proteome Discoverer Application Open the Proteome Discoverer application by choosing a Start menu command or clicking a desktop icon To start the Proteome Discoverer application From the Start menu choose Programs gt Thermo Proteome Discoverer or click the Proteome Discoverer icon age on your desktop The Proteome Discoverer main window opens as shown in Figure 4 Thermo Scientific Proteome Discoverer User Guide 19 2 Getting Started Closing the Proteome Discoverer Application Figure 4 Proteome Discoverer main window File Search Report Quantification Processing Workflow Editor Administration Tools Window Help POE BEB ST SBRBROBH BD BB ni KR sequesthT GH Mascot A AREL ABSEBTRIARE For information on the features of this window and how to customize them refer to the Help For instructions on opening an MSF file refer to the Help Closing the Proteome Discoverer Application Save your changes before you exit the Proteome Discoverer application because it does not prompt you To close the Proteome Discoverer application e Choose File gt Exit The Proteome Discoverer application closes 20 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Configuring Search Engine Parameters Configuring Search Engine Parameters Before you execute the search you can configure c
472. tcut menu and choose Show Peptide Groups To group the peptides in your search results and set grouping options 1 Open the MSF file 2 Click the Results Filters tab 3 On the Results Filters page click Settings beneath Peptide Grouping Peptide grouping options appear in the Filter or Grouping Settings area as shown in Figure 136 Proteome Discoverer User Guide 185 5 Filtering Data Calculating False Discovery Rates Figure 136 Peptide grouping options File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OF BSE BST SOROS H zomm R SequethT Kms AA LMG REBEREIARE Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf x Sequest TMT 6 plex Benchmark 1 4 0 175 msf x 4 Proteins Peptides Search Input Result Filters Peptide Confidence Search Summary Quantification Summary Filter and Grouping Set O mw E oa A Seve A Factory Defauts Based on Filter Set Filter or Grouping Settings Peptide Filters Peptide Grouping Options Active Filter Add a Filter E Show peptide groups Protein Filters Group peptides by Mass and x Active Filter j Add a Filter Peptide Grouping Disabled Settings Protein Grouping Enabled Settings Ready 292 1120 Protein Group s 2266 2266 Protein s 3286 3286 Peptide s 7721 7721 PSM s 1541 1541 Search Input s 4 If you want peptides to be grouped on the Pepti
473. te To work properly Percolator needs a sufficient number of PSMs from the target and the decoy search If the search identified fewer than 200 target or decoy PSMs or if fewer than 20 percent decoy PSMs are available compared to the number of target matches Percolator rejects them for processing and displays an appropriate message in the Proteome Discoverer job queue or in the Search Summary of an open report Figure 140 Workflow with Percolator attached to two different search nodes File Search Report Quantification Processing Workflow Editor Administration Tools Window Help Ba BA BOT SPB TTH COTA Ks Kut ARAM AGSEBTARSARB Sequest iTRAQ 8 plex Benchmark 1 4 0 175 msf xj Sequest TMT 6 plex Benchmark 1 4 0 175 msf x Celegans_FT_6ITDDDT_01_decoy msf X Workflow Editor x 4b Workflow Nodes g q Name Data Input a E Based on template ii Spectrum Files Description Spectrum amp Feature Retrieval A Event Detector J Spectrum Selector Spectrum Processing tug Noise Peak Filter E J oom ig Non Fragment Filter bes PEE i Spectrum Grouper Y ig Spectrum Normalizer ug Top N Peaks Filter a Spectrum Filters ee if Scan Event Filter Selector 1 T Spectrum Confidence Filter Ti Spectrum Properties Filter Sequence Database Search Mascot D SEQUEST log Sequest HT Spectral Library Search W MsPepSearch W SpectrasT PSM Validation lih Fixed ValueP
474. ted with one identification spectrum and two ea unique to one protein Claw Not ere Claw Shared C wepectuma Y C wepectuma Y A C ne nani Not C ne roten Case 7 Quantification Result Associated with Two Spectra Two Peptides and Two Proteins Case 7 shown in Figure 220 is a variant of case 6 Either the same search engine or different search engines identify different identification spectra for example CID and ETD spectra As in case 6 the peptides are unique but the quantification result is not The result depends on whether you specified in the quantification method to use only unique peptides or all peptides Figure 220 Case 7 Quantification result associated with two identification spectra and two different peptides unique to one protein Peptide A Protein A o Not Unique Peptide B Calculating Ratio Count and Variability Quantification result A Shared The Proteins page of search reports with precursor ion quantification results displays columns called Heavy Light Variability and Heavy Light Count Similarly the Proteins page of search reports containing reporter ion quantification results displays columns called Ratio Variability for example 114 113 Variability and Ratio Count for example 114 113 Count The way the Proteome Discoverer application calculates and displays the values in these columns depends on whether you want the results treated as replicates or as treatments Replic
475. tein quantification you would not use peptide A in this case If the quantification method specifies using all peptides for protein quantification the quantification result of peptide A would be divided equally between both proteins Figure 218 Case 5 Quantification result associated with one identification spectrum one peptide and two proteins Quantification result A e Case 6 Quantification Result Associated with One Spectrum Two Peptides and Two Proteins Peptide A Not Unique In case 6 shown in Figure 219 the quantification result is associated with one identification spectrum from which two different peptides are identified either by the same search engine as different ranks or by different search engines The two different peptides are contained in two different proteins The two different peptides are both unique to just one protein Nevertheless the associated quantification result is the same and you do not want to use it for Thermo Scientific Proteome Discoverer User Guide 323 7 Quantification Calculating Ratio Count and Variability the calculation of the protein ratios if you specified in the quantification method to use only unique peptides Only if you specify using all peptides can you use them for protein quantification This case illustrates the discrepancy between the uniqueness of peptides and the uniqueness of the quantification results Figure 219 Case 6 Quantification result associa
476. tein of interest Related peptides Proteins related to the selected protein The Is Master Protein column in the Protein Group Members view indicates whether the protein is the master protein of a protein group For some peptides a list of proteins might contain this peptide sequence but none of them is a master protein This situation can occur if the peptide contains isoleucine at a position where the master protein has leucine or vice versa To turn off protein grouping 1 On the Result Filters page click Settings below Protein Grouping Enabled and clear the Enable Protein Grouping check box or On the Proteins or Peptides page right click a protein grid cell or row to access the shortcut menu and clear the check mark for Enable Protein Grouping shown in Figure 133 The proteins are no longer grouped 178 Proteome Discoverer User Guide Thermo Scientific 5 Filtering Data Grouping Proteins Figure 133 Enable Protein Grouping command on the Proteins page shortcut menu Show Peptide Groups Show Filtered Out Rows Enable Protein Grouping 2 To regroup proteins reselect the Enable Protein Grouping check box on the Result Filters page or Right click a protein grid cell or row in the Proteins or Peptides page and choose Enable Protein Grouping from the shortcut menu Protein Grouping Algorithm The Proteome Discoverer application uses a protein grouping inference process to group p
477. teins page as shown in Figure 157 All proteins derived from the same gene have the same gene ID You can 214 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Displaying the Annotated Protein Results use this information to group or cluster biologically meaningful proteins together Because not all genes are stored in the Entrez gene database some proteins do not have a valid gene identification In this case the column is empty For more information on the Entrez gene identifications see Entrez Gene Database Annotation on page 204 To display Entrez gene identifications 1 Open the MSF file by following the instructions in the Help 2 In the Column Chooser dialog box of the Proteins page select the Gene IDs column For information on the Column Chooser dialog box refer to the Help The Proteome Discoverer application displays the gene identifications on the Proteins page of the MSF report in the Gene IDs column as shown in Figure 157 Figure 157 Gene IDs column and Pfam IDs column on the Proteins page Pfam IDs column Gene IDs column File Search Report Quantification Processing Workflow Editor Administration Tools Window Help
478. th links to detailed information at the International Union of Biochemistry and Molecular Biology Cellular Components Page The Cellular Components page of the ProteinCard page shown in Figure 167 summarizes information about where the protein carries out its function in the cell Figure 167 Cellular Components page of the ProteinCard page IIIN Protein Identification Details tobas Coverage ProteinCard General Keys Features Molecular Functions Biological Processes Diseases External Links Cellular Components GO Id Evidence codes PMIDs Go Slim Name GO 0005737 IEA cytoplasm cytoplasm GO 0005783 IDA IEA NAS TAS 18029348 7656593 cytoplasm endoplasmic reticulum endoplasmic reticulum GO 0005788 TEA TAS cytoplasm endoplasmic reticulum lumen organelle lumen endoplasmic reticulum GO 0005793 IDA 15308636 cytoplasm endoplasmic reticulum Golgi intermediate compartment Table 15 lists the parameters on the Cellular Components page of the ProteinCard page 228 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation ProteinCard Parameters Table 15 Parameters on the Cellular Components page of the ProteinCard page Command GO Id Description Lists the GO code for each of the protein s molecular functions Each code is linked to the QuickGO browser of the EBI which hosts a number of databases and services Evidence Codes Lists the evidence codes for each of the protein s cellula
479. thods to create your own quantification templates iTRAQ and TMT use the Reporter Ions quantifier node in the Workflow Editor For detailed information about isobarically and isotopically labeled quantification see Performing Reporter Ion Quantification on page 249 and Performing Precursor Ion Quantification on page 243 The Proteome Discoverer application also offers peak area calculation quantification which you can use to determine the area for any quantified peptide This type of quantification uses the Precursor Ions Area Detector node For more information about peak area calculation quantification see Performing Peak Area Calculation Quantification on page 259 The Qual Browser Application With the Qual Browser application you can view the entire ion chromatogram and browse individual precursor and MS data You can filter the results in a variety of ways for example to produce a selected ion chromatogram When you select a peptide and choose Tools gt Open QualBrowser the Proteome Discoverer application passes the currently active raw file for Qual Browser operations For more information about the Qual Browser application see Using the Qual Browser Application on page 149 Peptides and Fragment lons Thermo Scientific The types of fragment ions observed in an MS MS spectrum depend on several factors such as the primary sequence the energy source and the charge state Fragment ions of peptides a
480. tification in the Proteome Discoverer application Contents e Activating the Quantification Menu e Proteins Included in the Quantification e Performing Precursor Jon Quantification e Performing Reporter Ion Quantification e Performing Peak Area Calculation Quantification e Searching for Quantification Modifications with Mascot e Setting Up the Quantification Method e Adding a Quantification Method e Changing a Quantification Method e Removing a Quantification Method e Importing a Quantification Method e Exporting a Quantification Method e Summarizing the Quantification e Displaying Quantification Spectra e Displaying the Quantification Channel Values Chart e Displaying the Quantification Spectrum Chart e Using Reporter Ion Isotopic Distribution Values To Correct for Impurities e Excluding Peptides from the Protein Quantification Results e Excluding Peptides with High Levels of Co Isolation e Calculating Peptide Ratios Thermo Scientific Proteome Discoverer User Guide 241 7 Quantification Activating the Quantification Menu Contents continued e Calculating Protein Ratios from Peptide Ratios e Calculating Ratio Count and Variability e Calculating and Displaying Protein Ratios for Multiconsensus Reports e Identifying Isotope Patterns in Precursor Ion Quantification e Troubleshooting Quantification Activating the Quantification Menu In the Proteome Discoverer application the Quantification menu becomes availab
481. tification method 264 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Setting Up the Quantification Method You can also access the Quantification Method Editor dialog box by choosing Administration gt Maintain Quantification Methods or by clicking the Maintain Quantification Methods icon to open the Quantification Methods view shown in Figure 177 This view lists all ot the available methods for both precursor ion and reporter ion quantification Double click the appropriate method in the Method Name column Figure 177 Quantification Methods view File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BE aTEoegr onnan Zomm K Sweth K Macot ALKA REe BEBSBZBRPARE Administration x ZE dh Add 3 Remove P Edit Gi Import fy Export oora 8 Status Method Name Description Is Active la are Job Queue gt vY Dimethylation 3plex C2H6 C2H2D4 13C2D6 Dimethylation 3plex C2H4 C2D4 13C2D4 Method Vv SS v Full 180 Labeling 02 1802 180 labeling method for fully labeled samples Vv v Incomplete 180 Labeling 02 0180 1802 180 labeling method for incompletely labeled samples Vv A vY iTRAQ 4plex Method foriTRAQ 4 plex mass tags by Applied Biosystems Vv Content Management a wv iTRAQ 4plex Thermo Scientific Instruments Method for iTRAQ 4 plex mass tags by Applied Biosystems optimize Vv v iTRAQ 8plex Method for iTRAQ 8 plexmass
482. tio Distributions chart 313 315 peptide ratios calculating 313 315 deriving protein ratios from 320 displaying number used to calculate protein ratios 279 handling missing and extreme values 317 setting up 275 Peptide Score filter 197 Peptide Score peptide filter Mascot reports 197 SEQUEST reports 197 PeptideAtlas home page 129 131 peptides C terminus 40 calculating ratios 313 315 classifying for protein quantification 311 confidence indicators 188 defining uniqueness 243 displaying filtered out rows 170 excluding from protein quantification 309 excluding those with high levels of coisolation 310 expanding identified 175 filtering by Delta Cn 161 by rank 157 161 by search engine rank 163 deactiving filters 167 for site localization scores from phosphoRS 172 removing 166 result filters 154 155 row filters 167 with peptide filters 156 grouping on Peptides or Proteins page shortcut menu 185 Proteome Discoverer User Guide 355 Index P on the Result Filters page 185 on the Results Filters page 185 options 186 high levels of co isolation 311 high confidence Peptide Validator node 191 Percolator node 193 Result Filters page 176 search wizards 190 low confidence Result Filters page 176 medium confidence Peptide Validator node 191 Percolator node 193 Result Filters page 176 search wizards 190 N terminus 39 40 number displayed on status bar 185 Peptides page displaying filtered out rows 170 Ratio columns 273 275 row
483. tion e Updates the columns that are inaccessible to you e Leaves the modification name and abbreviation unchanged e Adds any new amino acids found in unimod org e Adds any amino acids that you removed if they are defined in unimod org e Leaves unchanged any amino acids that you added Deleting Amino Acids You can also delete amino acids from chemical modifications To delete an amino acid from a chemical modification 1 Choose Administration gt Maintain Chemical Modifications The Chemical Modifications view appears as shown in Figure 103 on page 142 2 Click to the left of the modification row that you want to delete The row expands and the associated amino acids appear 3 Select the amino acid row that you want to delete 4 Click the Remove icon Remove 5 In the Delete Row dialog box click Yes The row is removed from the chemical modifications table 148 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using the Qual Browser Application Using the Qual Browser Application Thermo Scientific The Proteome Discoverer application includes the Qual Browser application which you can use to examine spectra and chromatograms in detail With the Qual Browser application you can view the entire ion chromatogram and browse individual precursor and MS data You can filter the results in a variety of ways for example to produce a selected ion chromatogram The Qual Browser application a
484. tion 260 Figure 175 Beginning of the workflow for area calculation quantification fz spectrum Files 0 EventDetector 13 eS Precursor lons Area Detector Fixed Value PSM Validator 17 9 Add any other nodes that you would like and connect them For general information about creating a workflow in the Workflow Editor see Starting a New Search by Using the Workflow Editor on page 42 10 Click the Spectrum Files node and specify the raw file in the Parameters pane 11 Click the Event Detector node and set the parameters for it in the Parameters pane a In the Mass Precision box specify the expected standard deviation of the mass precision To create extracted ion chromatograms use three times the standard deviation The minimum value is 1 ppm The maximum value is 4 ppm The default is 2 ppm In the S N Threshold box specify a threshold signal to noise value that determines whether the Proteome Discoverer application removes peaks from the spectrum The application removes peaks with a signal to noise value below this threshold The minimum value is 0 0 and there is no maximum value The default is 1 12 Click the Spectrum Selector node and set the parameters for it in the Parameters pane a Proteome Discoverer User Guide Change the setting in the Max Precursor Mass box to an appropriate setting For example you could set this option to 6500 Change the setting in the S N Threshol
485. tion Calculating and Displaying Protein Ratios for Multiconsensus Reports Figure 225 Opening a multiconsensus report from three iTRAQ and two SILAC files in replicate mode File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE SABOTSATOTAH LOAD suth Kut AA AMS RCOESTAFARE i Input Files Result Filters I Open i Add 86 Remove Selected Files Filename Status Loading Progress Task Progress Current Task 4 a v Program Files Proteome Di C Program Files Proteome Di Program Files Proteome Di erer source Gldlalo Program Files Proteome Discoverer source files ke_Test_Files Itraq 4 plexWesper_S W Treat quan results as replicates T Show quan spectra on separate tab Result Items Per File Filename Spectra Processing Nodes Proteins Peptides Unfiltered Peptides Filtered 9mix_iTRAQ_A_PickFrit_2us5e5_2hrGr1_1 5857 SEQUEST 2 400 1000 1000 9mix_ITRAQ_A _PickFrit_2us5e5_2hrGr1_2 5857 SEQUEST 2 400 1000 1000 9mix_iTRAQ_A_PickFrit_2usSe5_2hrGr1_3 5857 SEQUEST 2 400 1000 1000 Jesper_SILAC_HeLa_SequestSilacQuan_1 1597 SEQUEST 2 3000 3000 3000 Jesper_SILAC_HeLa_SequestSilacQuan_2 1597 SEQUEST 2 3000 3000 3000 Ready Figure 226 shows the opened multiconsensus report loaded in Figure 225 The combined ratios from the i1TRAQ and the SILAC quantification are disp
486. tion Method Editor dialog box see Setting Up the Ratio Calculation on page 275 or refer to the Help If one or more of the reporter or mass tags are missing in the quantification spectrum the calculated ratios are either zero or infinity depending on which tag intensity is the numerator and which is the denominator Even if all tags are present the calculated ratios might be very high or very low You can use the Maximum Allowed Fold Change option on the Ratio Calculation page of the Quantification Method Editor dialog box to replace such extremely high or extremely low ratios with the maximum allowed number of times that the ratios can be multiplied In the example in Figure 202 the maximum allowed number of times that the ratio can be multiplied is 100 That is extremely high ratios are replaced by 100 and extremely low ratios are replaced by 0 01 You can expect an inherent dynamic range to be valid or detectable with the given instrumentation and method The Use Ratios Above Maximum Allowed Fold Change for Quantification option in the Ratio Calculation dialog box specifies whether such maximum calculated ratios should be considered when the Proteome Discoverer application calculates the protein ratios You can use this option to automatically include extreme values when the application calculates the protein ratios Since the protein ratios are calculated as the median outlier protein ratios are likely to o
487. tion perform batch processing and process Multidimensional Protein Identification Technology MudPIT samples You can select a server to connect to start workflows and monitor the execution of jobs on the configured server Unlike the search wizards which can only perform searches on one raw data file at a time the Proteome Discoverer Daemon application can perform multiple searches on multiple raw data files at any given time It can perform searches on multiple raw data files taken from multiple samples or from one sample You can run the Proteome Discoverer Daemon application on the command line or in a window interface Contents e Starting the Proteome Discoverer Daemon Application in a Window e Selecting the Server e Starting a Workflow e Creating a Parameter File That the Discoverer Daemon Application Uses e Monitoring Job Execution in the Proteome Discoverer Daemon Application e Logging On to a Remote Server e Running the Proteome Discoverer Daemon Application from the Xcalibur Data System e Running the Proteome Discoverer Daemon Application on the Command Line For information about MudPIT and creating a MudPIT workflow see Creating a Search Workflow for Multiple Raw Files from the Same Sample on page 53 Thermo Scientific Proteome Discoverer User Guide 69 3 Using the Proteome Discoverer Daemon Utility Starting the Proteome Discoverer Daemon Application in a Window Starting the Proteome Discoverer Daemon App
488. tion 337 Discoverer Daemon See Proteome Discoverer Daemon application Diseases page 222 231 Display Temporary icon 103 DNA 233 234 DNA binding GO Slim category 233 documentation survey xiv dot bias score 138 dot score 138 139 DTA files exporting spectra through Spectrum Exporter node 66 output file type 13 dynamic modifications definition 141 selecting in Sequest HT wizard 38 Thermo Scientific E EC number 228 230 ECD activation type analyzed by Sequest 3 description 8 fragmenting ions 347 ion factors 37 selecting in search wizards 33 specifying in Reporter Ions Quantifier node 256 Edit Configuration icon 21 204 Edit Quantification Method command icon 289 Enable Protein Grouping command 174 endoplasmatic reticulum GO Slim category 235 endosome GO Slim category 235 Ensembl database 225 340 Entrez gene database description 16 204 displaying annotation results from ProteinCenter in MSF file 214 retrieving information from 202 retrieving information from ProteinCenter 206 208 Web site 224 Enzyme Category EC information 227 enzyme regulator activity GO Slim category 233 enzymes 346 ESI fragment ions 347 peptides and fragment ions 7 with PQD 9 ETD activation type analyzed by Sequest 3 description 8 fragmenting ions 347 ion factors 37 selecting in search wizards 33 specifying in Reporter Ions Quantifier node 256 European Bioinformatics Institute EBI 227 Event Detector node attaching quantification node
489. tion Method for Multiple Input Files When you load multiple MSF files you can apply the settings of the Ratio Calculation Protein Quantification and Experimental Bias pages of the Quantification Method Editor dialog box to all the loaded input files by selecting Common Quan Parameters from the list at the top of the dialog box as shown in Figure 187 These pages contain the same options as those for single file processing Figure 187 Quantification Method Editor dialog box for multiple input files r Quantification Method Editor Ea Common Quan Parameters Mi General Ratio Calculation Protein Quantification Experimental Bias E Show the Raw Quan Values Minimum Quan Value Threshold 0 0 E Replace Missing Quan Values With Minimum Intensity E Use Single Peak Quan Channels V Apply Quan Value Corrections E Reject All Quan Values If Not All Quan Channels Are Present Fold Change Threshold for Up Down Regulation 2 0 Maximum Allowed Fold Change 100 Use Ratios Above Maximum Allowed Fold Change for Quantification Percent Co lsolation Excluding Peptides from Quantification 100 0K _Garcat Hee 282 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Setting Up the Quantification Method Although you cannot apply the settings of the Quan Channels and Ratio Reporting pages to multiple loaded MSF files you can apply them to individual MSF files as shown in Fi
490. tion creates preliminary protein groups from the PSMs collected in the first step It combines all proteins into one protein group that contains the same subset of peptides The Proteome Discoverer application takes the next steps in the protein grouping process if you select the Apply Strict Maximum Parsimony Principle parameter in the Result Filters page In the third step the application removes all protein groups that have no unique peptides among the peptides that it considers for the protein grouping process If a protein group does not contain at least one unique peptide all of its peptides are also included by other protein groups so there is no supporting evidence for the existence of this protein group At this point the application explicitly retains all protein groups that form circular rings of overlapping shared peptides For example suppose a circular ring is composed of the protein groups e ABCD identified by peptides a b c and d e CDEF identified by peptides c d e and f e EFAB identified by peptides e f a and b To explain all identified peptides only two of the three protein groups are needed but at this point it is not clear which to take and which to reject The application postpones the resolution of this issue until step 5 Proteome Discoverer User Guide 181 5 Filtering Data Grouping Proteins 4 In the fourth step the application first collects all spectra with more than one peptide match
491. tios of all peptides considered in calculating the protein ratio e The third section red circles displays the distribution of the ratios of all peptides that were not considered in calculating the protein ratio for example the peptide ratio was considered too extreme or this peptide is not unique to this protein or this protein group according to the rules defined in the quantification method 316 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 7 Quantification Calculating Peptide Ratios Figure 212 Peptide Ratio Distributions chart for reporter ion quantification 1297126 blue line Median of the distribution 95 percent of the peptide Distribution of the ratios are below peptide ratios this point considered in calculating the 50 percent of the protein ratio peptide ratios are between the 25th and 75th percentile lines Distribution of peptide ratios not considered in calculating the protein ratio 5 percent of peptide ratios are below this point In addition each chart displays the median ratio R and the inter quartile range IQR in linear and logarithmic format The header of the chart identifies the protein that the peptide belongs to Right click the chart and choose Show Legend for the identity of other notations on the chart Figure 213 shows the Peptide Ratio Distributions chart for precursor ion quantification Proteome Discoverer User Guide 317 7 Quantification Cal
492. to consider for the protein grouping process It then resolves these ambiguous cases and selects one of the PSMs to use for the protein grouping process while rejecting the remaining peptide matches of a spectrum In cases where more than one PSM is considered for a spectrum it resolves this ambiguity by selecting the PSM that is connected to the best protein group and rejecting the other PSMs The best protein group is the group with the highest number of unambiguous and unique peptides and the highest protein score 5 In the fifth step the application resolves the cases where protein groups form circular rings of overlapping identified peptides This step is the last step of the protein group inference process resulting in the final list of protein groups that are reported in the Proteins page of the MSF file The PSM Ambiguity column on the Peptides and Search Input pages can help you understand the process of selecting PSMs for the protein group This column is available for every PSM every search input entry representing the searched spectra and every peptide group For the search input entries and the peptide groups this column displays the best PSM ambiguity from all connected PSMs Refer to the Help for a description of the categories of ambiguity in this column Note If you want to investigate the protein grouping mechanism in detail set the Group Peptides By option in the Peptide Grouping Enabled area of the Result
493. to your workflow as shown in Figure 43 Set the Export Format parameter on the first Spectrum Exporter node to one format and the Export Format parameter on the next node to another format and so forth Figure 43 Workflow set to export data in two different formats File Search Report Quantification Processing Workflow Editor Administration Tools Window Help POA SOB OTISBRDOO BH g BW DIS Seuetht Mst i ARARE EDBERG Workflow Editor x 4 SA Name Parameters Saat E Data Input a i B 4 a Based on template ol Show Advanced Parameters i Spectrum Files Description E Spectrum amp Feature Retrieval Event Detector i Spectrum Selector a V Merge results of equal search nodes Spectrum Processing ig Noise Peak Filter ij Non Fragment Filter i Spectrum Grouper ig Spectrum Normalizer ig Top N Peaks Filter Spectrum Filters im Scan Event Filter TY Spectrum Confidence Filter Ti Spectrum Properties Filter Sequence Database Search Q Mascot SEQUEST W Sequest HT Spectral Library Search W MsPepSearch W SpectrasT PSM Validation lih Fixed ValuePSM Validator Percolator lih Target Decoy PSM Validator PTM Analysis S phosphors Quantification 2 Precursor Ions Area Detector Precursor Ions Quantifier Reporter Ions Quantifier Data Export m i Spectrum Exporter Annotation
494. ttern 304 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Displaying the Quantification Spectrum Chart Figure 204 Quantification Spectrum chart for precursor ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OA BB BOT SATO AH g a mm K sett Kut ARAM AGEBRABARE Sequest SILAC Dimethylation Search 1 3 0 297 msf x v4 Search Engine Rank QuanResultID MissingChannels Precursor Area Quan Channel Quan Info Quan Usage Heavy Light Medium Light Peptides Matched Probability 1 5 9 317e5 Heavy Unique Used 1 094 1 192 333 5 23 vou 1 48 6 101e5 Heavy Unique Used 0 786 0 985 66 4 70 Ot 1 9 618e5 Inconsistently Labeled NotUsed 760 461 o 1 9 618e5 Inconsistently Labeled NotUsed 856 4 61 0 0 z 9 618e5 Inconsistently Labeled NotUsed 877 4 61 ne SS ee ee ee eee eee 1 89 8 810e4 Light Unique Used 0 639 1 044 93 445 o4 5 8 520e5 Light Redundant NotUsed 1 094 1 192 96 442 Ot 1 46 5 215e6 Heavy Unique Used 0 873 0 990 47 4 36 ot E 87 2 630e5 Light Redundant NotUsed 0 801 0 966 98 4 23 ne E 299 1 199e8 Light Unique Used 0 978 1 058 119 4 17 0 1 1 407e6 Inconsistently Labeled NotUsed 697 411 0 0 1 1 4076 Inconsistently Labeled NotUsed 420 4 11 Ot 1 1 407e6 Inconsistently Labeled NotUsed 356 4 11 0 0 1 7 663e4 Inconsistently Labeled NotUsed 675 4 03 ne 1 7 663e4 Inconsistently Labeled Not
495. ttings with wider tolerances 58 Proteome Discoverer User Guide Thermo Scientific Figure 36 Non Fragment Filter node settings for data taken from LTO Orbitrap instruments 40163 ILCELNLCLCE Workflow Editor x 2 Getting Started Starting a New Search by Using the Workflow Editor File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BROT R HB Zamm g sequethT g Mascot A A hk BL amp ope asaRE Workflow Nodes X E Data Input Qi Spectrum Files Spectrum amp Feature Retrieval Event Detector Spectrum Selector Spectrum Processing iy Noise Peak Filter ug Non Fragment Filter ig Spectrum Grouper iu Spectrum Normalizer ij Top N Peaks Filter E Spectrum Filters Ty Scan Event Filter Tf Spectrum Confidence Filter Ty Spectrum Properties Filter Sequence Database Search Q Mascot W SEQUEST Q Sequest HT E Spectral Library Search W MsPepSearch W SpectrasT PSM Validation Fixed ValuePSM Validator Percolator Target Decoy PSM Validator E PTM Analysis i phosphors Quantification amp Precursor Ions Area Detector amp Precursor Ions Quantifier D Reporter Ions Quantifier Data Export us Spectrum Exporter Annotation Annotation Ready Thermo Scientific Parameters A 44 Hide Advanced Parameters 4 1 Precursor Peaks Remove Precursor Peak True Mass Windo
496. u can use this information to annotate the proteins in your results report Magellan storage file or MSF ProteinCenter is a Web based application that you can use to download biologically enriched annotation information for a single protein such as molecular functions cellular components and biological processes from the GO database For information see Protein Annotation on page 201 You can also upload search results directly from the Proteome Discoverer application to ProteinCenter e Proteome Discoverer Daemon which can perform multiple searches on multiple raw files at any given time You can use it to perform searches on multiple raw files taken from multiple samples or replicates from the same sample See Using the Proteome Discoverer Daemon Utility on page 69 e A number of graphical views that contain detailed information about the selected peptides and proteins You can display more than one view to perform a comparative analysis of your selected peptide or proteins For more information refer to the Help e The presentation of database search results available from multiple raw files in a single protein or peptide report For more information refer to the Help e Support for FASTA databases and indexes See Using FASTA Databases on page 101 e The ability to import protein and peptide reports in standard spectrum data formats such as MZDATA MZXML MZML and MGF See Importing Raw Data Files in Other Form
497. uest HT Search Engine Thermo Scientific Proteome Discoverer User Guide 21 2 Getting Started Configuring Search Engine Parameters Configuring the Sequest HT Search Engine 22 Follow these steps to configure the Sequest HT search engine 1 bd I To configure the Sequest HT search engine On the Administration page click Sequest HT under Workflow Nodes in the Configuration section In the Automatic box specify whether you want the Proteome Discoverer application to automatically estimate the workload level The default is True which means that the application automatically estimates the workload level Optional If you set the Automatic parameter to False do the following a In the Number of Spectra Processed At Once box specify the maximum number of spectra that the Sequest HT search engine can process at once The minimum value is 1000 and there is no maximum The default is 3000 The larger the value the more memory is required b In the Number of Parallel Tasks box specify the number of search tasks that Sequest HT can perform at the same time The minimum value is 0 and there is no maximum The default is 0 If you set this parameter to 0 this search engine performs as many parallel tasks as the number of available CPUs can handle If you are using the Sequest HT search engine to search low resolution data set the XCorr confidence thresholds under the XCorr Confidence Thresholds low resol
498. ult methods when you initially set up a workflow and first access the Quantification Method Editor dialog box Click the Quan Channels tab of the Quantification Method Editor dialog box if it is not In the top list select the name of the method to use For reporter ion quantification you Note If you are installing the Proteome Discoverer application for the first time the TMT 6plex quantification method does not appear in the application The TMTe 6plex method replaces it iTRAQ 4plex iTRAQ 4plex Thermo Scientific Instruments iTRAQ 8plex iTRAQ 8plex Thermo Scientific Instruments TMT 2plex TMT 6plex iodo TMT 6plex TMTe 6plex TMT 10plex Proteome Discoverer User Guide 271 7 Quantification Setting Up the Quantification Method 272 Proteome Discoverer User Guide Figure 181 Quan Channels page of the Quantification Method Editor dialog box for reporter ion quantification Quan Channels Ratio Reporting Ratio Calculation Protein Quantification Experimental Bias Residue Modification _ iTRA 4plex 144 102 Da KY r N Terminal Modification iTRAQ4plex 144 102 Da v 115 Tag Name 114 116 117 Monoisotopic miz 114 1106798 Average m z 114 17347 Reporter lon Isotopic Distribution R Name 2 1 zj 42 Isotope Shift 4 2 Isotope Intensity 0 0 m m From the Residue Modification lis
499. ults To filter PSMs and peptides for site localization scores from phosphoRS 1 On the Peptides page of the MSF file right click and choose Enable Row Filters to turn on the row filters 2 In the phosphoRS Site Probabilities column of an MSF file containing results from a phosphoRS search click the down arrow icon xl The filters shown in Figure 129 appear Figure 129 Row filters in the PhosphoAS Site Probabilities column File Search Report Quantification Processing Workflow Editor Administration Tools Window Help iva aonje evon Z BB GB R SequetHT Mascot i A R fy 3 Q eBteavanre Administration gt WorkflowEditor x C18 2mgPBMC Sample A 200uLbeads 01 PhosPhoNode msf x Scale Peptides Search Input Result Filters Peptide Confidence Search Summary A3 Sequence PSMs Proteins Protein Groups Protein Group Accessions Modifications phosphoRS Site Probabilities Probability Charge T T T H 1 I TGGGGGAsGsDEDEVSVES 10 5 1 1P100797397 1 S8 Phospho S10 Phospho 0 0000 T 1 20 1 S 8 88 6 Min probability Ze 3 E 72 I SLEETLHTVDLssDDDLPHD 10 1 1 1P100005809 7 12 Phospho 13 Phosph 0 0000 S 1 0 1 T 5 0 1 T 8 Target acids 3 E al I7 LDDAHsLGsGAGEGYEPSSD 4 3 1 1P100016472 4 S6 Phospho S9 Phospho 0 0000 S 6 100 0 S 9 99 9 3 ic al 7 KVEEDLKADEPssEEsDLEDK 5 3 1 1PI00455527 3 12 Phospho 13 Phos
500. up or cluster together the proteins that are biologically meaningful Because not all genes are stored in the Entrez gene database some proteins do not have a valid 8 8 Pp gene identification In this case the value displayed in the Gene IDs column on the Proteins page of the results file is 0 UniProt Database Annotation From ProteinCenter you can retrieve information on known PTMs from the UniProt database and compare it with information on found PTMs For details on this feature refer to the Help Configuring the Proteome Discoverer Application for Protein Annotation Before you can start a search that includes protein annotation in the results or display ProteinCards for proteins you must configure the Proteome Discoverer application for protein annotation To configure the Proteome Discoverer application for protein annotation 1 Choose Administration gt Configuration or click the Edit Configuration icon The Administration page changes to the Configuration view 2 Under Workflow Nodes in the Configuration section of the left pane click Annotation if it is not already selected The Annotation view appears as shown in Figure 147 204 Proteome Discoverer User Guide Thermo Scientific Figure 147 Annotation view 6 Protein Annotation Configuring the Proteome Discoverer Application for Protein Annotation File Search Report Quantification Processing Workflow Editor Administration Tools Window Help
501. use these settings to specify which PSMs to consider for the inference of the protein groups For example if you set the Consider Only PSMs with Confidence at Least parameter to Medium the Proteome Discoverer application considers only PSMs with a medium or high identification confidence when it creates the protein groups and ignores PSMs with a low identification confidence You can further use the Consider Only PSMs with Delta Cn Better Than parameter to filter out PSMs over a normalized score and consider the remaining PSMs for inclusion in the protein group inference process if their confidence levels fit Note Setting the Consider Only PSMs with Confidence at Least parameter to Low and the Consider Only PSMs with Delta Cn Better Than parameter to 1 and leaving the Apply Strict Maximum Parsimony Principle option unselected creates the same protein groups as the previous release of the Proteome Discoverer application This first step prevents protein groups from including low scoring low confidence PSMs Even if the Proteome Discoverer application loads all PSMs initially identified by the search engines without applying further result filters it considers only those PSMs meeting the specified criteria when inferring protein groups If the set result filters filter out PSMs the application does not consider them for the protein grouping process even if they would otherwise fit the set grouping criteria In the second step the applica
502. ution data parameter The default values appear in Figure 6 Proteome Discoverer User Guide Thermo Scientific 2 Getting Started Configuring Search Engine Parameters Figure 6 Sequest HT configuration parameters 4 1 Work Load Level Automatic True Number of Spectra Processed At Once 3000 Number of Parallel Tasks 0 4 2 XCor Confidence Thresholds low resolution data 15 z 1 Medium Confidence XCorr 07 z 2 High Confidence XCor 2 z 2 Medium Confidence XCorr 09 z 3 High Confidence XCorr 25 z 3 Medium Confidence XCor 12 z gt 4 High Confidence XCor 3 z gt 4 Medium Confidence XCorr 15 4 3 XCor Confidence Thresholds high resolution data z 1 High Confidence XCorr 12 z 1 Medium Confidence XCorr 0 7 z 2 High Confidence XCorr 19 z 2 Medium Confidence XCorr 08 z 3 High Confidence XCorr 23 z 3 Medium Confidence XCorr 1 z gt 4 High Confidence XCorr 26 z gt 4 Medium Confidence XCorr 12 For information on these parameters refer to the Help 5 If you are using the Sequest HT search engine to search high resolution data set the XCorr confidence thresholds under the XCorr Confidence Thresholds high resolution data parameter The default values appear in Figure 6 6 If you changed any settings click Apply The message box shown in Figure 7 appears Figure 7 Administration message box l E Changes were saved Ae They will have no effect for currently open workflows 7 Click OK
503. utomatically displays the elemental composition theoretical mass delta values and ring and double bond RDB equivalents for your high resolution data RDB equivalents measure the number of unsaturated bonds in a compound and limit the calculated formulas to only those that make sense chemically You must have the Xcalibur data system installed to use the Qual Browser application For information about using the Qual Browser application refer to the Thermo Xcalibur Qualitative Analysis User Guide You must also have a search results file open and a specific peptide or search input row selected before the Qual Browser application becomes available If you are viewing the Administration page the Qual Browser application does not open a raw file To open the Qual Browser application 1 In the Proteome Discoverer application choose Tools gt Open QualBrowser or click the Qual Browser icon hia gt or press CTRL SHIFT B to open the Spectrum window Note You must have a search results MSF file open and selected before the Open QualBrowser command becomes available on the Tools menu In addition the Open QualBrowser command is available only when peptides are ungrouped and you select at least a single peptide or a search input item first You cannot use QualBrowser if the original raw file or files are missing The MSF file and the raw file must reside in the same directory The Qual Browser application opens as shown in Figure
504. ver Location To connect to a Mascot server refer to the How to Connect to a Mascot Server section of the Proteome Discoverer release notes included on every Proteome Discoverer installation DVD To test the connection between the Proteome Discoverer application and the Mascot server refer to Testing the Connection to the Mascot Server in the Proteome Discoverer Installation Guide To direct Proteome Discoverer to the Mascot server location 1 Open a Web browser and try to access the Mascot server through its URL If you cannot access the Mascot server it might not be running or the URL might not be correct In this case contact your system administrator to assist you 2 If you can obtain Web access to the Mascot server test to see if the ping command which is used to reach the sever is blocked Do the following e Open a command shell and type ping Mascot_server_name If the ping command is successful the output should resemble that shown in Figure 10 Thermo Scientific Proteome Discoverer User Guide 25 2 Getting Started Configuring Search Engine Parameters Figure 10 Output of a successful ping command ommand Prompt Microsoft Windows XP Uersion 5 1 26661 lt C gt Copyright 1985 2661 Microsoft Corp C Documents and Settings ussjo prodeval2 gt cd C WINDOWS system32 C WINDOWS system32 gt ping www matrixscience com Pinging www matrixscience com 194 42 244 117 with 32 bytes of data Reply from 19
505. verer application currently uses It is a suitable approach for enzymatic MS MS searches IMPORTANT Reversing the database is not suitable for peptide mass fingerprinting or no enzyme MS MS searches especially for dynamic modifications You might see mass shifts at each end of a peptide sequence that transform a genuine y series match into a false b series match or vice versa You can perform the decoy database search in two ways e Perform two separate searches one against the non decoy database and one against the decoy database Then count the number of matches from both searches to determine the FDRs This approach is the more conservative approach e Create a concatenated database from the non decoy and the decoy database and then perform the search against this concatenated database The difference between the two approaches becomes clear in the case where you find two significant matches for a given spectrum The first match is from the non decoy database and the second one is from the decoy database Because the Proteome Discoverer application considers only the top matches when calculating the FDRs finding two significant matches for a given spectrum is not considered a false positive in the concatenated database approach but it counts in the separate databases approach The latter case is considered the more conservative one and is the approach that the application currently uses To calculate the FDR the application counts
506. w Editor gt Start Workflow or click the Start Workflow icon Thermo Scientific Proteome Discoverer User Guide 257 7 Quantification Performing Reporter lon Quantification Demonstrating How to Create a Workflow for Reporter lon Quantification The following demonstration shows you how to set up a workflow for reporter ion quantification and how to specify the quantification method Click the button below to view the demonstration 258 Proteome Discoverer User Guide Thermo Scientific 7 Quantification Performing Peak Area Calculation Quantification Performing Peak Area Calculation Quantification If you want to determine the area for any quantified peptide you can use peak area calculation quantification You might want to use this quantification method to obtain an idea of the relative quantities of all peptides in a sample If the Proteome Discoverer application calculates peptide areas during processing it uses them to automatically calculate protein areas for the proteins in the MSF report It calculates the area of any given protein as the average of the three most abundant distinct peptides identified for the protein The peptides must have different sequences to be considered distinct Peptides with different charge states or modification variants of the same sequence are considered the same peptide If you apply result filters the application recalculates the protein areas To create a workflow for peak area ca
507. w Help BAH BB 8 SS TISBDOOSS amp BB GB KR sequethT Q Mso AA LUG A2SERBERRIARGE Administration x TAP Import 3 Remove Apply a iS Is Active Modification Abbreviation DeltaMass DeltaAverage Mass Substitution Leaving Group Position al Fre 4 Job Queue F l a Si Al al a 3 Add a Modification Content Management a ie 4 4 a M Acetyl Acetyl 42 010565 42 0367 H 2 C 2 0 Protein N preis Z Acetyl Acetyl 42 010565 42 0367 H 2 C 2 0 Any_N_Te amidated Amidated 0 984016 0 9848 HN O 1 Protein _C is FSA mans V Amidated Amidated 0 984016 0 9848 HN O 1 Any_C_Te T Spectral Libraries iv Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 NO Any M Carbamidomethyl Carbamidomethyl 57 021464 57 0513 H 3 C 2 N O Any_N_Te a Chemical Modifications iv Carbamyl Carbamyl 43 005814 43 0247 HCNO Any 2 Vv Carbamyl Carbamyl 43 005814 43 0247 HCNO Any_N_Te 3 Cleavage Reagents EIE A Carboxymethyl Carboxymethyl 58 005479 58 0361 H 2 C 2 0 2 Any_N_Te a E eee Vv Carboxymethyl Carboxymethyl 58 005479 58 0361 H 2 C 2 0 2 Any M Deamidated Deamidated 0 984016 0 9848 H 1 N 1 0 Any iv Deamidated Deamidated 0 984016 0 9848 H 1 N 1 0 Protein_N License Management te M Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Any R Licenses E M Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Any_N_Te M Dimethyl Dimethyl 28 0313 28 0532 H 4 C 2 Protein_N z M Dimethyl
508. w Offset 8Da 4 2 Charge Reduced Precursors Remove Charge Reduced True Mass Window Offset 4Da 4 3 Neutral Losses From Charge Reduced Precursors Remove Neutral Loss Pea True Mass Window Offset 4Da Remove Only Known Mas True ENN nee eenoe 130 Da X 4 4 FT Overtones Remove Precursor Overtoi True Mass Window Offset 0 5 Da Maximum Neutral Loss Mass The maximum mass of a known neutral losses to be considered Minimum value 0 0 Da Maximum value Peaks arising from overtones are rarely seen within Orbitrap spectra but are prominent peaks in spectra from the LT FT instruments The range in which neutral loss peaks from the charge reduced precursor peaks are removed is scaled by the charge of the charge reduced peak Therefore if you specify a value of 130 Da as in Figure 36 the Proteome Discoverer application removes neutral loss peaks within a 130 Da range for 1 peaks a 65 Da range for 2 peaks and so forth To remove neutral losses you can remove either every peak within the specified range or only those peaks from an internal table of known neutral loss masses from charge reduced precursor ions such as those shown in Table 2 Proteome Discoverer User Guide 59 2 Getting Started Starting a New Search by Using the Workflow Editor Table 2 Mass of known neutral losses from charge reduced precursor ions Mass Neutral loss 17 027 NH3 18 011 H20
509. w ewe Bw N 5 Neurnsvnovwe Rue bobBubo 5S amp NeownN oN UH eHNan Slelele lel lelet l lelel_lelelel_ aE 5 Thermo Scientific 1367 1709 Protein Group s 3398 3398 Protein s 9767 9767 Peptide s 11743 11743 PSM s 4766 4766 Search Input s Each aspect of the annotation biological processes cellular components and molecular functions is represented in a separate column Each box represents a GO Slim category which is a selected subset of the Gene Ontology annotations If the protein annotation is included in one of these subsets the corresponding box is highlighted by a color specific to this GO Slim category Figure 151 provides the column names and shows the meaning of the GO Slim category colors Proteome Discoverer User Guide 209 6 Protein Annotation Displaying the Annotated Protein Results Figure 151 GO Slim cate
510. w nore karen es 167 Proteome Discoverer User Guide v Contents vi Chapter 6 Proteome Discoverer User Guide Grouping Proteins 6 iia ee kia nee na EE ETE A eee eharede 174 Protein Grouping Algotthitis so s00 cai acedouw ster nanru 179 Proteins Containing Peptides with Sequences Not Belonging to a Master Protein sisu tiere Ka e E a E ce a E a 183 Protein Groups in the Status Bar uuueuaauaauuauuunrr uneneen 184 Proteins Grouped by the Grouping Algorithm in Previous Releases 184 Number of Unique Peptides Column on the Proteins Page 184 PSMs Identified by Multiple Workflow Nodes 0020000 184 Grouping Peptides ip ecreris cirein eens ie eens EEEE eee ts 185 Calculating False Discovery Rates o nis narwvkes hee eke eee eee eee 186 Target EDRs conta st hata eects ON ape eee Ce E ibe che 187 Peptide Confidence Indicate ats doe ncn ndehe at phate ends ee 188 Setting Up FDRs in Search Wizards and the Workflow Editor 189 Viewing the Results on the Peptide Confidence Page 194 Recalc lating the PORE oink uh oe ent hee eee epee eee 197 Changing the Target Rate and Filter Settings 000 197 Protein Annotation fii cncdarnediaten odniten wren kaei e 201 Protein Genter Moves be danad tt Nobu er ie Eves eed peeked ube ae 201 Gene Ontology GO Annotation auas assanar earned ene teed ean ees 202 Piim Annotation saisis vee laow ane ier aa ene daek
511. wing steps to ensure that the interface between the Proteome Discoverer Daemon application and the Xcalibur data system is optimal To prepare to run the Proteome Discoverer Daemon application from the Xcalibur data system 1 Before you start the Proteome Discoverer Daemon application install the Proteome Discoverer application on a remote computer to decouple data processing from data acquisition 78 Proteome Discoverer User Guide Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility Running the Proteome Discoverer Daemon Application from the Xcalibur Data System Thermo Scientific strongly recommends that you perform data analysis and data acquisition on two different computers to avoid disturbing the data acquisition by resource consuming data processing 2 Start the Proteome Discoverer application 3 Install the Proteome Discoverer Daemon application on the same computer that the Xcalibur data system is running on 4 In the Proteome Discoverer application prepare the workflow to be used by the Proteome Discoverer Daemon application as shown in Figure 52 Save this workflow Figure 52 Simple workflow used for the samples File Search Report Quantification Processing Workflow Editor Administration Tools Window Help OE aog er SOG OSH LORD smth Kut ALAM AZEBSTABAR Administration X Workflow Editor x 4b Workflow Nodes a EER Parameters q Data Input a Boeod on tanwhase G4 Show Advanced Parameter
512. x Ile6 Uses isoleucine 6 SILAC 3plex Arg6 Lys4 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 4 for medium labels Proteome Discoverer User Guide 337 SILAC 3plex Arg6 Lys6 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 6 for medium labels Dimethylation 3plex Chemically adds isotopically labeled dimethyl groups to the N terminus and to the g amino group of lysine 180 labeling Introduces 2 or 4 Da mass tags through the enzyme catalyzed exchange reaction of C terminal oxygen atoms with 18O Note Low mass accuracy cannot be used for precursor ion quantification or precursor ion area detection Check your tolerance window If you get too many results decrease the size of the window For too few results increase the size of the window Make sure you chose the right database Check the species listed to make sure the samples came from that species Verify that the activation type used is correct Verify that the instrument type in the Mascot search engine is correct Use only the ETD Spectrum Charger node for low mass resolution ETD data a B FASTA Reference This appendix lists the most important FASTA databases and parsing rules that the Proteome Discoverer application uses to obtain protein sequences accession numbers and descriptions Contents e FASTA Databases e Custom Database Support
513. x type the login name of the server The Configuration page now resembles Figure 45 Proteome Discoverer User Guide Thermo Scientific Thermo Scientific 3 Using the Proteome Discoverer Daemon Utility 4 Click Apply to activate the newly entered settings 5 To return to the previous settings click Reset Starting a Workflow R kod To start a workflow 1 Click the Start Jobs tab The Start Jobs page appears as shown in Figure 46 Starting a Workflow Figure 45 Configuration page of the Proteome Discoverer Daemon application Ef Discoverer Daemon pce foe faa Start Jobs Configuration Job Queue Server Host localhost X User leonardo davinci Aob Reset You can start a workflow for batch processing or MudPIT processing Proteome Discoverer User Guide n 3 Using the Proteome Discoverer Daemon Utility Starting a Workflow Figure 46 Start Jobs page of the Proteome Discoverer Daemon application Sq Discoverer Daemor Cole ee Start Jobs Configuration Job Queue Spectrum Files Workflow Batch processing MudPIT 2 J Load Files Export Parameter File Server Output Directory Add Remove Start local connection CollectionName Output Filename 2 Click the Load Files tab if it is not already selected 3 Click Add 4 In the Open dialog box locate the file folder containing your raw data select the spectrum r
514. xport in the Quantification Methods view by clicking in the leftmost column 3 Click ej Export 4 In the Export Quan Method dialog box select the name of the method file containing the quantification method to be exported and click Save Thermo Scientific Proteome Discoverer User Guide 291 7 Quantification Summarizing the Quantification Summarizing the Quantification The Quantification Summary page summarizes the settings that you chose for the Precursor Ions Quantifier node or the Reporter Ions Quantifier node in the parameters pane of the Workflow Editor It also shows the settings that you chose on the pages of the Quantification Method Editor for precursor ion and reporter ion quantification You must conduct a search with a workflow that includes a quantification node for this page to appear To display the Quantification Summary page e In an open MSF file click the Quantification Summary tab Figure 195 shows the Quantification Summary for precursor ion quantification and Figure 196 shows the Quantification Summary for reporter ion quantification Figure 195 Quantification Summary page for precursor ion quantification File Search Report Quantification Processing Workflow Editor Administration Tools Window Help BH BS BOT SGBHDOO BH amp BD WD RM SequethT g Mascot hk LHe RBEBTRAPARE __ Sequest SILAC Dimethylation Search 13 0 297 msf x 4p Proteins Peptides Search input Result Filt
515. y Workflow Editor x Data Input iu Spectrum Files E Spectrum amp Feature Retrieval A Event Detector J Spectrum Selector Spectrum Processing ig Noise Peak Filter i Non Fragment Filter iu Spectrum Grouper ix Spectrum Normalizer ig Top N Peaks Filter E Spectrum Filters Ti Scan Event Filter Ti Spectrum Confidence Filter Ti Spectrum Properties Filter Sequence Database Search KR Mascot W SEQUEST 0k Sequest HT E Spectral Library Search W MSPepSearch SpectrasT E PSM Validation i Fixed ValuePSM Validator Percolator lih Target Decoy PSM Validator PTM Analysis i phosphoRS Quantification Workflow Nodes a m Parameters l Show Advanced Parameters A Name ce Based on template Sequest Decoy Benchmark 1 3 Description OR20070924_S_mix7_06 RAW 18mix092106_hinf fasta E Merge results of equal search nodes Workflow Failures Error Information The specified value is not valid for this parameter SEQUEST Version 1 20 is not available It was replaced by Version 1 21 One of the identified items was in an invalid format Target Decoy PSM Validator Version 1 9 is not available It was replaced by Versi The processing node has no parameter with that name a Parameter File Name s Value CADocuments and Settings ussjo prodeval2 My Documents Smoke_Test_ Protein Database 18mix092106_hinf fasta UseDe
516. y particular precursor ion is observed This parameter limits the maximum retention time difference between scans to be considered for grouping In general if the precursor masses of spectra are within the tolerance and the maximum retention time window they are grouped into a single spectrum The default is 1 5 8 Click Next The Sequest HT Search Parameters page appears as shown in Figure 18 Proteome Discoverer User Guide 35 2 Getting Started Starting a New Search by Using the Search Wizards Figure 18 Sequest HT Search Parameters page Sequest HT Search Parameters Please select the parameters for the Sequest HT search General Search Parameters Decoy Database Search Database X V Search Against Decoy Database Enzyme Trypsin gt Full X Target FDR Strict 0 01 Missed Cleavages 2 Target FDR Relaxed 0 05 Search Tolerances E Use Average Precursor Mass Precursor Mass Tolerance 10 0 E Use Average Fragment Mass Fragment Mass Tolerance 06 lon Series Calculated a lons Factor 0 x lons Factor 0 b lons Factor 1 y lons Factor 1 c lons Factor 0 z lons Factor 0 9 Set the Sequest HT search parameters In the Database list in the General Search Parameters area select one of the FASTA In the Enzyme list select the enzyme used for digestion and indicate whether the The default enzyme is trypsin and the default cleavage is Full a databases that you registered b
517. y temporary FASTA files 1 Choose Administration gt Maintain FASTA file The Administration page appears with the FASTA files view shown in Figure 74 on page 102 2 Select the Display Temporary check box Display Temporary You now see any temporary FASTA files for example Figure 75 shows Temporary for two files in the Status column Thermo Scientific Proteome Discoverer User Guide 105 4 Searching for Data Using FASTA Databases Figure 75 Displaying temporary FASTA files i WP Add 3 O lt a Refresh BP Compact Display Temporary Name bovine fasta human_ref Fasta ecoli fasta ipi HUMAN v3 26 Fasta NCBInr_CElegans fasta bovine fasta 01 30 2009 TMT 10Proteinsf fasta MascotS_MaxQuant_Human File Size kB Sequences Residues Status Last Modified a Tas BIS valle aa 671 1058 242630 Available 02 17 2009 19240 33819 15162721 Available 03 19 2007 7785 4996 1571284 Available 03 22 2007 36756 67665 28462007 Available 02 21 2007 19177 29447 13184841 Available 11 09 2008 NJA 462 160685 Temporary 01 30 2009 3 10 3051 Available 04 22 2008 NJA 377 234558 Temporary 02 12 2010 Adding a Protein Sequence and Reference to a FASTA Database File You can add a protein sequence and a protein reference to a registered FASTA database file The protein sequence refers to the sequence of amino acids that constitute the protein and the protein reference refers to the name or reference of the protein kod
518. ying UniProt Annotation Data Displaying GO Protein Annotation Results Follow these procedures to display GO protein categories in the MSF file e To display the GO protein annotation results e To filter the identified proteins by GO Slim categories To display the GO protein annotation results 1 Open the generated MSF file by following the instructions in the Help 2 In the Column Chooser dialog box of the proteins page select the Molecular Function Cellular Component and Biological Processes columns For information on the Column Chooser dialog box refer to the Help The Proteome Discoverer application displays the results on the Proteins page of the MSF report as colored boxes similar to those shown in ProteinCenter Figure 150 gives an example If the application does not find the requested protein in ProteinCenter it displays a protein not found message in the annotation columns If the annotation retrieval failed because of issues with the Web request you see an error message in the annotation columns 208 Proteome Discoverer User Guide Thermo Scientific 6 Protein Annotation Displaying the Annotated Protein Results Figure 150 GO Slim category boxes for the protein groups shown in the results of an annotation search File Search Report Quantification Processing Workflow Editor Administration Tools Window Help LOE BE BOTSAO OTH LOAD K squetht Kmart ARAMA AGEBTEBARB Celegans_FT_6ITDDDT_01_01 msf_ann
519. ypsin Full Trypsin Full 5 PM 31 AM 886116 10 04 2012 11 50 AM 1278 10 04 2012 11 51 AM Click the plus sign to the left of a database name to vertically display the settings for that database as shown for the uniprot fasta database in Figure 88 Figure 88 Database settings in the FASTA Indexes view i dh Add 9 Remove Apply A Restore Refresh g Options Auto Remove Indexed FASTA File Index Size kB Last Access Time _ combined fa Trypsin Full 58310 10 03 Trypsin Full 9996 10 04 2012 11 31 AM uniprot_sprot_2011_05 fasta Trypsin Full 886116 10 04 2012 11 50 AM Name FASTA Database uniprot_sprot_2011_05 fasta Enzyme Trypsin Full i File Size kB 886116 Last Access Time 10 4 2012 11 50 45 AM i Max Missed Cleavage Sit 2 Precursor Mass Range Da 350 5000 i Use Average Precursor M False Auto Remove Indexed FASTA File Enzyme Index Size kB Last Access Time Iv bovine fasta Trypsin Full 1278 10 04 2012 11 51 AM 120 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Using FASTA Databases Specifying the Columns to Display Use the Column Chooser to specify the columns that you want to display To set the columns that you want to display Click the Column Chooser icon 1 In the Column Chooser dialog box shown in Figure 89 select the check boxes corresponding to the columns t
520. ys the processed spectrum library properties such as the file name file size the number of proteins stored and the library type which determines the search node to use The Proteome Discoverer application processes the spectrum library and makes it available for use Figure 95 Spectrum libraries view Add icon Remove icon File Search Report Report File Quantification Processing Workflow Editer Adminijtration Tools Window Help u BIO T G2ORD vualo yE 2B 7 g EQUEST Q Mascot AK LHR Ee ABZEBERRPARE Administration x 4 Wp Add E Remove Process Management Name File Size kB Spectra Type Last Modified ce Job Queue gt 2011_05_24 yeast_spectrasttargz 338702 78825 SpectraST 03 13 2012 a 2011_05_24 yeast_nist tar az 275519 996484 Nist 03 13 2012 Content Management rY a FASTA Files A FASTA Indexes Uy a Chemical Modifications 3 Cleavage Reagents a Quantification Methods License Management R Licenses amp Discoverer Daemon amp FASTA Indexes Processing progress of 2011_05 24 yeast_nistiaraz Ready 130 Proteome Discoverer User Guide Thermo Scientific 4 Searching for Data Searching Spectrum Libraries Spectrum Libraries View Parameters Table 4 describes the options and columns in the Spectrum Libraries view in the Proteome Discoverer application Table 4 Options and columns in the Spectrum Libraries view Parameter Description Activates the Select a Spect
521. ys4 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 4 for medium labels Proteome Discoverer User Guide 265 Thermo Scientific 7 Quantification Setting Up the Quantification Method e SILAC 3plex Arg6 Lys6 Arg10 Lys8 Uses arginine 10 and lysine 8 for heavy labels and arginine 6 and lysine 6 for medium labels e Dimethylation 3plex Chemically adds isotopically labeled dimethyl groups to the N terminus and to the amino group of lysine e 180 labeling Introduces 2 or 4 Da mass tags through the enzyme catalyzed exchange reaction of C terminal oxygen atoms with 18O For more information on these methods see Performing Precursor Ion Quantification on page 243 For reporter ion quantification you can choose from the following methods when you initially set up a workflow and first access the Quantification Method Editor dialog box Note If you are installing the Proteome Discoverer application for the first time the TMT 6plex quantification method is no longer available The TMTe 6plex method replaces it e iTRAQ 4plex e iTRAQ 4plex Thermo Scientific Instruments e iTRAQ 8plex e iTRAQ 8plex Thermo Scientific Instruments TMT 2plex e TMT 6plex e iodo TMT 6plex e TMTe 6plex e TMT 10plex The two methods labeled Thermo Scientific Instruments have purity corrections optimized for the way Thermo Scientific mass spectrometers process samp

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