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PEAKS 7 User Manual - Bioinformatics Solutions Inc.

1. 164 Configuration and Preferences PTM name Carbamidomethylation PTM abbreviation Mass Monoisotopic 57 021465 Residues that can be modified C Anywhere peptide protein N term C term H 3 2 N 1 O 1 Create a new PTM Click on the New button to display the New PTM dialog Provide the information about the PTM The required information about the PTM are listed below PTM name PTM abbreviation Mass Monoisotopic Residues that can be modified a peptide protein e PTM Name This name will appear in the PTM list for future use after it is saved e PTM abbreviation PTM expressed in shortened form e Mass Monoisotopic The mass that the residue gains or loses as a result of the PTM e Residues that can be modified Enter residues that can be modified anywhere or residues that can only be modified if they are at the N or C terminus e Formula The chemical formula of the PTM This should correspond to the mass listed above e Rule This field can be used to enter a comment about the PTM to be used for your own reference Click the OK button to save the changes The new PTM will now appear in the Customized PTM list where it can be accessed later To delete a customized PTM select the appropriate PTM from the list and click the Delete button Note For inform
2. File Tools Window Help BH EBX sBKXOOW 2 FS Project View Start Page X gt gt gt Click to open a sample project lt lt lt 4 2 PEAKS Main GUI The main graphical user interface GUI of PEAKS is divided into the following areas see screenshot below 1 The project view shows all the opened projects Each project may include multiple samples and each sample may include multiple fractions LC MS runs The analysis results are also displayed as result nodes under the project 2 The menu and toolbar Selecting a node project sample fraction or result in an opened project will highlight the analysis tool icons available to the selected node 3 A result node in a project can be opened by double clicking the node Opened result nodes are shown in tabs 4 Each opened result node provides several different views as different tabs In particular the summary view shows the result statistics The summary view is also the central place to filter and export the results 5 The information pane shows useful information such as the node properties and the progress of running tasks Overview A x eae Nindow Help m Ss j J gt 2 4 Zid id DHANA 4H 8 00Qy lt 21 act tie Mert Page Y PEAKS 5 2013 10 04 09 31 x 3 LS D test PedksStudio7Testing BSA 3 Enzyme y DENOVO 201
3. Ic 4 Filtering PEAKS Search Results PEAKS Search results can be filtered to keep only the high quality peptides proteins and de novo only peptides Filtering parameters can be specified using the control panel in the Summary View Click the Apply Filters button to filter the results that will be updated accordingly in the Summary Protein Peptide and LC MS views The filtering options in the control panel are described in the rest of this section StartPage X 3K SPIDER 8 2013 10 28 12 08 x PB z Peptides i0lgP 15 2 y Proteins 109P 0 land 0 unique peptides A Denovoonly ALC 50 land 10lgp 115 2 Apply Filters E Note Whenever a filtering parameter is changed the Apply Filters button will be highlighted in red as a reminder that the changes have NOT taken effect yet Peptides Peptide spectrum matches are filtered by peptide 10lgP score The peptide 10lgP threshold affects the results shown in Peptide and Protein views Only the peptides with PSMs above the 10lgP score threshold are listed in the Peptide View These filtered peptides are used as supporting peptides to infer protein identifications and therefore affect the proteins shown in Protein View If the Estimate FDR with decoy fusion option is selected in the PEAKS search parameters the peptide 10lgP score threshold can be easily set using the estimated FDR Clicking the FDR button shows an interactive FDR curve Moving
4. Controli_1 0 0 E f Q Pairwise RT alignment Control1_1 w vs Control2_1 y Controli _1 Control2_1 73 7 100 Treatmenti2_1 73 6 Treatment24_1 73 6 0 0 100 Control2_1 3 4 Protein View The Protein view lists all the quantified proteins present in the sample characterizes each protein at the amino acid level and lists the supporting peptide features of each protein It has three components e Protein table Lists all the quantified proteins present in the sample e Coverage Characterizes the protein sequence at the amino acid level All the PTMs and mutations occurred on the protein sequence will be displayed together with the MS MS data supporting the inference e Features Lists the peptide features identified from the selected protein in the protein table 5 Show top w proteins in each group v accession contains search Q O no results E a Accession Significance Y Coverage Peptides Unique PTM Sample Profile Group Profile Avg Mass Description Mark 2 Proteins Y a oO E Q72NH3IRL5S_LEPIC 8 20620 505 ribosomal protein L5 OS Leptospira interrogans ser p P61442 DNAK_LEPIC 112 39 Bill I 1 18 10 5 Chaperone protein DnaK OS Leptospira interrogans ser v ao g Q72V72 RL25_LEPIC 106 57 Mn Ma itt 50 z 7 d
5. 4 6 Conducting an Analysis To conduct an analysis 1 select a project sample or result node in the project view 2 Click the desired analysis tool button Here PEAKS Search is shown as an example PEAKS Search is a database search workflow for a complete identification analysis 10 Overview File Tools Window Help SAUB A axe S QW FY Project View D test PeaksStudio7Testing BSA 3 Enzyme fig DENOVO 4 2013 10 04 09 31 4 Wf PEAKS 5 2013 10 04 09 31 A PEAKS PTM 8 2013 10 04 09 31 SPIDER 9 2013 10 04 09 31 oa L Sample 1 e F1 BSA GluC 1 RAW DATA REFINE 1 2013 10 04 09 26 L Sample 2 Ely F2 BSALysC LRAW i DATA REFINE 2 2013 10 04 09 26 A ji 3 fun A search parameter pane will pop up Most search options for PEAKS DB are standard and straightforward More details are provided in the following see screenshot below 1 If the proteolysis enzyme was specified for each sample at the project creation step one can choose to use the enzyme specified in each sample This makes it possible to use multiple enzymes in a single project and a single search Specify the fixed PTMs and a few common variable PTMs expected in the sample Select a protein sequence database or copy and paste the protein sequences for the database search Conduct de novo sequencing using the same parameters or base the search on an existing de novo sequencing result node Estimate the false discov
6. 1 112 04 94 03 95 01 56 52 Q 17 03 2 2 241 08 223 07 224 06 121 04 E 2413 11 2395 10 2396 08 1207 05 20 E 0 0 mee eo nooo bh tre a 3 338 14 320 12 321 11 169 57 2284 06 2266 06 2267 05 1142 53 19 4 467 18 449 17 450 15 234 09 E 2187 01 2169 00 2169 99 1094 01 18 0 05 5 623 28 605 27 606 25 312 14 R 2057 95 2039 96 2040 94 1029 49 17 s00 1000 1500 2500 6 737 32 719 31 720 29 369 16 N 1901 87 1883 86 1884 84 951 43 16 Bl EN ie 7 866 36 848 35 849 34 433 68 E 1787 83 1769 82 1770 80 894 41 15 Ftp a ira rs Pipe aS x 8 1026 39 1008 38 1009 37 513 70 C 57 02 1658 78 1640 77 1641 76 829 89 14 eh eer ed i 9 1173 46 1155 45 1156 44 587 23 F 1498 75 1480 74 1481 73 749 88 13 zi i oe oe Log 10 1286 54 1268 54 1269 52 643 77 L 1351 68 1333 67 1334 66 676 34 12 Si po 11 1373 58 1355 57 1356 55 687 33 E 1238 60 1220 59 1221 57 619 80 11 Si 12 1510 64 1492 63 1493 61 755 82 H 1151 57 1133 56 1134 54 576 28 10 500 1000 1500 2000 2500 3 1 1638 77 1690 72 1671 71 219 A7 K 1014 SN 296 SN 997 4A S07 7A Z 3 5 De Novo Only View The De novo Only View displays the filtered de novo only sequences A de novo sequence is de novo only if it is from an MS MS spectrum that is not confidently assigned to any database peptide The tabl
7. 2000 1900 1600 1400 1200 1000 p 600 400 004 1000 eri oa oS Ex 2 D roy A Ft 0 E o i har i ie 1 AA i e i A i 3 _ _ H i H i i i i i i i E i i i i Ff umber of residues Peptide De Novo Sequencing ALC 0 410 25 50 75 90 95 98 99 907 999 959 sea local confidence ALC 3 a mumbar of residues 90 95 98 09 7 99 9 re AN e confidence il l T 40 25 50 75 90 95 9899 997 999 50 60 65 96 65 947 9949 na ave confidence W I l e none Li on d LN dq L um Gi j k iT p 2 Exporting De Novo Results 30D B00 e 700 500 500 400 E 300 Cc 200 100 LI 300 400 3010 200 100 idh ALC 50 10 25 5 90 95 98 gg 997 99 9 a la al confidence W0 25 50 75 90 95 990 99 997 999 95 DE 99 997 999 Pr e confidence ALC 75 dl a 90 95 99 99 99 7 99 9 aaa lt a confidence W The Export button in the title bar of the Summary View allows exporting of the filtered results into various formats It provides the opportunity to share the report in HTML and to also use the result as supplemental information in a publication Please refer to Section 3 Exporting De Novo Results for details 6 De Novo Sequencing using Spectral Pairs or Triplets PEAKS supports de novo sequencing using spectral pairs or triples
8. A unique peptide is a peptide with its 10lgP score above the peptide filtering threshold that can be mapped to only one protein group PTM Profile Table 4 The table shows modifications identified in the filtered result and the number of PSMs containing each modification 3 2 5 Experiment Control Figures 4 a and 4 b in the Summary page show the precursor mass errors of PSMs in filtered results Figure 4 a shows the distribution of the precursor mass errors in a histogram Figure 4 b shows precursor m z versus precursor mass error in a scatter plot The precursor mass error is calculated in ppm as 10 x precursor mass peptide mass peptide mass Figure 4 a and 4 b help examine whether or not the mass spectrometer is properly calibrated For a well calibrated instrument precursor mass errors should center at O ppm across the range of m z number of PSMs a 45 0 5 o amp 40 r 5 a 0 5 0 15 mass error ppm mass error ppm b miz Table 5 shows the number of identified peptides in each sample with the number of missed cleavages indicating the enzyme digestion efficiency Table 5 Number of peptides by number of missed cleavages Missed Cleavages 0 sample 1 78 sample 2 62 sample 3 107 3 3 Protein View 1 56 38 55 2 23 3 4 16 0 4 0 6 0 The Protein view lists protein identifications that have been filtered by the current settings in Summary view It also visualizes the protein seq
9. Add sample button Select the Instrument type Frag mentation method and Enzyme name that was used to generate the experimental data from the drop down lists To apply the same sample details to the whole project select the sample with the correct settings and click the Copy to whole project button Note Select Mixed as fragmentation mode if more than one fragmentation mode is used and the information about the fragmentation mode of each spectrum is encoded in the data file PEAKS loads the fragmentation mode for each spectrum during analysis If the data file does not provide such information these spectra will be ignored during analysis button to add data files to the corresponding sample Browse to the directory of the files select the files you wish to load and click Open To remove a sample or data file select the appropriate node sample or data file in the project structure and press button To delete the file entries from the list on the left select the files and click Remove To change the order of the samples within a project or data files within a sample use the Up and Down buttons Click the Finish button to exit the Project Wizard without specifying parameters for further analysis otherwise click the Data Refinement button to proceed to set up parameters for data refinement step See Section 3 Data Refinement Setting Parameters for more details on data refinement parame
10. Note Peptides identified by SPIDER may contain residue substitution insertion and deletion The pepXML for mat does not specify the encoding for substitution insertion and deletion and the mzIdentML format only specifies the encoding for substitution Therefore when exporting from a SPIDER result those peptides may not be correctly interpreted by third party software e For Skyline peptides pepXML In addition to exporting the peptide spectrum matches to pepXML format PEAKS will export the spectrum data in mzXML format The exported pepXML file and mzXML files can be loaded into Skyline for spectrum library generation 154 Exporting Data Reports and Printing 4 2 Exporting Images From the Peptide view and the De novo only view the Annotated Spectrum lon Match table Error Map or Spectrum Alignment can be exported to an image file To do so position the cursor on any of those items in the result panel right click and select the Export Image command from the menu Refer to Section 3 2 Exporting Images for details 4 2 1 Exporting Protein Coverage To export the protein coverage pane click the tools button of the coverage pane and select the Save image as command from the menu Select the resolution of the image and the location to save Click the Export button The PTM legends can be included in the exported image Scale 01 Ox ORK 8x include PTM legends F Save to D test Sky
11. matched to a database peptide Residues in a verifiable de novo sequence can be validated using the database peptide as a reference 2 De novo only sequences A de novo sequence is de novo only if the associated MS MS spectrum is not confidently matched to any database peptide De novo only sequences may suggest novel peptides peptides with unknown modifications or other interesting research subjects 91 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Note An MS MS spectrum is considered to be confidently matched to a database peptide if there are PSMs to the spectrum with peptide 10lgP score above the current peptide filtering threshold De novo only sequences are crucial for a complete proteomic analysis However it is often necessary to remove the low quality sequences De novo only peptides can be filtered by de novo ALC score which is the average local confidence score of residues in the de novo sequence Figures 3 a and 3 b provide a guideline for determining a proper ALC score threshold The two figures show the local confidence score distribution of residues in de novo sequences that are filtered by the current ALC score threshold Figure 3 a shows the score distribution of residues in verifiable de novo sequences These residues are validated by aligning the de novo sequence with the database peptide A residue is considered correct if it is consistent with the database peptide Oth
12. 162 Configuration and Preferences Built in Enzymes All of the built in enzymes within PEAKS are listed in the Enzyme List Click on one of these built in enzymes to view the information about that enzyme in the Enzyme Details panel Note A built in enzyme cannot be deleted or edited Create a New Enzyme Provide the name of the new enzyme in the Enzyme Name field and specify how the custom enzyme will cleave the protein between two amino acids to create peptides in the Enzyme Details panel The letter X denotes any amino acid at this position while amino acids within the and brackets indicate any amino acid except for the one in the brackets Choose where the cleavage sites are by selecting after or not after and before or not before to specify the range Add multiple amino acids to indicate that cleavage happens before or after any of the stated amino acids For example after RK means after R or K not after R and K Click the Add Update button to save the changes The new enzyme will now appear in the Enzyme List where it can be accessed later To delete a customized enzyme select the appropriate enzyme and click the Delete button Note For information on defining new enzymes on the fly for PEAKS de novo or PEAKS DB refer to Section 2 2 Enzyme 2 2 PTM Configuration From the Configuration window select PTM from the left side menu to change the PTM configuration 163
13. 17 03 21 N Peptide Spectrum Matches 2908 SS Peptide Sequences 1025 Pyro glu from Q 17 03 l N term Protein Groups FI Acetylation 42 01 10 N term Proteins 2 Ubiquitin 114 04 9 ST FDR Peptide Spectrum Matches 1 0 eee E Dihydroxy 31 99 8 KPW FDR Peptide Sequences 2 4 Acetylation 42 01 6 K FDR Protein 58 8 Carboxymethyl 58 01 5 N terr De Novo Only Spectra 165 ES ii Formylation 27 99 3 K_N term Methyl ester 14 02 4 DE C term Phosphorylation 79 97 4 SY Glu 129 04 E Pyro glu from E 18 01 4 N term Carboxylation 43 09 DK Most entries in these tables are self explanatory The following entries are explained e Peptide Sequences Table 3 This is the number of distinct peptides in the filtered result Peptides with the same primary sequence but different PTMs are counted separately However if several peptides differentiated only by leucine L and isoleucine I they are counted as one Since the same peptide may be identified from multiple spectra due to data redundancy and differing charge states this number is usually smaller than the number of Peptide Spec trum Matches e Protein Groups Table 3 PEAKS groups proteins identified by a common set of peptides This number shows the number of protein groups in the filtered result 94 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Proteins Unique Peptides Table 3 This entry counts the number of proteins identified with unique peptides
14. 31 99 Dihydroxy y e n v E 58 01 Carboxymet 5 JE 42 01 Acetylation 5 v E 18 01 Pyro glu fro 4 m i JB 43 99 Carboxylati 4 81 83 88 88 91 a 9859 101 110 114151617 125 140 144 147 155 Y Ei 27 99 Formylation 4 EE E a Be E a Ae E a d Jm 129 04 Monoglutamyl 4 21 ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK vB 155 12 4hydroxyn 3 _ e o z gt ME 37 95 Replacemen 3 3 3 1 Protein Table Each row in the table represents a group of proteins that are supported by a common set of peptides A dark blue node indicates that the group has multiple proteins To expand the group click the button at the left The Show drop down list located above the protein table specifies the way the proteins in each group are displayed all shows every protein in the group top shows the top proteins in the group determined by the number of high confidence supporting peptides first shows only the first of the top proteins in the group Note A supporting peptide is considered as of high confidence if it has a peptide 10lgP score above the filtering threshold The columns in the protein table are e Accession The accession number of the protein as in FASTA database e 10lgP The protein confidence score e Coverage The percentage of the protein sequence covered by supporting peptides The coverage is visualized
15. Each computer can only have one free trial Requesting a trial license repetitively will not extend the trial period automatically Please contact lt support bioinfor com gt to discuss a trial license extension 4 3 Use PEAKS as a viewer BSI has consolidated the PEAKS product line PEAKS Studio and Viewer are now one product Unlicensed PEAKS Studio can be used in the viewer mode Researchers around the world are now able to take advantage of PEAKS most advanced user interface to share PEAKS results In the viewer mode all non analytical features such as read display raw data open navigate through existing PEAKS results exporting etc work the same way as the full PEAKS Studio version 4 4 Activate PEAKS manually In certain situations when the computer does not have an Internet connection or is behind a firewall that blocks the activation the activation process requires the assistance of another computer with an Internet connection or outside the firewall Manual activation can be accessed via the link on the bottom right corner of the wizard Manual activation consists of the following steps 1 Provide the license key and user information required to the license wizard on the computer that will be running PEAKS The option I have already received the license file should be check when the license file from BSI is already at hand Click Next and go to step 5 16 Gather Information Save License Request File Upl
16. Mascot v2 4 X Tandem v2010 12 01 1 and OMSSA v2 1 8 A uniform FDR also known as inChorus FDR is estimated to assist the filtering of the combined result In this chapter it is assumed that you are already familiar with PEAKS database search tools Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER The usage of PEAKS inChorus is outlined in the overview For details of each step please refer to later sections in this chapter 1 th Select a project a sample or a fraction node Click the PEAKS inChorus button on the tool bar MY PEAKS Studio File Tools Window Help 2 In the inChorus parameter dialog there are drop down lists for each supported database search engine Using the drop down lists you can choose the search results to be combined You can either e Choose an existing result The list shows search results that already exist in the PEAKS project Simply choose an existing result from the list e Import a result from a file If the result by the search engine is exported to a file that file can imported by choosing Import from the list Specify the file and the searched database used from the pop up dialog e Launch a new search A new database search can be launched using the particular search engine by choosing New Run from the list Specify the search parameters in a pop up dialog similar to the search engine s native interface Please refer to the particular
17. Y ft BSI Products Online Registration Request file Brow No file selected Visual verification Input the characters shown below case sensitive 4 If the license request is sent successfully an automated BSI service will generate the license file 1icense 1cs and send an email from lt support bioinfor com gt to the email address provided to the License Wizard Either save the attached license file or copy the content between gt and lt in the email to the Windows clipboard 5 Transfer the license file to the computer running PEAKS and import the license file into the license wizard 18 Installation and Activation Gather O Paste the license Information Save License Request File Upload License Request File Import the license file the email attachment Import License Activation Complete 4 5 Re registering PEAKS Re registering PEAKS may be necessary when an additional software module was purchased or SPS was renewed BSI will modify the license information accordingly on the server side A new license file 1s required to make the changes effective Select About PEAKS from the Help menu The About BSI PEAKS Studio dialogue box will appear 19 Installation and Activation About BSI PEAKS Studio PEAKS Studio 7 0 build 20120921 Copyright 2000 2013 Bioinformatics Solutions Inc All rights reserved Bioinformatics Solutions Inc BSI acknowle
18. dialog will appear Converted Projects BampleProject_version7_0 Project Location D testiConvertedProjects Browse 07o Choose the converted project name and location Click Start to begin the conversion process A new version of the project will be created at the new location The old project will not be modified Note For PEAKS 5 x projects you need PEAKS 6 to convert these projects into PEAKS 6 projects first then use PEAKS 7 to convert the PEAKS 6 projects to PEAKS 7 projects Note The project converter is designed to convert PEAKS 6 projects so that they can be viewed in PEAKS 7 but further analysis on the converted projects are strongly discouraged 9 Changing the Default Project Location If many projects are to be created it is convenient to change the default project location to the directory where all the projects are stored Please make sure this folder is readable writable by PEAKS 1 Click from the toolbar The following Preference dialog pops up General General Display Options Default Input File Directory RMI Connections O Di best Browse Derby Database Default Project Folder Raw file convertor Di best Browse ABI wiff 2 Select General in the in the Preference dialog and click the Browse button below Default Project Folder to specify the default project location 42 Chapter 5 Data Visualization 1 Overview After the project is created the spectral data can be vi
19. tification section The Attach identification results dialog lists all available identification results Select the identi fication results to be attached and the FDR threshold to be used then click the Attach button All selected results will be listed in the Attach Identification section If multiple identification results for the same fraction are selected then only the first identification result in the list will be used Use the Move Up and Move Down buttons to set the order in which the identifications will be attached Results with FDR validation of PEAKS 4 uf PEAKS 6 of PEAKS 8 FOR Threshold 1 0 a Cancel 3 Understanding the LFQ Results Once completed a label free quantification result node will be added to the project tree Double click on this node to open the LFQ result that contains multiple tabs as described below e Summary Outline of PEAKS LFO search results with statistics This is the place to examine the overall perfor mance of the experiment and adjust filters LC MS The overall mass signal view for each MS run marked with detected features and MS MS spectra SeeSec tion 4 LC MS View for details on LC MS view Features The quantified peptide feature vectors listed in a table One feature vector is a list of features from different samples aligned together for one peptide Extracted ion chromatogram XIC 2D 3D LC MS view and retention time alignment are also displayed for each feat
20. 0 206 0 073 0 00 0 00 0 00 0 00 Y 2 H Q2PMQ9 PSBB_SOYBN 286 00 E I I BEB 24m 13 1 56035 Photosystem II CP47 chlorophyll apoprotein OS Gly 1 0 747 0 702 0 927 0 00 0 00 0 00 0 00 Y E O QOB655 ASR1_SOLLC 285 39 pr 30 19 19 13130 Abscisic stress ripening protein 10S Solanumlycop 1 0 921 4 277 3 579 0 00 0 22 18 14 13 27 Y P28460 RBL_VITAE 283 60 MMMM IIE I Ez 17 1 51600 bisphosphate carboxylase large chain Fra 0 0 0 o 0 00 0 00 0 00 0 00 Y E A7PRIG METK4_VITVI 278 72 SEM Ml 11 Mag 14 6 42931 5 adenosylmethionine synthase 4 OS Vitis vinifera 1 1 316 0 218 0 155 0 71 69 34 0 15 0 11 Y 3 P53492 ACT7_ARATH 270 96 MMM MMMM Mil 629 19 0 41736 _ Actin 7 OS Arabidopsis thaliana GN ACT7 PE 15V 0 0 0 o 0 00 0 00 0 00 0 00 Y gt P29685 ATPBM_HEVBR 265 41 MM MEN LINMINE 43 20 2 60259 ATP synthase subunit beta mitochondrial OS Heve 0 0 0 o 0 00 0 00 0 00 0 00 Y QO6GN4 PSsBB_PIPcE 264 92 E I I WEE I 23 13 1 56150 tosystem II CP47 chlorophyll apoprotein OS Pip 1 0 781 0 643 0 415 0 00 0 00 0 00 0 00 Y O P43643JEF1A_TOBAC 264 32 I EEIE MMM 29 17 0 49282 ation factor 1 alpha OS Nicotiana tabacum PE O 0 0 o 0 00 0 00 0 00 0 00 Y amp P24459 ATPAM_PHAVU 263 17 B IN I ILE HE M E 38 20 5 55345 ATP synthase subunit alpha mitochondrial OS Phas 1 1 031 0 685 0 693 0 00 0 6
21. 30 May 12 18 50 Show top proteins in each group ef PEAKS 5 30 May 12 18 50 a i 12 54 EET SPIDER 7 30 May 12 18 50 i E AL Trypsi El FLESA Trypsin 1 RAW dj DATA REFINE 1 30 May 12 18 50 GJ Lysc Ely F2 BSALysC LRAW i DATA REFINE 3 30 May 12 18 50 E Jk Gluc El F3 BSA GluC 1 RAW DATA REFINE 2 30 May 12 18 50 Accession El Proteins BN PO2769 ALBU BOVIN 473 04 0 P14639 ALBU_SHEEP _ PO8835 ALBU_PIG Peptide Protein Summary O P49064 ALBU_FELCA H A2v9Z4 ALBU_MACFA HlO P49822 ALBU_CANFA P35747 ALBU_HORSE i P49065 ALBU_RABIT O Q5XLE4ALBU_EQUAS De novo only Coverage Denovo Tags 2 Setting PEAKS Search Parameters After selecting a project node a sample node or a fraction node in Project View and clicking the PEAKS DB Search toolbar icon E the PEAKS DB parameters dialog will appear 85 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER pS SS Error Tolerance Precursor mass 15 0 ppm 7 using monoisotopic mass 7 Fragmention 0 05 Enzyme Specified by each sample Allow non specific deavage at one end of the peptide Maximum missed cdeavages per peptide 35 3 Carbamidomethylation Maximum allowed variable PTM per peptide 3 Database Select database Database Swiss Prot 5 Paste sequence Taxa Bos taurus cow De Novo Tag Options Available de novo tags de nove with current parameter General Options
22. C PO2829 HSP82_YEAST 0 protein selected uot olor fo show popup menu 3 2 LC MS View This view displays the distribution of LC MS signals features in the fractions SeeSection 4 LC MS View for details on LC MS view 3 3 Feature View The Feature view lists detailed information about all the quantified feature vectors It has four components e Feature Table Lists all the quantified feature vectors present in the samples 140 LC MS Summary Features Proteins 3 PEAKS Q Label Free Feature Details Provides detailed information about the selected feature vector Sample Features Displays detected features in each sample at the LC MS level RT Alignment Shows retention time alignment among the samples highlighting the RT range of the selected feature da 1 10000f 1002 vy Mm seg contains search Q noresults Peptide Quality Significance m z RT range z Avg Area Sample Profle Group Profle RT mean Accession PERA 812 4773 72 13 76 44 2 P61438 CH60_LEPIC A 2 ALEGDESEIGMPAILK 20 00 65 75 70 01 2 3 487 mm 67 55 Q9XD38 EFTU_LEPIN 3 LENTTLQMLGR 19 31 60 00 638 3441 33 13 38 87 2 1367 36 24 P61438 CH60_LEPIC 4 LLDQAEAGDNIGALLR 19 01 60 00 834 9579 67 87 72 91 2 3 367 a 70 17 Q9XD38 EFTU_LEPIN ILEVPVGPELLGR 17 33 60 00 696 4226 76 69 81 01 2 SES ma 78 35 Q72SY1 ATPA_LEPIC 6 VIAEGGSPAESVPILLGLTK 17 18
23. Combining Multiple Database Search Engines with PEAKS inChorus for details on inChorus parameters Refine Dat B inChorus OK Cancel 147 Chapter 15 Exporting Data Reports and Printing PEAKS offers a rich collection of exporting functions to allow users to create reports and share the analysis results with collaborators colleagues and clients The supported formats include HTML Comma Separated Values CSV pepXML mzIdentML and various image formats for image exporting Labs with in house software can easily make use of the CSV files in their own analysis workflow The exported results in HTML can be viewed with a web browser The entire exported result directory can be zipped and emailed to colleagues or the whole directory can be put directly on a website 1 Exporting Data Spectrum data can be exported to a number of file formats including mzXML DTA MGF and PKL To do so right click on the sample node or the data file that is to be exported and select the desired exporting format FN Project View hpi D test Peaks60 PeaksProjects test1 S Sample 1 E oe AN i DATA RE Data Refine MUERO De novo i fl PEAKS 10 o Peaks Database Search Spider Search inChorus Search Export DTA file Export PEL file Export MGF file Export MzxML file Export Result to Excel Export Result to Html Delete fraction s Selecting the Export DTA file command will open a dialog prompting for the folder name and
24. D MassLynx Converter massWolf exe Erowse Options centroid nolockspray 8 3 Agilent Data Agilent Q TOF data can be loaded provided that MassHunter software is installed on the same computer as PEAKS Agilent Ion Trap data can be loaded provided that CompassXtract is installed on the same computer as PEAKS The spectral data will be contained in the yep baf or fia file Select the p folder containing yep and baf file and the 1IFT folder containing fia file during the data loading step To load all fid files at once select the top level folder to load them CompassXtract 3 1 is readily available on the Bruker Daltonics web site You may need to contact your Bruker repre sentative to obtain CompassXtract 3 1 See Section 8 5 Bruker Data 37 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow 8 4 Applied Biosystems Sciex Data 8 4 1 QSTAR or QTRAP ABI data can be loaded into PEAKS provided the required converters are installed Analyst QS is required for QSTAR data Analyst 1 4 is required for QTRAP data and AB SCIEX MS Data Converter is required for ABI 5600 data PEAKS Configuration Wizard can download and install mzWiff automatically AB SCIEX MS Data Converter can convert MS data from any AB SCIEX instruments to mzML or MGF format that PEAKS can load readily AB SCIEX MS Data Converter can be downloaded from the following link Please see the specified software package to find out
25. ont 0 IIS E YT J 4 LC MS View The LC MS view shows the distribution of LC MS signals features as a heat map Placing the cursor on the heat map will show the m z value and retention time of that point 45 Data Visualization AA Navigation Operations 1144 Right Button Drag Select view range dd a boli Left Sutton Drag Move view range RT 25 55 Mouse Wheel Zoom miz and RT Locate m 2 AT Ci heel Change contrast Fealres Drag on Thumbnail Move mizand RT Mee on TIC Zoom RT FP MS MS Spectra Drag an TIC Move RT 1E8 ee an Axs Zoom m z or RT 1E7 IEF Drag an Axis Move m z or RT 1E6 1F4 The LC MS view provides various convenient ways to zoom and navigate the LC MS features in the data Move to a specific Heat Map area click on the heat map hold and drag the cursor until the desired area of the heat map is reached release the mouse button Zoom to a specific Heat Map area right click on the heat map hold and drag the cursor to select the desired area of the heat map release the mouse button Move to a specific Heat Map area from Thumbnail current view range of the heat map is shown as a rectangle in the Thumbnail Click on the thumbnail view range rectangle hold and drag the cursor until the desired area of the heat map is reached release the mouse button Jump to a specific Heat Map area provide approximate m z and RT values separated by a space in the Locate m z RT box
26. the requirements for it http www absciex com downloads software downloads 8 4 2 Converters for WIFF Three Wiff converters are supported by PEAKS AB SCIEX MS Data Converter mzWiff and MSX Note Before defining the converters in PEAKS please make sure these software packages are installed correctly on your computer and that they convert successfully from command line on your computer To set WIFF related preferences in PEAKS open the Preferences window by clicking the Preferences toolbar icon Po e or selecting Preferences from the Window menu to open the Preferences window Select ABI wiff from the Raw file convertor section on the left side to display the preferences for ABI instruments 38 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow ABI witf a AB SCIEX MS Data Converter Location D AB SCIEX MS Data Converter AB SCIEX MS_Converter exe Browse Options Centroid Profile E mzWIFF mzWiff Location Browse Options Survey Spectrum Centroiding E Product Spectrum Centroiding Product Spectrum Deisotope Precursor Charge Determine 5 MSX converter Location D MSX msx exe Browse Options ABI raw filed may contain serveral samples do you want to merge all the samples into one data set 0 Yes No 7 Survey Spectrum Centroid W Product Spectrum Centroid Select one of the converters as the default
27. 2 Spectrum Annotation The spectrum annotation displays a graphical representation of the peptide spectrum match Sequence LLKHKPKATE Tag Length 10 ALC 99 ppm 3 6 cerco YR AT Y7 810 45 50 Y5 545 29 ve 673 39 va b4 H20 y 938 54 474 32 2H20 Y2 H20 a 792 44 _ be 1017 65 231 12 va oi as 016 61 448 24 miz 00 400 600 oO 1000 1200 Ma yli tet 2c 2Y ErrTol 0 05Da Y preprocess Y low intens label The title bar shows the information of the peptide sequence that is being displayed in the spectrum Press the All candidates button to open a pop up window that displays all alternative peptides Click on a peptide sequence in the pop up window to select and display the annotation Sequence LLKHKPKATE Tag Length 10 ALC 99 ppm 3 6 ety i fi Tag mk ALC 5 ppm Mode meme 8 o OS MEN E AE EO 50 LKLKPKATE OO o o 62 36 OC ue A as a Ya 938 54 Y2 H20 ba 1017 65 231 12 016 61 miz 00 400 600 oO 1000 12700 Sly 131 2x 2 ErrTol 0 05Da Y preprocess Y low intens label Moving the mouse cursor over the peptide sequence in the spectrum will show the mass transitions for a particular amino acid residue 67 Peptide De Novo Sequencing Moving the mouse cursor over a peak in the spectrum will show the annotation and the peak s m z ratio and relative intensity The m z ratio and the relative intensity can also be found on the right bottom corner of the spectrum annotation p
28. 3 637 3579 91 59 12840 14 Q07955 SRSF1_HUMAN z cdd 2 a GGGGNFGPGPGSNF 4 03 R 6 02 50 96 1386 6674 9 3 694 3474 48 16 6103 1 P22626 ROA2_HUMAN 5 a x de O 9 SHEGETAY 4 03 IR 5 02 50 49 1171 5867 7 7 586 7961 24 40 2433 3 Q07955 SRSF 1_HUMAN 1 1 074 0 123 BE 10 DGYDYDGY 4 03 R 6 02 48 23 1132 4706 3 6 567 2405 44 78 5544 3 QO7955ISRSF1_HuMaN de 3 4 LC MS View The LC MS view shows the distribution of LC MS signals features and identified peptides in an interactive LC MS heat map Check the option Identified peptides to show the locations of the MS MS spectra with peptide identifica tions as indicated by blue squares Refer to Section 4 LC MS View for a list of supported interactions in the LC MS heat map 3 5 Filtering Quantification Results Quantification results can be filtered based on the number of fold changes between samples To do this use the with fold change drop down list in the title bar of the Summary view panel To change the reference sample select 121 PEAKS Q MS Level the appropriate sample eg ratio to light from the Show drop down list To change the normalization factor of the protein ratio select auto manual or no from the Normalization factor drop down list For manual normalization enter the normalization factors in the textbox to the right Click the Apply Filters button to fi
29. 435 7363 24 38 869 4607 3 0 1 99 38 EHME 29 06 0 90 21 06 7 1E 2 E 10 NPTAFKK 142 403 2322 17 77 804 4494 0 5 1 99 25 20 29 0 91 35 37 11 AALEAARDSK 405 516 2780 22 17 1030 5408 0 6 2 99 16 20 95 0 83 32 96 12 VISQDTQPHQQK 557 704 8610 24 33 1407 7107 2 3 1 97 93 P 34 71 Individual Engine s Score In the Peptide View each engine s own score is displayed A dash symbol indicates that the peptide is not found by the engine FDR curves The FDR curves for all selected search engines are placed together in a single figure in the Summary View The black marks on the search engines FDR curves indicate the score thresholds of the corresponding engines The red mark indicates the inChorus FDR threshold FDR 0 500 1000 1500 2000 2500 3000 3500 4000 number of peptide spectrum matches PEAKS Mascot OMSSA Sequest Inchorus Engine Threshold 115 Combining Multiple Database Search Engines with PEAKS inChorus Note Since there are overlaps in the peptides identified by different engines the FDR threshold for the inChorus result is higher than the respective FDR threshold for each individual engine Venn Diagram A Venn diagram shows the number of identified PSMs by the three engines that identified the most PSMs under the current FDR threshold setting PEAKS Mascot 595 0 lt 0 1 1612 18 109 10 11 a
30. 59036 atin type II cytoskeletal 75 OS Bos taurus GN KRT75 Y i Q29521 K2C7_BOVIN 100 69 I IIl 6 3 2 Eh 51578 atin type II cytoskeletal 7 OS Bos taurus GN KRT7P Y e A2TIN1ISPA35 ROVIN 39 65 I i 3 3 Lam 46397 in A3 5 PE 3 SV 1 fw i Coverage Peptides Denovo Only Tags gt sp P02769 ALBU_BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 va 2 O outline 0 coverage 63 76 a CS 3 25 q 3 235 237 41 58 0839 73 7577 80 E 30 m Epa line El ii e n B E E am E Ecu E Show de novo only sequences 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD F d v de novo tags sharing 5 AAs a _ O SUS IS Y de novo peptides fully matched A B Show necator SS Y minimal ion intensity 5 2 Y AM P rs 57 02 Carbamidom 1584 Y 57 02 Carbamidom 581 7 mu Y 8l 0 98 Deamidation 88 H ma vd 18 01 Dehydration 56 4 E 48 00 Dethiomethyl 30 E v 6 43 01 Carbamylation 29 es v B 21 98 Sodium adduct 21 JB 17 03 Ammoniaos 21 B 15 99 Oxidation M 19 sll 718 17 03 Pyro glu fro 14 E Y 11 114 04 Ubiquitin 9 j ja Y 162 05 Hexose 9 eee B 410
31. A E E 125 33 Understandine the Results ir ii ie R 124 Sl SUMMA VION SA ES IAE A RI 124 Dies OL MAS 125 IPEPE VI Wi o ft 126 ithe WC AVI SV ee Wo ii o io deena 126 30 Piero Ouantiricat on ESSE NAAA A AS RI 126 a EXporu o Quantification Resulta si A e 127 IS PEAKS O Babe Pree ii A AA aa 128 PONEN ea ide eos dee os Mis io ce o eee eer eee 128 PEAKS 7 User Manual ES A O A A AONE 128 3 Wnderstandine the LEO RESUS aussi is 130 SS VION AA AS O A Suances ced Nl ronan kes Meade ated ices 131 Sol Result IE OM PARAR OOO AO on TA 131 5 lee SUIMIMALY REPO abia 135 331 3 Exporting Q uantificanom Results ad ts 139 SNA Adit Ona PUNCUONS casnar A A AS ai 139 De CINES NTE E Aaa 140 MECA VIEW A ete Mehta ohonlm A 140 Dele Cale A canola tena sl 141 1 CAMPS STAINS a SA AE AS RN Manette nacelle 142 O E EAS sanas 142 VEK IR E I eei E A 143 E a E a E R E sees A A T E O E came AT 143 Sl Pre Table A A A a as 143 SAR DO A O O O ean cen etue ala ET 144 SAO IS A O 144 AM rios 145 1 TARA Cation INCOLORO AA RAS Ri 145 2 Oane aon W OLK POW isla dd aga liawineh 146 AE NOUS WOW asd waco ieanltnes A tied iwendiaiine 147 15 Exportine Data Reports and Ponts dd cia 148 EXPO Dati A TA AA Tare nt 148 2 EXPONE Result Prom Project VIEW 149 3 EXportino De NOyo Results ua ib 149 Sol BX porn summary and Peptides asii oa 149 SL ER DORMS A TES A A N an 150 4 Exportine Database Search Results sad 152 4 1 Exporting Summary Proteins
32. Configuration and Preferences Name 7 Mono mass Residue site 4 hydroxynonenal HNE 156 1150 CHK A ao OM Acetylation N term Acetylation Protein N term Amidation Ammoniadoss C N term C N Ammoniatoss Protein N term TS N Beta me thyithiolaton Biotinylation Carbamidomethylation 57 0215 C Carbamylation 43 0058 Carboxymethyl Citrullination Deamidation NQ Dehydration Dimethylation Dioxidation M FAD Farnesylation Formylation Formylation Protein Niem 2798 l Geranyl geranyl 272 25 MT Guanidination x Built in PTMs The built in standard PTMs within PEAKS are listed in three separate PTM lists under Common Uncommon and Artificial tabs The Common list contains the most commonly used PTMs the Uncommon list contains less frequently used PTMs and the Artificial list contains the PTMs that can be induced only artificially The most recently used PTMs are listed in the Recent tab and the Customized tab lists all the user defined PTMs Double clicking on any of these PTMs will display the PTM details in the PTM Info popup dialog The same information can be viewed by selecting a PTM from a list and clicking the View button For details on PTMs refer to Section 2 3 PTM
33. DB PEAKS PTM and SPIDER e M Mascot S SEQUEST X X Tandem O OMSSA Also the following appearances of the icon indicates e Blue background The engine identified the peptide with high confidence above the engine s own score threshold See Section 3 Filtering PEAKS inChorus Results e White background The engine identified the peptide with low confidence below the engine s own score threshold e Dash The engine did not identify the peptide gt 1 280f28 y gt w scan seach QUIM noresul E 3 Peptide Scan m z RT Mass ppm Spec Score PEAKS SequestE Mascotil XiTandemEd Omssafe PTM c IN AVARAYN 98 TTK 533 365 8648 24 04 1094 5720 0 6 2 99 96 BET E 41 02 2 73 40 92 7 31E 5 a 2 2 ASEALKPDSQK 194 391 8754 18 59 1172 6038 0 5 8 99 95 HERK E 56 42 2 43 39 99 5E 4 a 3 SEVAHRFK 414 487 2649 22 28 972 5142 A 2 99 92 HSR 45 42 1 33 42 05 v 4 LVAETEDRK 465 354 1932 23 07 1059 5559 1 7 3 99 92 aSK E 28 04 2 27 45 01 1 01E 2 a 5 QGVTEAAEK 167 466 7378 18 14 931 4611 0 1 3 99 92 asii E 27 83 1 11 40 80 5 2E 3 a 6 IGYGSN 98 KK 220 434 2328 18 96 866 4498 1 4 2 99 91 g i 44 77 37 32 d 7 Q 17 03 AKHEEIDTK 568 394 5337 24 46 1180 5725 5 7 1 99 66 iJ ae 40 05 24 58 P 8 EAHEIVSK 17 456 7428 15 30 911 4713 0 2 1 99 54 iJ mwe 24 01 30 76 3 9 PGGGOVEVK 561
34. Data Refinement gt Finish Cancel For spectral pairs and triplets PEAKS de novo sequencing algorithm analyzes each individual spectrum and then constructs a better sequence with a higher ALC score In the Peptide Table a de novo sequencing result of a spectral pair or triplet is presented in a single row When viewing the sequence PEAKS shows all spectra annotated by the sequence Clicking the All candidates button will show the other de novo sequences generated for the individual spectrum 74 Start Page x la DENOVO 2 2013 10 17 16 30 Peptide De Novo Sequencing qe 1 1000 of 1035 gt ab 0 sam ss QA morents E Scan Peptide Tag mies ALC db a RT Mass pom PTM Mode 54 23542355 VTTTLSK 375 2240 33 26 MB oe D7 ETDHCO 95 29682369 AELHOR 6 st zz 6842 Z J sa o ETD HCO g 2 2370 2371 VELAMHK ETH I a7 2372 2373 LESSHR 2 sE ETD HCD g 23772378 REQNGM 15 99 C 57 02 3 125 EM ETD HCD g 9 23812382 LEYEN 98 ELNeR 3 1307 6357 aa E ETDHCD 100 2400 2401 SKAQ 98 AESLYQSK E 447 5618 3 33 90 1339 6619 13 E ETD HCD 301 24062407 S5c 57 02 C 57 02 C 45 6 01 1414 2 800 2615 ETH 102 2418 2419 VOWSPSLGKHSEK 12 418 5672 3 1252 6775 0 ETD HOD 103 2421 2422 LTYM 15 99 51KYPGX n 439 5637 3 34 ae 11 7 o ETO HCD 104 2426 2427 LGKEPSR 393 7257 2 34 25 EDHO gt Sequence WTTTLSK Tag Lengi
35. Estimate FDR with decoy fusion Find unspecified PTMs and common mutations with PEAKS PTM Advanced Setting Find more mutations with SPIDER Error Tolerance Precursor mass Precursor mass tolerance PEAKS Search identifies peptides within the precur sor mass tolerance Precursor mass tolerance can be specified in either Daltons or ppm Fragment ion Fragment ion m z tolerance PEAKS Search uses this value when scoring peptide spectrum matches PEAKS considers that a fragment ion is matched if the calculated m z is within the tolerance Error tolerance parameters need to be set consistently with the mass accuracy of the instrument The default parameters for the instrument generating the data can be loaded by choosing default in Predefined parameters Note When the samples in a project are generated by different instruments and default 1s chosen from the Pre defined parameters drop down list the default parameters of the first sample are loaded However it is rec ommended to set the tolerance parameters according to the least accurate instrument 86 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Enzyme PEAKS search digests the searched protein database in silico to generate peptide candidates It is necessary to specify the enzyme for protein digestion whether or not non specific cleavages are allowed and the number of missed cleavage sites allowed in a peptide Enzyme Selection
36. RT and precursor charge state information in a pop up window Click an MS MS spectra indicator to view the spectrum in a pop up window The indicator for that selected spectra will turn into a small red square 48 Data Visualization A 21 0 Intensity 9 100 54 0 m z 656 3992 30 RT 49 37 z 2 1000 m Sa my 1 1 2x 2Y ErrTol low intens label oa i jik mi h o boo 660 boo 6 0 4 6 Intensity View In addition to the heat map view PEAKS also provides a 3D intensity view for visual inspection of the LC MS data To switch between the heat map view and the intensity view click the A button in the top right LC MS control options The intensity view has the same set of controls to zoom and navigate easily 49 Data Visualization RT 3 E TFN Te e f ae y Pane 4 7 Export LC MS 900 miz Click 2 button on the LC MS control options to export the LC MS heat map or intensity view in PNG format m Ox OA D testiicms png 50 Chapter 6 Adding a Sequence Database 1 Configuring Sequence Databases PEAKS has the ability to search through a database to identify proteins using the PEAKS DB function In order to use this function PEAKS must have access to a protein or EST database in FASTA format the standard format for popular public sequence databases PEAKS can be configured to use existing databases on the system or download them from public servers Additionally taxon
37. Sample 505 ribosomal protein L25 OS Leptospira interrogans se v F QD4PT7IRS10_LEPBJ 103 26 Ea E HE 30 3 3 Control 1 1 00 305 ribosomal protein 510 OS Leptospira borgpeterseni Y Q9XD19 RS5_LEPIN 102 88 E 8 40 5 5 Control 12 0 96 305 ribosomal protein S5 OS Leptospira interrogans GN v a 5 P61437 CH10_LEPIN 102 65 E E ME 30 3 3 Control2_1 0 82 10 kDa chaperonin OS Leptospira interrogans GN groS V E P52277 RECA_LEPIN 99 27 El E EIn I 8 31 10 8 Control2_2 otein RecA OS Leptospira interrogans GN recA PE v Q72VM5 EFG_LEPIC 96 03 A l I M IM 24 11 11 cd ation factor G OS Leptospira interrogans serogro W Q72UA5 RS7_LEPIC 95 56 E 32 5 5 el 305 ribosomal protein S7 OS Leptospira interrogans ser Y y t 1 Treatment24_1 z Coverage Features Treatment24_2 gt sp Q72NH3 RL5_LEPIC 505 ribosomal protein L5 OS Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni GN rplE PE 3 SV 1 E a oe outline 8 coverage 1 MAARLRTKYK KEIVPELNKK FKFSSIMQVP RLEKIVLNVG MGEAHTNPKA LEAAVEELAL ITGORPVKTK AKKSIAGFKI 80 4 Asperline 10AA gap p P q _ _ x _ x orRrrroo Show de novo only sequences 81 REGMSLGCMV TLRGDYMYEF LDRILVNVALP RVRDFKGVSE KGFDGRGNYN MSIKEQIIFP EIKVDKINTL YGINMTEVIN RANDE _ _ da Y de novo peptides fully matched 161 SKSNEEAYSL LAAFGMPYRN OK ee Show modifications minimum ion intensity 551 3 4 1 Protein Ta
38. Select the enzyme used for protein digestion from a drop down list If the enzyme used for protein digestion is not in the list a new enzyme can be added Please refer to Section 2 2 Enzyme for further details Non specific Cleavages Specify whether or not non specific cleavages are allowed at a peptide terminus If None is selected the specificity of the selected enzyme is strictly enforced at both termini of a peptide One allows one terminus to disobey the enzyme specificity Both allows both peptide termini to disobey the enzyme specificity Missed Cleavages Specify the number of missed cleavage sites allowed in a peptide Note When the selected data node is a project of multiple samples Use Sample Enzyme allows samples to be analyzed separately using their respective enzyme specified during project creation Note None is a special enzyme allowing non specific cleavage at both ends of the peptide The None enzyme can cut at every residue generating peptides with length up to 65 amino acids Its usage 1s recommended when the digestion enzyme exhibits a high degree of non specificity PTM To select PTMs for PEAKS DB search click the Set PTM button to open the PTM Options dialog PEAKS DB search supports a limited number of variable PTMs Refer to Section 2 3 PTM for details Note To prevent long search times select only the most frequent PTMs in the sample for PEAKS DB and check the PEAKS PTM opt
39. Server Client and Worker will appear The port numbers can be changed if conflicts arise Contact technical support at BSI lt support bioinfor com gt for more information 1 1 3 Derby Database Click Derby Database from the menu on the left side to show the derby database preferences on the right side 158 Configuration and Preferences Derby Host localhost Port 15270 Derby Server StartMemory 512 Derby Jar Location fib Derby Host The name of the Derby Host as well as the Port number will come up by default The port number can be changed Derby Server Start Memory The amount of memory used to start the derby server will also come up by default but can be changed if more memory is available however it is not recommended to change this from the default setting To increase performance use the performance configuration utility see Section 5 PEAKS Performance Configuration Derby Jar Location The Derby Jar Location panel will list the location of the derby jar file by default This is displayed to find its location This location cannot be changed 1 2 Raw File Converter Preferences This section allows users to change the preferences for the raw file converters of the following instruments AB SCIEX Bruker Shimadzu Varian and Waters 1 2 1 ABI wiff Click the ABI SCIEX wiff option under the Raw file converter section from the menu on the left side to show th
40. and hit the Enter key to zoom in to an area centered at the specified m z and RT value Zoom in out from Thumbnail place the cursor on Thumbnail scroll the mouse wheel button Zoom in out smoothly place the mouse pointer at a particular m z value RT position scroll the mouse wheel button Move to a specific RT range click the mouse cursor on TIC or RT axis hold and drag the cursor until the desired range 1s reached 46 Data Visualization e Zoom to a specific RT range place the mouse cursor on TIC or RT axis scroll the mouse wheel button e Move to a specific m z range when m z axis is zoomed in click the mouse cursor on m z axis hold and drag the cursor until the desired range is reached e Zoom to a specific m z range place the mouse cursor on m z axis scroll the mouse wheel button e Change contrast of the Heat Map place the mouse pointer on the heat map press and hold Ctrl key and scroll the mouse wheel button e Change peak height in the 3D intensity mode place the mouse pointer on the heat map press and hold Ctrlkey and scroll the mouse wheel button e View the whole Heat Map click the 1 1 button in the control e Move Back Forward to previously specified Heat Map area use the Back and Forward buttons in the control to navigate the previously specified heat map areas The Heat Map view offers various controls to study the LC MS data features The following sections describe the control options and the
41. by a color bar Light blue blocks indicate the parts of the sequence covered by low confidence peptides Dark blue blocks indicate the parts covered by high confidence peptides e Peptides The number of high confidence supporting peptides e Unique The number of high confidence supporting peptides that are uniquely mapped to the protein group Unique peptides with same sequence but different modifications are only counted once in this number e PTM The identified modifications displayed with color coded icons e Avg Mass Protein mass calculated using average mass 96 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER e Description Protein s header information as in FASTA database e Mark Allows the selection of specific proteins The marked proteins are exported when using the exporting func tions in the summary view Note For Peptides and Unique two peptides with the same starting and ending positions in the protein are count ed as one regardless of their PTM forms This is to follow the MCP Molecular Cellular Proteomics guidelines 3 3 2 Protein Coverage The protein coverage visualizes the mapping of supporting peptides and de novo tags to the protein selected in the protein table The coverage view also shows all identified sites with modifications or mutations to assist with protein characterization at the amino acid level The coverage view has three major components e Prote
42. corrected to e Filter Scans Precursor Mass Range The precursor mass region to select scans for further analysis Retention Time Range The retention time region in minutes to select scans for further analysis Quality Threshold The spectrum quality threshold to select scans for further analysis The recommended value is 0 65 This is a percentage 56 Data Refinement Note Data pre processing centroiding deisotope deconvolution is performed on all the data sets Once all parameters are set press the OK button to initiate the data refinement process 2 1 Saving the Parameters for Future Use After setting up the desired parameters you can save them for future use Click the drop down list at the top right of the window select Save as and define a name for these preferences for future use reference when prompted Any parameters that are saved will be available in the drop down list at the top of the window To examine the contents select a saved parameters file and the parameter values will be automatically displayed 57 Chapter 8 Peptide De Novo Sequencing 1 Overview De novo sequencing derives the peptide sequence from the tandem mass spectrum without the need of a sequence database It is the preferred method for identifying novel peptides and the study of unsequenced organisms PEAKS provides a reliable and comprehensive solution for automated peptide de novo sequencing It features Accurate de novo sequenc
43. g F1 441 LTKVHKE 7 39 427 7569 2 10 41 853 5021 3 4 CID oC F1 574 LLKHKPKATEE 11 ES 647 3784 2 12 95 1292 7451 2 3 CID F1 2142 VSVLLRLAKE 10 za 564 3594 2 37 80 1126 7073 2 7 CID F1 1451 KKFWGKYLYE 10 z3 681 3644 2 27 54 1360 7180 2 7 CID F1 391 KVTKC 57 02 C 57 02 TE 5 sa 513 2394 2 9 45 1024 4681 3 8 cc CID F3 2456 MPC 57 02 TEDYLSLLLNR 14 sa 862 9199 2 42 42 1723 8274 1 2 e CID F3 1707 KQTALVELLK 10 s3 571 8597 2 30 59 141 7070 1 8 CID Sequence LLKHKPKATE Tag Length 10 ALC 99 pom 3 6 Al candidates pa pa pa pa p p p d mtensty L L K H K p K A T E N 100 810 45 50 6 673 39 ya 938 54 Y7 H20 ba b7 bs 1017 6 845 57 916 61 miz 100 200 300 400 500 600 700 800 900 1000 1100 1200 5a0 Sas miz Aa dl 1 2X 2Y ErrTol 0 05Da Y preprocess V low intens label Fraction D Vyang_deta Xisowen Liu 6SA GluC LRAW fon Match Survey Fragmentation Mode CID CAD y and b ions ric 1 766 b bH20 bNH3 b Seg y y H20 y NH3 y 24 n 1 114 09 96 08 97 06 57 55 10 2 227 17 209 17 210 15 114 09 L 1051 63 1033 61 1034 60 526 34 9 0 aa aage E 44 2 s n ape Y 3 355 27 337 26 338 26 178 14 K 933 54 920 53 921 53 469 77 8 m a E 4 492 33 474 32 475 30 245 66 H 810 45 792 44 793 42 405 72 7 0 05 T 0 06 5 620 42 602 41 603 40 310 71 K 673 39 655 33 656 36 337 20 6 200 400 600 300 1000 1200 miz 6 717 48 699 47 700 45 359 24 545 29 527 23 5
44. in blue and C terminal ions are shown in red A fragment ion is found when there is a 68 Peptide De Novo Sequencing matching peak within the mass error tolerance as defined in the de novo sequencing parameters and relative intensity of the matching peak is at least 2 The ion types displayed in the table can be configured in Spectrum Annotation Preferences Section 3 2 2 Spectrum Annotation Fragment ions can be selected by clicking the headers tation and error map b 116 03 b NH3 99 01 b H20 98 02 Selected fragment ions are highlighted in the spectrum anno yH20 yNH3 230 08 212 07 327 17 455 19 213 05 1 2 3 4 5 6 7 B 1019 43 1001 43 1002 40 1090 47 1072 47 1073 43 an 1440 20 1441 67 3 2 4 Error Map e 327 18 338 16 533 23 539 20 aos ear a T 995 49 Y 749 35 1189 53 1171 51 1172 50 Y 515 35 497 34 we a 1489 76 1471 77 1472 73 1375 71 1357 71 1358 71 1278 69 1260 56 1261 66 T 1150 64 e 1133 60 1 H 1049 60 1032 55 The Error Map shows the mass errors of matched fragment ions The m z ratio is displayed on the x axis and the error is listed on the y axis in Daltons Each matched fragment ion is represented by a dot The ion types displayed can be configured in Spectrum Annotation Preferenc
45. of the noteworthy new features in PEAKS 7 Highlights New Label Free Quantification module A new EM based algorithm for feature detection a new optimization model based solution for feature matching and RT alignment 10lgP significance score on peptide features and proteins and normalization using TIC or selected protein s Project Wizard for project creation and easy workflow setup New project wizard to facilitate the creation of a complex project with a large number of samples and fractions and set up an easy workflow for Identification or quantification LC MS view for data 2D 3D heat map visualization and navigation New LC MS view provides much improved data 2D 3D heat map visualization and navigation Visually analyse the LC MS data with great details and ease and verify the feature detection and identification results Complementary fragmentations support in de novo sequencing PEAKS allows de novo sequencing on spectral pairs or triplets that are generated using CID HCD ETD comple mentary fragmentations Automatic de novo result validation New statistical charts allow for accurate filtration of de novo sequencing results Algorithm Improvements Improved de novo residue local confidence score Improved speed for de novo sequencing especially on high charge spectra Improved speed for PEAKS DB PEAKS PTM and SPIDER Overview e Reduced false discovery on PEAKS PTM algorithm Better Community Support e Bette
46. peptide ratios in the protein The sample on which the ratio is based on can be changed in the Show ratio to drop down menu in the Summary view Normalization factors can be selected in the Normalization factor drop down list in the Summary view The coverage of the selected protein and the peptides of the selected protein together with their ratios are displayed at the bottom half of the Protein view Refer to Section 3 3 2 Protein Coverage for details on protein coverage pane StartPage X d QUANTITATION 11 2013 10 25 10 40 x 5 Show top w proteins in each group v accession contains search Q no results Accession 10lgP Coverage Peptides Unique Avg Mass Description Light Medium Heavy SD Light SD Medium SD Heavy Mark c amp Proteins Vila 007955 SRSF1_HUMAN 6 6 Serine arginine rich splicing factor 1 OS Homo sapie G P10412 H14_ HUMAN 84 74 ME MN El 29 7 7 21865 Histone H1 4 OS Homo sapiens GN HIST1H1E PE 1 1 0 974 0 076 0 00 0 61 0 34 Y O P15880 RS2_HUMAN 84 26 M IEE isy 5 5 31324 405 ribosomal protein 52 OS Homo sapiens GN RPS 1 1 38 0 073 0 00 0 65 0 19 Y E 0 P61247 RS3A_HUMAN 76 13 IEL E ma 21 7 7 29945 405 ribosomal protein 53a OS Homo sapiens GN RP 1 0 857 0 101 0 00 0 53 0 00 Y P22626 ROA2_HUMAN 70 90 I ma 13 4 3 37430 Heterogeneous nudear
47. ribonudeoproteins A2 B1 OS 1 2 805 0 181 0 00 0 00 0 00 Y P62424 RL7A_HUMAN 66 26 m E Ei 4 4 29996 605 ribosomal protein L7a OS Homo sapiens GN RP 1 0 623 0 342 0 00 0 60 0 26 Y S 1 0 Q13733 AT1A4 HUMAN 65 93 l Io l 5 4 4 114166 Sodium potassium transporting ATPase subunit alpha 1 0 137 0 061 0 00 0 00 0 00 Y Lal P35612 ADDB_HUMAN 65 20 1 5 2 2 80854 ta adducin OS Homo sapiens GN ADD2 PE 1 SV 3 0 0 0 0 00 0 00 0 00 Y i Q13642 FHL1_HUMAN 60 66 nn E 11 3 3 36263 our and a half LIM domains protein 1 OS Homo sapi 1 1 591 0 16 0 00 0 00 0 00 Y H LIA ARI 5537 aa m RESE 5 5 asas il i i ops n L n n nam ana nan TralY Coverage Peptides gt sp Q07955 SRSF 1_HUMAN Serine arginine rich splicing factor 1 OS Homo sapiens GN SRSF 1 PE 1 SV 2 k n C outline coverage 80 AAs per line 10AA gap 1 MSGGGVIRGP AGNNDCRIYV GNLPPDIRTK DIEDVFYKYG AIRDIDLKNR RGGPPFAFVE FEDPRDAEDA VYGRDGYDYD Show de novo only sequences Y de novo tags sharing 6 gt AAs 81 GYRLRVEFPR SGRGTGRGGG GGGGGGAPRG RYGPPSRRSE NRVVVSGLPP SGSWQDLKDH MREAGDVCYA DVYRDGTGVV Y de novo peptides fully matched a0 Show modifications E pu E Y minimum ion intensity 5 161 EFVRKEDMTY AVRKLDNTKF RSHEGETAYI RVKVDGPRSP SYGRSRSRSR SRSRSRSRSN SRSRSYSPRR SRGSPRYSPR pa ae P i Y B 4 03 4 4 5 5 D4Lysine 27 241 HSRSRSRT E 8 01 13C 6 15N 2 Sila 17 vE 6 02 13C 6 Silac label 12 3 3 Peptid
48. search engine s user manual for instructions For the PEAKS database search please refer to Section 2 Setting PEAKS Search Parameters 111 Combining Multiple Database Search Engines with PEAKS inChorus Mascot Please select XiTandem Please select Precursor Error Fragment Error Missed Cleavages PEAKS is not selected inchorus combination will not be performed Note A PEAKS result must be selected for combining results in inChorus Note When launching a new search if the data in the PEAKS project is not yet refined data refinement will be performed first with default parameters Important To enable the calculation of the inChorus FDR the same target decoy database needs to be searched by all the engines For PEAKS X Tandem and OMSSA this target decoy database is generated automatically For SEQUEST and Mascot a target decoy database needs to be exported from PEAKS and added to their database list Use the tool from Configuration gt Database to export the target decoy database 112 Combining Multiple Database Search Engines with PEAKS inChorus Database Instrument Database Details AA FASTA format database export Decoy 08 Y ee ee Basic Options Path D PeaksStudio5 3 DatalSampleDB fasta or F EST database Advanced Options Fasta Tite Format Rule to parse accession id from FASTA title p Rule to parse description from FASTA title Accession id URL http
49. sequences 1 Result filtering Filtering rules can be set up at the top of the summary view Low quality de novo sequences can be filtered out by specifying a minimal threshold of the ALC score More detail about how to properly set the ALC threshold is discussed in Section 4 Filtering De Novo Sequencing Results 2 Result exporting De novo sequencing results can be exported to various formats by clicking the Export button at the top of the summary view More detail is discussed in Section 5 Exporting De Novo Results The report is composed of three sections 1 Notes Header of the report Experiment description or other information can be added to this section The notes can be edited by clicking the Notes button 2 Result Statistics Key statistics shown in figures and tables 3 Other Information Search parameters and MS instrument information 63 Peptide De Novo Sequencing Denove Ac 2 5 iris ise Ce Denowo Summary 1 Notes icms 2 Result Statistics Figure 1 a Scatterplot of peptide ALC score versus precursor mass error b Distribution of residue local confidence in filtered result a b ap 25 50 75 SO 95 9809 997 999 35 936 49 89 7 093 ALC score residue ae confidence number of residues Table 1 Statistics of data and result Table 2 Result filtration parameters 2 of MS Scans ALC score of MSMS Scans Peptides after filteration 3 Other Inform
50. the Control Sample Control Sample is marked with 14617 AESH r Bi T y tr logAratio 5 0 0 0 5 0 Default group 1 2 Notes 3 Result Statistics Table 1 Statistics of data and unfiltered result Table 2 Result filtration parameters of MS Scans 5591 Protein fold change gt 5 of MS MS Scans 12443 4 Other Information Table 4 Search parameters Quantification Type ICAT SILAC Quantification Mass Tolerance 0 1Da Quantification RT Range 1 0min Upper Bound Charge 4 Peptide Score Threshold 15 0 No Label K 100 0 K 100 0 Light K 4 03 100 0 R 6 02 100 0 Heavy K 8 01 100 0 K 10 01 100 0 3 2 Protein View Table 3 Instrument parameters Fractions VPS4B1J RAW Ton Source ESI nano spray Fragmentation Mode CID CAD IRMPD y and b ions MS Scan Mode FT ICR Orbitrap MS MS Scan Mode Linear Ion Trap The Protein view shows a list of proteins that are identified in the database search together with the protein coverage of their identified peptides in the window below The relative intensity of those quantifiable proteins are displayed 120 PEAKS Q MS Level in the ratio columns with label names incorporated into the header e g Light Medium or Heavy The ratio is calculated from the unique peptides of the protein Proteins with no unique peptides will not be assigned a ratio SD represents the standard deviation of the
51. the cursor along the curve will show the estimated FDR at a specific score threshold At a desired FDR the corre 103 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER sponding peptide 10lgP score threshold can be set by right clicking and selecting Copy score threshold FDR selection from a list of predefined FDR values is also possible which will also set the score threshold Score selection 1 l 1 l l l 1 l l l l 1 l 1 l l 1 l J l l 1 10lgP 259 number of peptide spectrum matches If the Estimate FDR with decoy fusion option 1s not selected the peptide 10lgP score threshold needs to be set empirically A score threshold set at 20 1s a good choice to start with the equivalent p value will be 0 01 with a 10lgP score of 20 Note The concepts of P value and FDR are different In PEAKS Search p value is defined as the probability that a false identification in the current search achieves the same or better matching score A 1 p value does not automatically correspond to a 1 FDR For more details please see http www bioinfor com peaks tutorials peaksdbscore html Proteins Protein identifications are filtered by protein 10lgP score and the number of unique peptides It is impor tant to empirically set a protein 10lgP score threshold A protein score threshold of 20 or higher is recommended Unique peptides are supporting peptides that are unique to a group o
52. the database and the action to be taken by the wizard is displayed once selected If a public database is already configured using this wizard then configured text in green will appear beside its name in the list It can be selected again to overwrite the configuration 29 Configuration Wizard Configure Instruments and Public Databases Select Databases NCBI nr The nr database is compiled by the NCBI that contains non identical sequences from GenBank CDS translations PDB Swiss Prot PIR and PRF The Configuration Wizard will download extract and configure the database _ UniProtKB Swiss Prot configured UniProtKB TrEMBL _ IPI Database Path D FastaDB Browse lt lt Back Cancel Help The Database Path displays the location where the configured database will be stored Click the Browse button to change the default database path Click Back to go back to the instrument selection panel Click Next to proceed to the download information panel 4 Download Information The download information panel displays all the requested instrument software and database downloads in two tables Downloads and Selected Instrument Software 26 Configuration Wizard Configure Instruments and Public Databases Download Information Downloads Progress Installed 781 916 Downloaded eat 25 6MB Downloading 63 Selected Instrument Software Status Display Information AB SCIEX MS Dat
53. the features in the feature vector e Sample Profile The relative abundance is color coded as a heat map The tooltip displays the ratio to the base sample Press Ctrl to fix the tool tip window in case the sample list is too long and scrolling is needed e Group Profile The relative abundance is color codes as a heat map The tooltip displays the ratio to the base group e RT mean Each feature in the feature vector has its own retention time center This column displays the average retention time center of the feature vector e Accession The accession of the protein from which the peptide comes If the peptide has more than one protein associated with it only one will be displayed e PTM The post translational modifications appear in the peptide 3 3 2 Feature Details The Feature Details tab contains the eXtracted Ion Chromatogram XIC chart that displays the shape of the selected peptide feature vector over the retention time range where they were identified The table at the top left corner displays the area and ratio of the features in the feature vector This table also works as a control to select the features that will be used to draw the XIC plot and other plots in the Sample Features and RT Alignment tabs The button at the top right corner of the tab can be used to toggle the plot data between before and after RT alignment Note By default the first features from each group are selected to be displayed
54. up by selecting the similar database from the drop down list and edited to set the desired parsing rules Click the Add Update button to save the configured database The database name will appear in the Database List Note Apart from starting with a greater than gt symbol the precise syntax of the FASTA title line varies from database to database For this reason PEAKS uses Java Regular Expressions to define how the accession string and the description text should be parsed from the FASTA title line To be able to run PEAKS DB using a specific taxonomy corresponding files must be downloaded and then referenced by PEAKS in the Taxonomy Options section Taxonomy files for NCBI nr database are gi_taxid_prot dmp gz and taxdmp zip for UniProt Swiss Prot they are speclist txt and taxdmp zip l To download the taxonid file click the Download button A window will appear confirming the FTP or website URL which has been identified as the location of the desired taxonomy file To invoke the default FTP client software and download the file automatically click OK Click Cancel to copy the URL to the system clipboard If Cancel was selected click OK on the dialog detailing the copy to the clipboard Next open a browser and paste the URL into the address bar When the file download window opens click Save Use the same procedure to download the taxdmp file Be sure to save the file to a location that is accessibl
55. www uniprot org uniprot Deter Taxonomy Options mani O SOS tdo O Then check the Search decoy database from PEAKS checkbox for each third party engine in their para meter settings or importing dialogs for PEAKS check the Estimate FDR with decoy fusion checkbox 113 Combining Multiple Database Search Engines with PEAKS inChorus Mascot Predefined parameters Database Mascot Database f Local Database SampleDB Set View Taxa Enzyme Enzyme Trypsin PTM Fixed Carboxymethyl C Modification Acetyl N term Acetyl Protein N term Amidated C term Amidated Protein C term Ammoniatoss N term C Biotin K Biotin Variable Deamidated NQ Modification oxidation M Display all modifications Error Tolerance Peptide Tol 20 pom 4c oo MS MS Tol 0 8 Da Peptide Charge 24 3 and 4 Monoisotopic Average Misc Instrument ESI TRAP Report top ALTO hits 3 Wait for PEAKS inChorus to complete the analysis A new inChorus result node will appear in the Project View Double click the node to open the result 4 The result presentation is similar toa PEAKS Search result with additional columns in the peptide table table which show the search engine s that identified the peptide and their corresponding scores 2 Understanding PEAKS inChorus Results A inChorus result is displayed in a very similar format of a PEAKS Search results Section 3 Understanding PEAK
56. 0 2200 2400 2600 2800 Rank PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 PEAKS 12 27 Apr 11 19 54 Protein Score Distribution Protein Number Venn Diagram PEAKS 12 27 Apr 11 19 54 PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 D 500 1000 1500 2000 2500 3000 3500 4000 Rank PEAKS 12 27 Apr 11 19 54 PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 dr PEAKS 12 27 Apr 11 19 54 109 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 7 5 Exporting Comparison Result The peptide table protein table and statistical charts in the comparison result can be exported into a single spreadsheet Right click on the comparison result node and choose Export Excel Choose the items to be exported and set the image quality for statistical charts Export protein result Export statistics graph Image Options Scale 1005 File Format Png Save As compare result 110 Chapter 10 Combining Multiple Database Search Engines with PEAKS inChorus 1 PEAKS inChorus Overview Properly combining the results from different database search engines can enhance the accuracy and sensitivity of peptide identifications PEAKS inChorus is a tool for combining PEAKS result with search results from other engines PEAKS inChorus can either launch a database search or import a search result The other supported search engines are SEQUEST Proteome Discoverer versions 1 3 and 1 4
57. 05 1964 07 1946 06 1947 04 982 54 17 A e gt oe ere r 0 5 DENOVO 43 2013 10 02 13 47 has 5 G gt 666 2654 RT 29 1056 Scan 1437 3 5 528 30 510 29 511 29 545 33 264 65 L 1876 97 1850 97 1832 98 1833 96 925 99 16 500 atches ts 6 665 36 647 35 648 34 682 39 333 18 1763 88 1737 90 1719 89 1720 88 869 45 15 ies ers F a 7 793 46 775 45 776 43 810 38 397 21 K 1626 82 1600 82 1582 82 1583 82 800 92 14 m HRH i wis pen A at gt 668 8436 RT 29 1728 Scan 1440 2 8 890 40 872 43 873 48 907 54 445 75 P 1498 73 1472 75 1454 75 1455 74 736 87 13 E ort tat ys Identification gt 809 92413 RT 29 1883 Scan 1441 2 9 991 56 973 56 974 53 1008 49 496 28 T 1401 68 1375 70 1357 70 1358 68 688 35 12 Ee PEA Information di gt 877 9146 RT 29 2148 Scan 1442 2 10 1092 61 1074 60 1075 58 1109 63 546 80 T 1300 63 1274 65 1256 64 1257 62 637 37 11 Se ote tit da 5 Scan C lu i il i kja RT 20 2601 Scan 1444 2 11 1220 65 1202 65 1203 64 1237 69 610 85 Q 1199 58 1173 61 1155 60 1156 58 587 30 10 F 12 1333 78 1315 74 1316 72 1350 77 667 38 I 1071 52 1045 54 1027 49 1028 50 523 28 9 500 gt 571 85986 RT 29 2949 Scan 1445 2 13 1420 78 1402 77 1403 75 1437 74 711 36 G 958 44 932 46 914 45 915 44 466 73 8 y gt 491 58835 RT 29 3288 Scan 1446 3
58. 120 56 1150 64 50 1049 6 91252 226 12 246 18 2490 ei as 1604 81 1439 76 00 400 BO B00 1000 1200 1400 1600 Orbange miL m 2m2 802 90607 Ma le L1 2X 2 ErTol 0 503 F intensity threshold na 12350 5549 62 7 Selecting a peak To select a peak simply click on it A blue by default arrow called the Freeze Bar indicates the selected peak Alternatively an ion peak can be selected by clicking on its corresponding cell in the Ion Table Inkensity 5 100 1585 79 1375 71 50 1049 6 812 52 226 12 246 18 4996 1031 49 1604 81 1489 76 200 400 600 800 1000 OraiSample mi0ML me 9E m2 802 90607 r 2RT O0 0729 TIC 1 79E Sly EL 2x 2r ErrTol 0 5Da 4 intensity threshold Measure the m z difference between two peaks Select a peak blue arrow with the Freeze Bar and move the mouse to the left or right Hold the Position Bar green triangle above another peak A pop up window displays the difference between the two peaks in the example below the difference is 109 92297 78 Peptide De Novo Sequencing Tnkensity 07 100 1585 79 1375 71 109 92297 50 1049 6 913 52 226 12 246 18 749 46 021 40 1604 81 1489 76 Wa mf 200 400 600 600 1000 1200 1400 OrbiSample mzXML ms 2 mz 802 90607 Silly 1 2x 2r ErrTol 0 50a FF intensity threshold eeo e Deselect a peak The Freeze Bar cab be deselected by holding Ctrl and clicking anywhere in the spectrum Zoom in on part of t
59. 162 05 Hexose T X N term 11 114 04 Ubiquitin 7 31 99 Dihydroxy 81 82 88 88 EN E 839 101 110 111 114151617 125 140 144 147 155 42 01 Acetylation N term EE EE a EE E Eo HERE E 58 01 Carboxymethyl KW X 27 99 Formylation 18 01 Pyro glu from E 43 99 Carboxylation DKW 37 95 Replacement of 2 protons 58 01 Carboxymethyl 1 03 Lysine oxidation to amino 860 33 Hex 1HexNAc2dHex2 38 02 Acrolein addition 38 129 04 Monogalutamyl 42 01 Acetylation K 0 98 Amidation 226 08 Biotinylation 43 99 Carboxylation E an o 15 99 Hydroxylation 156 12 4hydroxynonenal HNE 14 02 Methyl ester 162 05 Hexose b2 H20 203 08 HexNAcylation N Y1 H20 b gt Y2 125 90 Iodination 200 400 600 800 46 01 ISD a series 56 03 Acrolein addition 56 ao aaa aa o aa aaa oa jo pea pea pa pea f pa pa pa f i f a f a jaja rr ia iia a Co oD mal will 1 1 2x 2Y ErrTol 0 05Da preprocess Y low intens label 28 03 Ethylation AR ZA S 43 04 Carboxyl modification wit 4 5 Creating a PEAKS Project To create anew PEAKS project from raw data files do the following steps see screenshot below Select New Project from the file menu or click the n
60. 28 27 273 15 5 TOTS ae 5 z LL a oy are Se a E K aj Tj Erha 7 845 57 827 56 828 55 423 29 e 443 24 430 23 431 21 22462 4 5 ET AH EH Pp _t H E H 144 L HiyMax a 916 61 898 60 399 60 458 80 A 320 14 302 13 303 12 160 57 3 Z TERE pos 9 1017 65 999 64 1000 63 509 33 T 249 11 231 12 232 08 125 05 2 5 E t f og 10 E 143 05 130 05 131 03 7453 1 f 200 400 600 600 1000 1200 3 2 1 Peptide Table The peptide table shows the filtered de novo sequencing result For each scan the best candidate is displayed The result can be sorted by any column For example to sort the peptide sequence candidates by the scan number just click on the title of the Scan column The following list describes the contents in each column e Scan Scan number The scan number is a unique index for tandem mass spectra in the data e Peptide Amino acid sequence of the peptide as determined by de novo sequencing A modified residue is followed by a pair of parentheses enclosing the modification mass e Tag Length Length of the longest sequence tag in the peptide When low confidence residues are reduced to mass tags the remaining residues become sequence tags The length of the longest tag is reported in this column Refer to Mass Tags e ALC Average local confidence ALC is calculated as the total of the residue local confidence scores in the peptide divided by the pepti
61. 3 10 04 09 31 Peptides 1019P 2117 1 y FDR Proteins 109P 20 yland 0 y unique peptides AMPEAKS 5 2 113 10 04 09 31 E 2 PEAKS PTM 4 2013 10 04 09 31 aA De novo only ALC 70 y and 109P 17 1 Apply Filters Export Notes 7K SPIDER 9 2043 10 04 09 31 z l D o 3 2 Result Statistics 3 Figure 1 False discovery rate FDR curve X axis is the number of peptide spectrum matches PSM being kept Y axis is the corresponding FDR Y E O O EEES 5 _ Identified Peptide Spectrum Matches 1864 pPS BSA Trypsin 1 RAW o yeep aca mam DATA REFINE 3 2013 10 04 09 26 2 0 v e 3 5 3 0 g 2 5 u 4 2 0 1 5 0 5 penning 0 0 PN 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Search details Instrument_default number of peptide spectrum matches Search Engine Name PEAKS 7 0 Parent Mass Error Tolerance 20 0 ppm Figure 2 PSM score distribution a Distribution of PEAKS peptide score Scatterplot of PEAKS peptide score versus precursor mass error Y igu pep erp pep pr Fragment Mass Error Tolerance 0 05Da Precursor Mass Search Type monoisotopic a Enzyme Specified by each sample Max Missed Cleavages 3 500 15 Non specific Cleavage one j Fixed Modifications 2 400 ff L 10 Carbamidomethylation 57 02 Y l 5 Variable Modifications 300 E Deamidation NQ 0 98 5 a 0 Oxidation M 15 99 E 200 5 ax variable PTM per peptide 3 10 Matabase Swiss Prot 100 4 A ds AAA i Seal hed En
62. 4 0 13 0 17 M Q96483 ACT2 SOLLC 262 55 MNN MN ME IEE MN ci 16 0 37175 Actin 51 Fragment OS Solanum lycopersicum PE 0 0 0 o 0 00 0 00 0 00 0 00 Y y Coverage Peptides gt sp P43311 PPO_VITVI Polyphenol oxidase chloroplastic OS Vitis vinifera PE 1 SV 1 ra Ooutine 0 coverage 1 MASLPWSLTT STAIANTTNI SAFPPSPLFQ RASHVPVARN RSRRFAPSKV SCNSANGDPN SDSTSDVRET SSGKLDRRNV de 80 5 AAs per line V 10AA gap E E Show de novo only sequences 81 LLGIGGLYGA AGGLGATKPL AFGAPIQAPD ISKCGTATVP DGVTPTNCCP PVTTKIIDFQ LPSSGSPMRT RPAAHLVSKE Y de novo tags sharing 6 5 AAs i oH m Y de novo peptides fully matched 222 4 a Show modifications 161 YLAKYKKAIE LQKALPDDDP RSFKQQANVH CTYCQGAYDQ VGYTDLELQV HASWLFLPFH RYYLYFNERI LAKLIDDPTF Y minimum ion intensity 5 5 G Gan AM P Y 144 10 TTRAQ 4plex K 860 Y 144 10 TRAQ 4plex Y 87 T v E 15 99 Oxidation M 29 241 ALPYWAWDNP DGMYMPTIYA SSPSSLYDEK RNAKHLPPTV IDLDYDGTEP TIPDDELKTD NLAIMYKOIV SGATTPKLFL 3 3 Peptide View The Peptide view displays all the identifiable peptides and their relative intensities The interface is similar to the peptide table in a PEAKS DB result see Section 3 4 1 Peptide Table The relative intensities of the quantifiable peptides are displayed in the intensity columns with label names as the headers eg 114 gt 1 10000f 10581 v gt w scan search QUA noresults Peptide 10l
63. 4ups IGF2 HUMAN UPS PA2O27 ups PPIS HUMAN UPS PAQG6 Sups TAO HUMAN UPS POAZ4 ups COS HUNAN OFS PO2TS7 ups TREE HUMAN UPS POd40ups CATA HUMAN UPS POS24 dups CATG HUMAN UPS PO4127 ups PDGFE_HUMAN UPS POOTOdups LALBA HUMAN UPS POT 2239 ups CATD HUMAN UPS F142081ups SYHC HUMAN UPS POZ753ups RETBP_HUMAN_UPS Pogoodups CvC HOMAN UPS P10145ups ILS_HUMAN_ OPS POOD1Sups CAA1 HUMAN UPS PO 144ups My G HUMAN UPS POOS4Sups CAH2 HUMAN UPS POCM67 ups C BS HUMAN UPS POS26ups GST44_ HUMAN UPS QOGS20ups PFRDX4 HUMAN UPS P5557 ups BID HUMAN UPS POO 44ups GSTP4_HUMAN UPS POAS7Sups TNFA_AUMAN OFS PORT 32 ups KCRM_ HUMAN UPS PE63165ups SUMOT_HUMAN_ UPS PRA76Gups B2hG AUMAN OPS 3 1 1 Result Filtration y RT 184 BaseSample Sample 3 m Fold change 1 Quality 0 Avg Area 0 1 Charges 10 detected in atleast 1 of 6 samples and with peptide ID wn un un un wn un fu lu Du lu lu lu 3 J J J J J T T T T a T T T T T T T gt gt 2 log2 ratio IS lll 40 0 0 4 0 PO Group 1 Grup 2 a Co Lae Figure 1 Protein profile heatmap Cell colour represents the log2 ratio to the average area across different samples Y PEAKS provides an extensive set of tools to filter LFQ results according to the users requirements 131 PEAKS Q Label Free F RT Range O y SRT 140 y Base Sample Controli_i ha Apply Filters Export Notes E A Peptide feature Significance 0 Fold change 1 Quality 0 Avg Area 0 1 Ch
64. 5 RT y MJ XITANDEM 9 08 Mar 11 11 00 i 2 507 30252 RT Jy DENOVO 16 09 Mar 11 13 52 Cappy 547 59247 RT P DENOVO 7 08 Mar 11 11 00 YY a PE e DENOVO 17 09 Mar 11 13 53 YD En pai E P PEAKS 10 08 Mar 11 11 00 YL a 798 0384 RT cp 9 PEAKS 3 07 Mar 11 14 24 YM Cancel 582 81006 RT INCHORUS 11 08 Mar 11 11 00 YA 537 2491 RT 5 PEAKS 5 07 Mar 11 17 00 ui 733 2819 RT E da DENOVO 2 07Mar 11 14 11 Intensity Yo 100 o 1692 29 1893 51 Spectrum Annotation Panel 1375 71 1260 56 1150 64 50 1049 6 Y1 1020 47 1189 53 226 12 246 18 749 46 1031 49 1604 81 1359 66 1489 76 b1 H20 miz 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 a y 1 12 2 ErTol 0 5Da Y intensity threshold _OrbiSample mzXML ms 2 mz 802 90607 2 2RT 0 0729 TIC 1 79 7 info Ion Match Survey b bH20 DNHS Seg y yH20 y NH3 1 693 30 675 28 676 27 692 29 2 2 893 51 912 52 894 51 895 50 1 aS as Error Map S a T r 1 0 5 lon Table l soo 1000 1500 m2 e et o r E E owe Error da in E t t t Intensty so Spectrum Alifnment 1500 The panels are briefly described below e Result Panel The Result Panel shows all sequencing results The results of manual de novo are listed under Manual De Novo e Spectrum Annotation Panel The Spectrum Annotat
65. 6 2 G gt 668 8438 RT 28 6133 Scan 1417 2 G 527 3181 RT 28 6376 Scan 1418 2 a9 i b19 2 27 0 571 8601 RT 28 6829 Scan 1420 2 Y9 26 0 H 755 760 mz gt 582 3178 RT 28 7774 Scan 1424 2 b6 Selected MS MS OrbiSample RAW G gt 475 89145 RT 28 7996 Scan 1425 3 miz 55 7366 RT 28 2609 Scan 1404 3 A Retention Time 28 261 gt 761 0547 RT 28 8344 Scan 1426 4 500 1000 1500 2000 Orbisample RAW mz 755 7366 2 3 0 laa H 627 9721 RT 28 8776 Scan 1428 3 Ma y 1 1 2x 2Y ErrTol 0 5Da Y preprocess Y low intens label a Peaks in raw spectrum 2518 RT 28 2609 TIC 2 96E6 Max intens 1 57E5 tation Type CID CAD y and b ions G gt 1014 40393 RT 28 8983 Scan 1429 3 ton Match a Se ep 941 4555 RT 28 9276 Scan 1430 2 Survey lon Match Table and Survey Scan E ee gt 998 89575 RT 28 9669 Scan 1432 2 b b H20 bNH3 c b 2 Seq x y yH20 yNH3 yQ goSs SPIDER 46 2013 10 02 13 47 has 1 1 116 03 98 02 99 01 132 61 58 52 D 2 atches gt 594 3011 RT 29 0018 Scan 1433 3 5 mes E 2 215 10 197 09 198 08 232 13 108 05 v 2176 15 2150 17 2132 16 2133 14 1075 59 19 o o al tag gt 868 9091 RT 29 0366 Scan 1434 2 3 302 13 284 12 285 13 319 16 151 57 s 2077 08 2051 10 2033 09 2034 08 1026 05 18 ie eer G gt 736 8788 RT 29 0824 Scan 1436 2 4 415 22 397 21 398 19 432 25 208 11 L 1990
66. 60 00 976 0763 94 70 100 2 2 766 97 44 Q8FOS3 RPOC_LEPIN 7 AGDDASGLAVSEK 17 08 60 00 610 2068 5 76 6 86 2 1 367 mm 6 20 P21989 FLA2 TREPH 3 VPTPN 98 VSVVDLTVR 16 88 45 76 748 9266 61 76 66 10 2 8 amp 6 m 63 57 B7LQ20 G3P1_ESCF3 3 GITIATSHQEYETANR 16 22 60 00 895 9464 7 42 10 27 2 187 8 63 Q9XD38JEFTU_LEPIN 10 QVGVPYIVVFLNK 15 59 60 00 738 4409 88 60 93 62 2 656 mr 90 88 Q5L3Z9JEFTU_GEOKA 11 LTLVEAADLVK 15 55 60 00 586 3547 65 95 71 04 2 466 m 67 72 Q8FOSIJRL7_LEPIN v Feature Details Sample Features RT Alignment m Sample Area Ratio AIC Intensity Control1_1 2 9 Controli1 3 037 1 00 A Saket EE L Control1_2 3 08E7 1 02 je Treatment24_1 Y Control2_1 2 9187 0 96 Y a SS 7 Control2_2 2 9187 0 96 7 Y Treatmenti2_1 4 587 1 51 Treatment12_2 4 5367 1 50 Y Treatment24 1 3 657 1 21 Treatment24_2 3 7E7 1 22 3 1 Feature Table The quantified features are listed in the features table This table contains the following columns Peptide The amino acid sequence of the peptide If there is any PTM on an amino acid the amino acid is followed by a pair of parentheses enclosing the delta mass of the PTM Quality This defines how well the peptide can be quantified If a peptide s abundance does not change across t
67. ANA ia telus 102 O LOIS VIEW cat toamtcennetse 102 A Fiene PEAKS Scarch Resulta aid id 103 gt Pxporine PEAKS Search Res ed 104 6 Running PEAKS PTM and SPIDER Separately ai AA AA AR tt 105 6 1 Running PEAKS PTM on a PEAKS DB Result ooooocooniccccccoccnncccorcnncccnccncancninncnc nadan 105 6 2 Running SPIDER on a PEAKS DB or PEAKS PTM Result oooocccccccccnccccncncnnoncncononcnannos 106 E Comparison Ol PEAKS RESUMS ti dd id 106 Tel Compar Son RESISTE A AAA E A 107 dee PEPE COMPAS ts ts sia 107 Ts ELO COMPA SOM dd ie 108 TAS TA CAL A e 109 HO EXPortino Comparison ROSIN sii AA AA Ai 110 10 Combining Multiple Database Search Engines with PEAKS inChorus ooocococcnccnconcnnoncnnoncononnoncnnancononos 111 1 PBAKS DEKOS ONELVIEW sai A O A 111 2 Understanding PEAKS nC hors Results sn tin 114 3 Filtenine PEAKS inChorus Resist vical nad wie A wees anne tannin ea 116 4 Exporiine inChors Results A A 117 t PEAKS OSS Vel NE TEN E E 118 OVENI O a E E E E E T ET E NT N E A 118 DAS LME AMES enia a a A ES A A N ced 118 Ss Understandi o he Result li 120 Sols SUMMA Vie Wi id umadssiete a Peake sine dail nied 120 Ore TOEI N TO E ER 120 PEPE NCW ASA AT EA AAA AS 121 E AA O O Necelan tania atalteamtice Suen dceeh A 121 552 bienine Quantification Results di tata 121 4 Exp rins Quantification Results tad 122 PEAKS OS MS MS LEVE luar i NNE E NN cana waa banca N EN 123 Me MOMCIV IG A E E N a a a a a a a eantet 123 ES A AA
68. C peaksdatabases Note that the folder C My Documents databases wouldn t work as it contains a space between My and Documents Using spaces in the database file name causes the same problem Once databases have been downloaded and extracted save the database file as ncbinr fas or ncbi_nr fas rather than ncbi nr fas Mascot The database used by Mascot has to be identical to the database configured in PEAKS in order for inChorus to parse Mascot results correctly 54 Chapter 7 Data Refinement 1 Overview Raw LC MS MS data often contains noise redundancy as well as errors due to sample preparation and instrument approximation The PEAKS Data Refinement tool can be used to improve the overall quality of the data All or some of the following functions can be applied to the data in a project according to the user s requirements Correct data refinement especially the precursor m z correction can often result in significant improvement in the final analysis result e Merging scans MS MS scans from the same precursor m z and at a similar retention time will be merged together e Precursor m z correction the precursor m z values given by some instruments are often not of the monoisotopic 10n This creates problems in downstream analysis By examining the isotope shapes in the corresponding MS scans this function can accurately adjust the precursor m z to be equal to the monoisotopic ion m z e Precursor charge correction occas
69. EAKS Studio supports Label Free quantification Reporter ion quantification with isotope labels at MS MS level and Precursor ion quantification with isotope labels at the MS level See Section 2 Setting Parameters Section 2 Setting Parameters and Section 2 Setting Parameters for more details on quantification parameters 33 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow Label Free Quantification Predefined parameters Basic Options Label Free Mass Error Tolerance 10 0 Retention Time Shift Tolerance 5 0 Sample Groups Attach Identification Reporter Ion e search sample list Select All Collapse Attach Remove eq TRAQ TMT Group Color Precursor lan C Quantification eg SILAC Move Up Move Down Upon clicking the Finish button PEAKS will create the project load the data run the data refinement on the fractions and then conduct de novo sequencing database searching and quantification on the data at project level See Chap ter 13 PEAKS Q Label Free Chapter 12 PEAKS Q MS MS Level and Chapter 11 PEAKS Q MS Level for more details on the quantification tools available in PEAKS 6 Adding Data to an Existing Project 1 To open a saved project select File Open Project command or select from the File Open Recent Project list or click laa button from the toolbar 2 To add data to an open project choose the project from the Project V
70. EOP NAD AA A tat eweu tists O 69 32 5 SPEC A onm eNe do dde 69 A PAN CIM SS A ON 69 SL ES MS Sap SOU A ts 70 CANES VIEW a dias 70 4 alten De Voyo Sequencino Results as un EE E E E E E EE O EEES 71 Ss EX PORES DeNOyO Reuler ARNEE TI AS E A AAA AENA 73 6 De Novo Sequencing using Spectral Pairs or Triplets cc cece cece eee ee ec ence eee eens eeeee eee eaeneeeeneenees 73 Ta De NOVO SEQUENCING OSa le Peces id 75 S Manual 290 60 Sequence ii counter tons gaunt ns id 76 1 Manual De Novo Graphical User Inter ace essai ils nd anita tein das ined dna E 76 9 2 Manual De Novo Operations ia 1 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER ee 84 A E O RRA 84 2 Setting PEAK See aC hy Parametros AA alam IAEA 85 Zo ay mosto Parameters tor Emure USE ai Ni Meantdanda aaa a 88 5 Understandine PEAKS Search Results cursis in 89 Sh Peptide and Proe SCOOT ii di a es 89 1v PEAKS 7 User Manual LS UA VIEW ia A AOA 89 32 1 Fae Discovery Rate DR Curves da o 90 3 2 2 PSM SCOPE DIS DION A AA AAA Me AA RS 90 3122 DE NOVO Result Valid ao 91 5 24 SLAUSUICS OF Data and IRCSUIUS a ds 93 LO Experiment CONTO dl leed 95 O NS A ON 95 Jolo Prote A e ae O 96 Olea CON Cla SC il ns 97 IS APPO Pe DMCS a aia 100 II SONO VAS AN A AA RN ri eNts 101 FAs BEpade VIEW us tt ias 101 JAP pide Table ula 101 32 Peptide Spectra Mate IES ai ii as 102 30 De NOVO ODIY VIEW as AE ESA
71. Formats The following exporting options are available for various text formats Proteins The list of protein identifications filtered by the protein filters in the Summary page and the Show filter in the Protein view above the protein table will be saved to proteins csv in Comma Separated Values CSV format Supporting peptides A list of supporting peptides of each protein identification filtered by the peptide filters and protein filters in the Summary page and the Show filter in the Protein view above the protein table will be exported to protein peptides csv This usually contains more entries than the Peptide table as a peptide can be identified in several proteins and hence can be reported multiple times in this file DB search peptide spectrum matches The selected peptide spectrum matches PSM filtered by the peptide filters in the Summary page will be exported to DB search psm csv Peptides differentiated with only I L isoform are represented by separate entries As a result the number of entries in this file might be bigger than the number of PSMs mentioned in the Summary page De novo only peptides A list of good de novo sequences that do not have good or any database matches will be saved to de novo only peptides csv Proteins fasta A list of protein identifications will be saved to proteins fasta Peptides mzidentml version 1 0 0 A list of peptide spectrum matches will be saved to peptides_1_0_0
72. Htm il D ftest Peaks60 Pea gg JL TRA Save A Copy As yy TRAQSample 4 DATA RE Close Project bo iy DENOVO Heat Map Compare af PEAKS 3 16 May 12 10 45 More function specific export options are available from the Summary view of the respective results The following sections describe in detail the function specific exporting options available in various formats 3 Exporting De Novo Results The PEAKS de novo sequencing result can be exported to CSV HTML and pepXML formats All exporting functions are available through the Summary view panel 3 1 Exporting Summary and Peptides To export results press the Export button in the title bar of the Summary view panel The following export dialog will appear Select the items that you want to export from the available options Select the output location and click the Export button to save the selected result components to the specified location 149 Exporting Data Reports and Printing HTML Report Text Formats HTML Report Text Formats Result summary summary html De novo peptides de novo peptides csv De novo peptides pepxml de novo peptides xml C All de novo candidates all de novo candidates csv Save into D test PeaksExports sample project_DENOVO_9 Browse Save into D test PeaksExports sample project_DENOVO_9 Browse Export Cancel Export Cancel The export options are grouped into HTML Report and Text Formats based on
73. KEC 57 02 57 02 HGD 57 0 76 58 2169 8989 1 3 724 3089 20 78 Fi x000 2 Poz7eslaL U BOVIN BBB PEAKSPT 8 WGTRC 57 02 C 57 02 TKPESERIP F2 2321 8 PO27ESIALBU BOVIN BEEE PEAKSDS GLVLIAFSQYLOQC 57 02 PFDE S7 2783 F2 2471 1 POZT6SIALBU BOVIN i PEAKSPTM VHKEC 57 02 C 57 02 HGD 57 0 58 1782 Fisizi2 2 PO276S AL6U_BOvIN BEBE PEAKSPTM GLVLIAFSQYLQQC 57 02 PFDEHVK 45 49 Fi 2624 51 PO2769 ALBUBOVIN E PEAKSDB Tip Right click on a peptide to show a pop up menu and select Show original spectrum This would jump to the spectrum in the data view The following list describes the contents in each column e Peptide The amino acid sequence of the peptide as determined in PEAKS Search A modified residue is followed by a pair of parentheses enclosing the modification mass e 10lgP Peptide 10lgP score The score indicates the scoring significance of a peptide spectrum match e Mass Monoisotopic mass of the peptide e ppm Precursor mass error calculated as 10 x precursor mass peptide mass peptide mass e m z Precursor mass to charge ratio 101 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER e RT Retention time elution time of the spectrum as recorded in the data e Scan Scan number e Spec Number of spectra assigned to the peptide e Accession Accession number of the highest scoring protein containing this peptide e PTM Types and nu
74. LIFT directories from Bruker mass spectrometers can be imported provided that the Compass Xtract Runtime library is installed on the same computer as PEAKS The spectral data will be contained in the yep baf or fid file Select the p folder containing yep and baf file and the 11FT folder containing fia file during the data loading step To load all fid files at once select a top level folder CompassXtract 3 1 can be downloaded from Bruker Daltonics web site You may need to contact your Bruker repre sentative to obtain CompassXtract 3 1 Note PEAKS 7 supports Compass Xtract 32 bit version only Please contact lt support bioinfor com gt for support on Bruker data loading Instrument Preferences for Bruker Data To set Bruker data related preferences in PEAKS open the Prefer ences window by clicking the Preferences toolbar icon or by selecting Preferences from the Window menu Click on Instrument and then Bruker yep baf fid on the left hand side This will show the Bruker instrument preferences on the right hand side Bruker yep baf fid Raw File Convertor Options Bruker file reader will export Raw data Line spectra Bruker fid file may contain several files do you want to merge them yes no CompassXtract by default will export raw data If the attempt to load raw data results in no spectra then choose Line spectra A Bruker fia file may contain several samples By default th
75. MSSA search engines However Mascot search depends on Mascot s databases only When using these third party software tools note the following with care X Tandem At the time of writing X Tandem has difficulty in searching through large databases and may crash It 1s therefore suggested that X Tandem only be used with small databases If used with a large database a taxon should be specified For example NCBI nr and SwissProt databases should be used with sub taxa selected when using X Tandem OMSSA At the time of writing OMSSA cannot be used with databases that are not in NCBI nr or SwissProt format in a way that is available to inChorus Also a bug in OMSSA results in a database stored in a folder that contains a space in its path to be unusable This creates problems when PEAKS creates temporary databases on your behalf To avoid this best practices suggest that all the databases should be put in a folder with no spaces such as C peaksdatabases Note that the folder C My Documents databases does not work as it contains a space between My and Documents Using spaces in the database file name causes the same problem For example once the NCBI nr database been downloaded and extracted save the database as ncbinr fas Or ncbi_nr fas rather than ncbi nr fas Mascot The database used by Mascot will have to be identical to the database configured in PEAKS in order for inChorus to parse Mascot results correctly 2 5 Instrume
76. N Serum albumin OS Bos taurus GN ALB PE 1 SV 4 25 31 33 35 3 41 58 ez 6339 73 ziz 20 c EEEE c c c de E ECE E 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD s 111 81 83 86 82 31 B sex 101 uo O 1141516 125 149 144 147 155 c c d ES d BEA 21 ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK 169 161 163 E 173 183 185 19132 196 20 22829 233 239 E a d E a ce e c cc E E 1 amp 1 YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA 242 254 257 281 265 2683970 276 288 230 298 200 Be 208 E 31112 21819 c E 8 c ge agg c Ba Sl EE c ce c 241 RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA 25 229 256 281 281 383 292 c e e c c c c 2321 IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL 402 480 489 47 405 412 415 420 421 42829 428 449 461 463 72 474 c E E E E HE c c E E p E c 211 KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL 48384 487 495 43900 502 518 52823 533 537 544 ri 555 558 Be E c Ne a c ce c e e a E 221 NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP in 564 573 in oo E 79 58182 cc e 8 561 KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA 583 590 e Control panel This panel controls what to display in the protein sequence coverage 97
77. New Project 12 m I SS oe SE IAN 2 Data Refinement Parameters After selecting a data node in the project tree click the data refinement toolbar icon a The Data Refinement Parameters dialogue window will appear 55 Data Refinement Data Refinement Predefined parameters default w Retention time window for raw files only min Precursor m z error tolerance se Da ppm Merge CID and HCD scans together Correct Precursor 8 Mass only recommended Mass and Charge states Min charge 1 Maxcharge 3 Filter Scans Only keep scans satisfying Precursor mass between and Da Retention time between and min Quality value greater than suggest 0 65 OK Cancel Help e Merge Scans Retention Time Window The maximum retention time difference between two spectra to be merged Precursor m z Error Tolerance The maximum difference in m z between two spectra to be merged Merge CID and HCD scans together When merging scans the scans of different fragment type will not be merged If users want to enforce merging CID and HCD scans please check this check box e Correct Precursor Mass only Correct the precursor s mass Mass and Charge states Correct the precursor s mass and charge It is suggested to use this option only when the precursor charge information is missing in data files Min Charge The minimum charge a precursor ion can be corrected to Max Charge The maximum charge a precursor ion can be
78. Note Advanced Settings for PEAKS PTM are not saved 3 Understanding PEAKS Search Results After PEAKS Search is complete several result nodes are generated One is the de novo sequencing result assuming the default option for on the fly de novo sequencing 1s enabled The other result nodes are identification results from the database search tools PEAKS DB PEAKS PTM and SPIDER If more than one database search tool is enabled the results from previous search tools are automatically merged Double click the node to examine the analysis report The analysis report 1s presented in five pages e Summary shows an outline of the PEAKS database search results with key statistics The overall quality of the experiment can be examined and the filters for peptide and protein identifications can be adjusted e Protein shows a list of protein identifications This view also visualizes protein sequence coverage and helps with protein characterization e Peptide shows a list of peptide identifications This view also provides spectrum annotation and other detailed information for peptide spectrum matches e De novo only shows a list of quality peptide sequences detected by de novo sequencing that remain unidentified by the PEAKS database search e LC MS shows peptide spectrum matches visualized in an interactive LC MS heat map 3 1 Peptide and Protein Scores PEAKS Peptide Score 10lgP The PEAKS peptide score 101gP is calculated
79. PEAKS 7 User Manual PEAKS Team PEAKS 7 User Manual PEAKS Team Publication date 2013 Table of Contents e OVVIE W oTa E E A AE a A acy teaser eeedaaduntmeaiotasdines l Howto Use This Manta E ic uta l A PEARES A AE I N Gtnieaetsecuue Geman avestes iets enlaaatacs l 5 What Is INew tm PEAKS 7 Frinn E A A E TE E E 3 A Oue A A S E N 4 AA Openean ESUN PTO CCl seora r da acid 4 AD PEAKS Mim GUI tii a dado ica siicictas 5 4 3 Result Summary and EETAS sna n aunt A O 6 4A Rolt VISUAIZ ALON aa 7 AS Cra a PEAKS PrO E cad a EA 9 AO Conduce AN AMA SIS ad a a ais ara odiada TE 10 2 Installation and ACV AMO A AA A AA EE 13 Backase COMMIS eta A O T atest oaks 13 2 SY Ste Ml URC Ure MEN S aana t shaenaiainae ie 13 5 iastallation Ona WIndows Compile tte a a is 13 A A asic E seh lo a E N EA een eedara ETO NA 14 4 1 Activate PEAKS with a trial or purchased license key cccccececeeceeec eens eeeeaeneeeeneenees 15 42 RESISter to Seta tree SU day trial license Key asian 16 40 Use PEAKS asa VIE Wer sets dilata dale cda cias 16 AAS Aevi PEARS MIOS A AS A A E 16 ADs Reret PRARS rte a arta 19 40 Common Errors duro Registano aa 20 de PEAKS Perlormance Contursi a ai E 20 dl PEAKS GEDE Vors ON EA A EA AAA AE 21 del PEAKS DIEVER O ri ra rinda 22 O WESEN AE uo anios 23 3 Configuration Wizard Configure Instruments and Public Databases oocooccccccnconcnnoncnnoncononnoncnnonannonos 24 A a E N E T T padiaracan eden ca Bue o
80. Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER A outline coverage Show de novo only sequences 4 de novo tags sharing 5H AAs 4 de novo peptides fully E Show modifications minimal ion intensity 5 4 ES o ca 18 01 Dehydration 56 48 00 Dethiomethyl 30 Se 53 01 Catan 22 B 21 98 Sodium adduct 21 _ MB 17 03 Ammonia os E 7 1599 oxoton 197 ME 17 03 Pyro glu fro 14 Zid 11904 Ubiqain 9 y fi 162 05 Hexos 9 alaste ohyo f 7 ME 58 01 Carboxymet 5 7 E e2ot acen S wm 129 04 Monoglutamyl 4 COMER MIA 37 95 Replacemen 3 MIA 162 05 Hexose NSY 2 MII 860 33 Hex1HexNA 2 Km 1 03 Lysine oxida NA YB 4202 acetylation 2 que 2 02 2 amino 3 e Mode control The protein sequence can be displayed in either outline or coverage mode e Outline mode In outline mode the protein sequence is displayed without supporting peptides and matched de novo tags However the sequence coverage at a specific position can be examined by left clicking a residue 98 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER ES 78 d c 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD 101 E 116 144 154 s Al s 3 DDSPDLPKLK PDPNTLCDEF KADEKKFWGK 1 spectrum i f l l l i x KVLT sub A SSARQR Scan F3 523 m z 573 3395 z 2 RT 12 06 10lgP 24 06 p
81. Plot Figure 1 a is a scatter plot showing de novo sequences ALC score versus pre cursor mass error in ppm In a typical de novo sequencing analysis peptides with higher ALC scores are more likely to have a small mass error indicating a higher chance for the peptide to be correctly sequenced This figure provides a rule of thumb for setting the ALC score threshold The threshold can be set at the score where the precursor mass error Starts to converge 71 Peptide De Novo Sequencing O 10 20 30 40 FO 60 70 go g0 100 ALC score Residue Local Confidence Distribution Figure 1 b shows the local confidence score distribution of residues in filtered de novo sequences It is essentially a mixed score distribution of both correct and incorrect residues This figure allows you to examine the proportion of correct and incorrect residues when adjusting the ALC threshold By increasing the ALC threshold the number of incorrect residues will decrease because poor sequences are filtered out first It is recommended to gradually raise the ALC threshold until the number of correct residues starts to significantly decrease Note In some cases the local confidence score may not be able to distinguishably separate incorrect and correct residues as shown in the user manual 72 rofresidues m i Ta numi number 01 residues number of residues 9 4000 3500 3000 2500 2000 1500 1000 500 A
82. S Search Results This section only highlights the differences Peptide Score The first noticeable difference is that the inChorus peptide score is not the 10lgP score used in PEAKS DB Instead a percentage confidence score is used to reflect the probability that this peptide spectrum match is correct The percentage score is calculated in accordance with the empirical calculation used in PeptideProphet Keller et al Anal Chem 2002 74 5383 92 1 The method of PeptideProphet is applied to each engine s result to estimate the probability of correctness for each peptide identification 2 If a peptide spectrum match is identified by multiple engines the scores for all those engines are added up to a weighted sum 3 The weighted sum scores of all peptides are converted to a probability by using the PeptideProphet method again 114 Combining Multiple Database Search Engines with PEAKS inChorus Protein Score The protein score is also a percentage score It is calculated using the following approach The scores of the peptides from a protein are added up to a weighted sum The PeptideProphet method is then applied to the weighted sum scores of all proteins to convert to a probability score Engines Icon For each peptide in the Peptide View the Engines column shows the engine s that identified the peptide Each engine is represented by a icon showing the initial letter of the engine s name P PEAKS Search PEAKS
83. Select a peptide and zoom in to the reporter ion region of the MS MS to examine the reporter ions 3 4 LC MS View The LC MS view shows the distribution of LC MS signals features and identified peptides in an interactive LC MS heat map Check the option Identified peptides to show the locations of the MS MS spectra with peptide identifica tions as indicated by blue squares Refer to Section 4 LC MS View for a list of supported interactions in the LC MS heat map 3 5 Filtering Quantification Results The Quantification result can be filtered based on the number of fold changes in the proteins You can set the appro priate values for the filters by changing the filtration parameter values from the drop down lists in the title bar of the Summary view panel and clicking on the Apply Filters button The result will be updated in the Summary view the Protein view and the Peptide view accordingly The intensity columns of the Protein and Peptide views display the absolute intensity or relative intensity of the quantifiable proteins and peptides To change the reference sample select the appropriate sample eg ratio to 114 from the dropdown list beside Show in the Summary 126 PEAKS Q MS MS Level view PEAKS also supports changing the normalization factor of the protein ratio Select auto manual or no from the dropdown list For manual normalization provide
84. a Converter Display Information lt lt Back Next gt gt Downloads The Downloads table shows all the downloadable requests their status and available actions on them The status of an ongoing download is displayed in the Progress column To cancel an ongoing download click the Cancel button in the Action column The databases will be configured automatically after the download finishes Once completely downloaded the Install button appears in the Action column for the instrument software The software can be installed by clicking the Install button The Next button remains disabled until all the downloads and the installations are completed Previously installed instrument software can be reinstalled by clicking the Reinstall button that appears in the Action column once the corresponding item is installed Selected Instrument Software The configuration wizard cannot download some vendor specific software the cor responding vendor needs to be contacted to get that software If any of those software packages were selected in the instrument selection panel then their information appears in the Selected Instrument Software table Clicking the Display Information button shows information on how to get the software from the vendor in a popup dialog Click Back to go back to instrument selection or the database selection panel to change the selected items Click Cancel to cancel the wizard anytime All ong
85. a Workflow Configuration Start the ABI 4700 converter tool Choose Settings from the File menu Configuration requires the following e 4700 SERVER Name or IP Address input localhost if the Extractor is running on the same computer as ABI 4700 Explorer this is the default value otherwise enter the IP address of the 4700 SERVER e The socket used by the 4700 SERVER the port that the Oracle database listens to the default is 1521 e Username to access the Oracle database most likely does not need to changed the default is tsquared e Password to access the Oracle database most likely does not need to changed Data Extraction Procedure The data extraction requires 1 Load Spot Set List from the database Do this via menu File Load Spot Set List The extractor will export the peak list of a spot set into a PKL file 2 Open a Spot Set menu File Open Spot Set Spot Set Chooser will help the user choose a spot set After selecting a spot set click OK to open it The job run information of the spot set will be shown 3 Select a job to run There is a button to select before each run Only the MS MS job run can be selected for export as the precursor information is needed Select a job run and click Convert to do the extraction 4 Choose a filename to save After clicking the Convert button the user needs to input a file name and the peak lists of the selected job run will be exported 8 5 Bruker Data D and
86. abels with the same sample name These labels will contribute to the same sample in the ratio The modification for each label must be selected from the PTM lists which can be accessed by clicking the button in the Modifications column 2 4 Database Configuration To use the PEAKS DB function to search through a database to identify proteins PEAKS must have access to a protein or EST database in FASTA format the standard format for popular public sequence databases PEAKS can be configured to use existing databases on the system or download them from public servers Additionally taxonomy options may be specified with certain databases From the Configuration window select Database from the left menu to change the database configuration The Database List on the top shows all the configured databases in the system Select a database from the list to view the detailed information about the database in the Database Details panel 167 Configuration and Preferences Enzyme Database List nl Labeled Q Method Database Instrument Database Details FASTA format database NCBI nr Basic Options Database name Validated EST database Advanced Options Fasta Title Format Rule to parse accessionjid from FASTA title iai I d Rule to parse description from FASTA title HL Accession id URL http fer ncbi nlm nih govw fentrez viewer fogi db proteinaval lt Accession ID gt Delimiter
87. ample Allow non specific deavage at one w end of the peptide Maximum missed deavages per peptide 35 PTM F Carbamidomethylation i Oxidation M i Deamidation NG Remove Switch type Maximum allowed variable PTM per peptide 3 lt Database Select database Database UniProt_SwissProt w View C Paste sequence Taxa Homo sapiens human Set View taxa General Options Estimate FDR with decoy fusion _ Find unspecified PTMs and common mutations with PEAKS PTM Advanced Setting Find more mutations with SPIDER Skip Identification Quantification gt Click the Finish button to exit the Project Wizard without specifying parameters for further analysis Upon clicking the Finish button PEAKS will create the project load the data run a data refinement on the fractions and then conduct denovo sequencing and database searching on the data at project level See Chapter 8 Peptide De Novo Sequencing and Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER for more details on denovo sequencing and database searching with PEAKS Alternately click the Quantification button to set up parameters for quantification 5 Quantification Setting Parameters To run quantification tools on the project immediately after the project creation data refinement and identification click the Quantification button to open the Quantification panel The optional PEAKS Q module of P
88. anMS tool The PKL data can be loaded into PEAKS readily Please contact your Varian representative to obtain WarianM5S tool Default xmirai exe File Location Browse lt lt Back Cancel Help 6 Commit the Changes After configuring the necessary downloaded vendor specific software and public databases click the Next button to get to the finalize panel 28 Configuration Wizard Configure Instruments and Public Databases Configuring Instruments and Databases The following FASTA databases and or Instrument software are installed successfully Thermo MSFileReader IPI The following FASTA databases and or Instrument software are downloaded but not installed ABI mzWiff A database or an instrument software will be configured for use with PEAKS only if you do so explicitly by clicking the Install button in the Download Information panel No further configuration of PEAKS is required for the following items But you must ensure that the relevant vendor software are installed on this computer ABI Analyst AB SCIEX MS Data Converter Please contact the Manufacturer representatives to obtain the required vendor softwares Press the Back button to return to downloads and selection panels where you can change your selection of databases or instrument software for configuration Click Finish button to commit the changes to configuration file lt lt Back Cancel Help A summary about the changes that the wi
89. and Peptides cai lid 152 AA EDO Mo TIDES aa T T OT 155 AD LA Expone Protein COVER 155 gt Ex portine Oudmerication Rest a E E A 155 5 1 Exporting Labeled Quantification Results aid ia 155 5 2 Exporting Label Free Quantification Results ii tacelaniaescbines 156 OAE PON mE Hors RESTE IS A A AA AA a 156 LO Contisuratonand Pretetences aa Sta 157 Le PEAKS Environment Ereterences siii dios 157 Pel Ge Neral Eretotonces usado li cio cdo id cotos 157 As Display ODIOS WS AS AA ASES Rd 158 EZ RM Eme CUONS it ali 158 Teo Derby Data Dase iniciadas 158 Le Rav File Converter Pre lerences iS 159 DD sca ABLA A AAN E AN EA AAA A NA 159 12 2 Bikr NEP Dabs Tid Gat ech oawia cae diaealtcanien E EE E eet ditens 159 123 Shimadzu AX IMA Gin dd id cap twem evened ees 159 DAA NV A al AAA nn A eee te cael nase hetes 159 VO WW UES TW AA E AAA AA OEE A OOA R 160 1 Staten Emoto Prelcrences ias 160 ESA a A O O O A AN 160 ESAV Fanden oe MN 2 q OO CORD O E E EE 160 1 3733 OMS SA SE UA IA AAA ANSIA 161 14 Spectrum Annotatiom Preferentes is atal a a Rataleaianiaies 161 Ze PRAKS AC ONE Suri E A 162 Za l Enzyme ONO ii di a id 162 vi PEAKS 7 User Manual 22e PEN COMPUTA ON seri ANETO E EAA N OO 163 23s Labeled O Method Con eraon carreres is TE AE Sie 166 24 Database ONT ura On A A A NT 167 Ze strument Oni UN dd 169 vil Chapter 1 Overview Welcome to PEAKS de novo sequencing PEAKS 7 1 How to Use This Manual Thi
90. anel The spectrum annotation panel provides convenient ways to zoom and navigate within the spectrum e Zoom to a m z region Drag horizontally from the start m z and to the end m z with left mouse button e Zoom in out smoothly Place the mouse cursor at a particular m z value right below the x axis zoom in out by scrolling the mouse wheel e Increase Decrease peak intensity Place the cursor in the spectrum and scroll the mouse wheel e See the whole spectrum Double click in the spectrum or click the 1 1 button The ErrTol is used to adjust the error tolerance to view the display of matched ions You can use the profile Mm and peak ll buttons to switch the Spectrum View between the profile mode and the peak mode The intensity threshold check box provides an option to annotate lower intensity peaks To change the Spectrum Annotation Preferences click the button to open the dialog Refer to Section 1 4 Spectrum Annotation Preferences for more details Show Decimal Places 25 m z on fragmenabon F m z on unannotated V sequence fragmentation 447 in place ion info Intensity 5 Low i Medium High Hide Reset default 3 2 3 lon Table The Ion Match tab at the bottom panel of the de novo view contains the Ion Table that shows the calculated mass of possible fragment ions If a fragment ion is found in the spectrum its mass value is displayed in color N terminal ions are shown
91. arges 10 detected in atleast 1 of 8 samples and with peptide ID Edit a Protein Significance 20 Fold change 2 has atleast 1 unique peptide Edit Normalization Factor Heatmap Sample Correlation Use the Edit button to pop up the corresponding filter dialogs to configure filters Feature filters Significance Fold change Quality Avg Area With peptide ID Charge between Have at least Usually three steps of filtration need to be performed to obtain meaningful results e Step 1 The measured intensity of a quantifiable peptide does not change random variations may be present when its abundance remains unchanged across the samples Several filters are designed to effectively locate quantifiable peptide feature vectors e RT range filter Feature vectors within the retention time range will be included in the results and everything else will be removed This filter can be used to remove useless head and tail in an MS runs e Quality filter The higher quality of a peptide indicates that the peptide is more quantifiable A proper cutoff value for quality can be selected by checking the ratio quality figure The rule of thumb is to set the quality threshold around a fold change of 8 as shown in the figure 132 PEAKS Q Label Free Ratio 182 1 4 1 8 1116 0 0 25 5 0 7 5 10 0 12 5 15 0 17 5 20 0 Guality e Avg Area filter This filter is applied directly to the MS signal intensity Using th
92. ate a new peptide candidate for manual de novo sequencing select the m z value in the Result Panel and right click to display up a pop up menu startPage x y OrbiSemple mzxML x E Orbisample mzx1 Z P 802 90607 I E Mew Candidate for Manual De Nowo 635 34015 f T Remove the selected Candidate by 507 30252 f a Config Error Tolerance in Manual De Nowo Es H 547 59247 Config PTM in Manual De Novo fe gt 820 8849 Ro H 798 0384 R Anes gt 582 81006 F Redo E E 537 2491 R 100 Add new sequence Can t Save i 733 2819 R Select New Candidate for Manual De Novo from the pop up menu A new candidate will be created under the Man ual De Novo heading The new candidate is initiated as unsequenced so it is represented by the total mass of the residues see an example below 802 90607 2 El Manual De Novo AT A amme m Pas se 802 910 Note The pop up menu will not be accessible 1f you have highlighted any of the results in the Result Panel The figure below shows the main panels related to manual de novo sequencing The five main panels are indicated in the figure below 76 Peptide De Novo Sequencing StartPage x y OrbiSample mzxm x wn OrbiSample mzXML gt 802 90507 2 Tag P f Searched Tags a ane Selected Tags Manual De Novo Result Panel a g PP OMSSA 8 08 Mar 11 11 00 A 695 3401
93. ation Table 3 Search parameters Table 4 Instrument parameters Parent Mass Error Tolerance 15 0 ppm Fractions BSA GlwC 1 RAW BSA LysC 1 RAW BSA Trypsin 1 RAW Fragment Mass Error Tolerance 0 05 Da Ton Source ESI nano spray Enzyme Specified by cach sample Fragmentation Mode CID CAD y and b ions Fined Modifications MS Scan Mode FT ICR Orbitrap Carbamidomethylation 57 02 MS MS Scan Mode FT ICR Orbitrap Variable Modifications Oxidation M 15 99 Deamidation NQ 0 98 Max variable FTM per peptide 3 Report peptides 5 Data Refine dependencies 1 2 3 Statistical figures in this report are discussed in a later section Section 4 Filtering De Novo Sequencing Results 3 2 De Novo Peptide View The de novo view displays the de novo sequencing results in greater detail as shown in the next figure The table on the top displays de novo sequences and the bottom section provides additional information about the peptide spectrum match 64 Peptide De Novo Sequencing StartPage X 4 DENOVO 4 2013 10 22 11 14 x 1 10000f106 Y sfo w scan 2 0 noresuts 5 Scan Peptide Tag Length ALC m z z RT Mass ppm PTM Mode k PATE 582 8565 2 12 23 1163 7026 3 6 CID 3 F3 893 C 57 02 C 57 02 TESLYNR G ES 569 7514 2 19 11 1137 4907 2 1 ES CID F F1 1490 KKFWGKYLYE 10 24 681 3651 2 28 05 1360 7180 1 8 CID F1 336 KVTKC 57 02 C 57 02 TE 3 33 513 2394 2 8 39 1024 4631 3 8 cc CID
94. ation method Selecting the Save as command from the Predefined parameters drop down list allows the users save parameters for the ease of use when regularly performing quantification with the same parameters 119 PEAKS Q MS Level 3 Understanding the Results E Once completed the protein quantification result will be displayed in the quantification node in the Project view Double click on this node to open the result that contains four views Summary view Protein view Peptide view and LC MS view The Summary view tab will appear by default 3 1 Summary View The MS level quantification result summary is presented in the Summary view The quantified proteins are clustered into a tree structure in the Heatmap Proteins are clustered according to their exhibition of a similar expression trend across samples Move the cursor to the tree to select a cluster and left click to show the variation trend chart for that cluster Other information displayed on the summary page is self explanatory 1 Heatmap View OSHCDS NCOAS_ HUMAN t1 050Z94 050734 HUMAN 075351 VPS48_HOMAN P12236 ADT3_HUMAN P18124 RL7_HUMAN P132637 ATIAS HUMAN t B4DN00 B4DN00_HUMAN P01817 KV204_ HUMAN tr O08N355 08N355_ HUMAN tr B2KNZ4 B3KNZ4 HUMAN P28232 CTNA2_ HUMAN tr 098B47 098847_HUMAN tr O81UK7 Q8 UK7_HUMAN tr CO JW88 C9JWB88_HUMIAN O7LOJ3 SV24_HUMAN t1 B4E254 B4E354 HUMAN tr DBRBUS DSRBUS HUMAN Cell colour represents the log ratio to
95. ation on defining new PTMs on the fly for PEAKS de novo or PEAKS DB refer to the section Section 2 3 PTM 165 Configuration and Preferences 2 3 Labeled Q Method Configuration From the Configuration window select Labeled Q Method from the left side menu to change the labeled Q method configuration E caro Quantification Method Detail Name TRAQ Aplex Method Type Reporter lon Quantification Modification Target N Terminal Modification C Terminal Modification Side Chain Modification at Modification Mass 144 10207 Label Options Name Reporter Ion Mass Da 114 114 1107 115 115 1077 116 116 1111 117 117 1144 The built in labeled Q methods are listed in the methods list Select a method from the list to view detailed information in the Quantification Method Detail panel Methods with the R icon beside the name are reporter level methods Methods with the P icon beside the name are precursor level methods Name and Method Type The name and the type of quantification method The method type can be Reporter Ion Quantification or Precursor Ion Quantification Modification Target The modification target and mass of the unfragmented modification for Reporter Ion Quan tification The modification targets can be N terminal C terminal or Side Chain Label Options List of quantifiable labels For reporter ion quantification methods the label options pane will contain the name and repo
96. ble The quantified proteins are listed in the protein table with homologous proteins clustered together This protein table is same as other protein tables found in the PEAKS DB PEAKS PTM and SPIDER results except the following columns as mentioned below 143 PEAKS Q Label Free e Significance The protein significance is inferred directly from its supporting peptides significance weighted by two factors the intensity rank and the correlation between the relative abundance of the protein and the relative abundance of its supporting peptides e Sample Profile The relative protein abundance among the samples is depicted as a heat map Place the cursor on a profile to view the abundance ratios in the tool tip window Press Ctrl to fix the tool tip window in case the sample list is too long and scrolling is needed e Group Profile Similar to the sample profile the group profile depicts the relative protein abundance between the groups See Section 3 3 1 Protein Table for more details on other entries of the protein table 3 4 2 Coverage Tab The coverage tab characterizes the selected protein sequences at the amino acid level Refer to Section 3 3 2 Protein Coverage for details 3 4 3 Features Tab The supporting peptide features assigned to the protein are shown under the Features tab Coverage Features Peptide Unique Quali
97. ble by PEAKS Default Log File Location The log files for PEAKS can be found at PEAKS_HOME_DIRECTORY by default These files locations cannot be changed 1 1 1 Display Options Click Display Options from the menu on the left side to display interface preferences on the right side Display Options Show Decoy Hits Show Percentage Score Show inChorus Score Show FTM with frequency greater than 3 in PTM profile table Show Decoy Hits Check this to display protein and peptide hits from the decoy database in PEAKS DB results Show Percentage Score PEAKS uses 10lgP to display its results by default Check this to view the percentage score along with the 10lgP score in peptide and protein view These changes are applied to the exported files of PEAKS DB results Show inChorus Score Check this to display the percentage score in the peptide and protein views of inChorus results Set the PTM display threshold by selecting the minimum PTM frequency in the PTM profile table If there are fewer instances of a PTM identification in a protein identification than the minimum it will not be displayed in the PTM profile table 1 1 2 RMI Connections Click RMI Connections from the menu in the left side to show the RMI Java Remote Method invocation connec tions preferences on the right side RMI Connections Server Host localhost Server Port 33003 Client Port 31003 Worker Port 35003 The default port numbers for the
98. ble through the Summary view panel 4 1 Exporting Summary Proteins and Peptides To export the result press the Export button in the title bar of the Summary view panel The following export dialog will appear 152 Exporting Data Reports and Printing HTML Report Text Formats For Third Party Report proteins in HTML with summary view protein coverage supporting peptides best unique PSM Collect all the above details in a single html HTML Report Text Formats For Third Party Proteins proteins csv Supporting peptides protein peptides csv DB search peptide spectrum matches DB search psm csv De novo only peptides de novo only peptides csv _ Proteins fasta proteins fasta Peptides mzidentml 1 0 0 peptides_1_0_0 mzid Peptides mzidentml 1 1 0 peptides_1_1_0 mzid _ Peptides pepxml peptides pep xml De novo only peptides pepoml de novo only peptides xml Save into D test PeaksExports Sample project_DBSEARCH_10 Browse Save into D test PeaksExports Sample project_DBSEARCH_10 Browse Export Cancel Export Cancel HTML Report Text Formats For Third Party For Scaffold PEAKS exports Identification results in mzIdentML format along with the Spectra data in MGF format for use with Scaffold peptides mzIdentML peptides_1_0_0 mzid peptides mzIdentML peptides_1_1 0 mzid For Skyline PEAKS exports results in pepXML format along with t
99. c position in TIC will display the corresponding survey scan The tandem scans associated with the current survey scan are shown in the bottom right panel LC MS MsimMs MS OT mmo me o P m a E 43 Data Visualization StartPage x y OrbiSample RAW x n TIC Intensity 100 a 549 26 w w 3 gt Survey Scan TE 678 30 654 78 50 w 524 03 o 632 29 872 71 Qu o miz 200 400 600 800 1000 1200 1400 1600 Ma yl 22 2 2r Errtol Y low intens label OrbiSample RAW ms 1 RT 25 3737 scan 1283 TIC 2 16E8 a 650 28296 4 866 7075 3 778 3211 2 Intensity 100 193 73 237 13 Tandem Scan a 50 651 33 S 331 23 916 33 166 06 787 26 1029 39 1016 39 717 21 miz 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 RT 0 0093 64 99 min Y ly 11 2x 2Y Errrol Y low intens label OrbiSample RAW ms 2 mz 650 28296 z 4 RT 25 3853 scan 1284 TIC 1 13E5 The survey scans and tandem scans provide a few convenient ways to zoom and navigate the spectrum e Zoom to an m z region click the desired start m z and drag horizontally to the desired end m z release the mouse button e Zoom in out smoothly place the mouse pointer at a particular m z value right below the x axis line scroll the mouse wheel button e Increase decrease the peak intensity place the mouse pointer on the spectrum scroll the mouse wheel button e See the whole spectrum double click on the
100. converter for WIFF file loading AB SCIEX MS Data Converter Click Browse to tell PEAKS the location of the raw file converter Select the preferred mode of raw data to load This 1s a useful option for ABI 5600 instruments mzWiff Click Browse to tell PEAKS the location of the raw file converter Select Survey Spectrum Centroiding and or Product Spectrum Centroiding if centroiding has been performed on the survey spectrum and the product spectrum respectively before loading it into PEAKS This is important to ensure PEAKS optimal performance MSX Click Browse to tell PEAKS the location of the raw file converter Select Survey Spectrum Centroid and or Product Spectrum Centroid if centroiding has been performed on the survey spectrum or the product spectrum respectively before loading it into PEAKS 8 4 3 ABI 4700 4800 T2D files can be extracted and imported into PEAKS with a free tool created by BSI The PEAKS Configuration Wizard can download and install the AB 4X00 Extractor automatically see Section 2 Instrument Selection System Requirements This extractor can be installed on the same machine as the ABI 4700 Explorer and the Oracle database or another machine that has direct network access to the 4700 SERVER There cannot be a firewall or proxy between the computers Windows 7 or Windows XP is recommended for use of this tool 39 Project Wizard Create a PEAKS Project Load Da ta and Set up
101. ct a data node in Project View The selected node can be a fraction a sample or a project PEAKS will analyze all fractions under the selected node Launch a PEAKS search by clicking the PEAKS DB Search button on the tool bar 84 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER hed Fi BSA Gluc LRAW Jl BSALysc hed F2 BSA LysC L RAW GJ BSA Trypsin p F3 B5A Trypsin LRAW Note Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for details on creating a PEAKS project 2 Specify the PEAKS DB parameters in the pop up dialog Most of the parameters are self explanatory and the default parameters provide a good starting point for the analysis PEAKS PTM and SPIDER can be enabled in the dialog to automatically run after PEAKS DB search Click OK to start PEAKS search Note If data is not yet refined a prompt to specify data refinement parameters appears Refer to Chapter 7 Data Refinement for details on refinement 3 Wait for the analysis to finish Search times range from minutes to hours depending on several factors including the dataset size and computer speed New result nodes for PEAKS DB PEAKS PTM and SPIDER will appear in Project View Double click the result nodes to examine analysis reports FA Project View 36 SPIDER 7 30 May 12 18 50 X E A D Peaksworkspace derbyServer serverDB In depth Protein Analysis y DENOVO 4
102. d algorithm is used for feature detection deconvolution and refinement PEAKS uses an optimization model for simultaneous feature matching and retention time alignment The efficient and accurate algorithms used for Label Free Quantification can even handle significant time shifts and distortions The quantification ratios can be normalized using TIC or selected protein s e Multiple Instrument Vendor Support Overview PEAKS supports most major instruments raw file formats which saves the trouble for file format conversion More importantly the algorithms have been tuned for different instrument type to ensure optimal accuracy and sensitivity Result Exporting The analysis result can be exported to a variety of text formats The HTML format makes it easy to share the results on a website whereas other text formats such as csv make it easy to view the result in spreadsheets and post analyze with the in house script or other third party software PEAKS also supports exporting to standard result formats such as pepXML and mzIdentML inChorus PEAKS can import the results from other database search engines already acquired by the lab and combine all the results together PEAKS can estimate FDR for the combined result and can allow for result filtration For each identified peptide PEAKS also displays all the engines that identified the peptide as well as each engine s identi fication score 3 What Is New in PEAKS 7 Here is a list
103. d the DVD drive and open it to access the disc Double click on PEAKS_Studio_Installation exe 4 A menu screen will appear Select the top item PEAKS Installer The installation utility will launch the installer When the PEAKS installation dialogue appears click the Next button PEAKS Complete Proteomic Software 5 Follow the on screen instructions to finish the installation 4 Activation All users are required to go through a software activation process in order to use PEAKS A license wizard will appear to guide users through the activation process the first time PEAKS is launched 14 Installation and Activation Welcome to PEAKS Thank you for using PEAKS The most accurate sensitive easy to use software package for complete proteomics analysis Activate PEAKS with a trial or purchased license k By entering the license key the features of PEAKS will be activated and the software will be ready for use Register to get a free 30 day trial license key Pa OY 0 By completing the online registration form a 30 day trial license key will be sent to you via email You will also gain access to free email and phone supports during the trial period Y Use PEAKS as a viewer ee PEAKS can be used as a viewer without activation Take advantage of the advanced user interface provided in PEAKS to share results with colleagues and collaborators Activate PEAKS manually Important Keep the license ke
104. de length e m z Precursor mass to charge ratio e z Precursor charge e RT Retention time elution time for the spectrum as recorded in the data e Mass Monoisotopic mass of the peptide e ppm Precursor mass error calculated as 10 x precursor mass peptide mass peptide mass e PTM Types and numbers of modifications present in the peptide shown as color coded icons e Mode Fragmentation mode in which de novo sequencing is performed by the algorithm 65 Peptide De Novo Sequencing Confidence Scores Amino acids in de novo sequences are color coded according to their local confidence scores Red represents a very high confidence greater than 90 purple represents a high confidence 80 to 90 blue represents a medium confidence 60 to 80 and black represents low confidence less than 60 Hover the mouse cursor over the sequence to view local confidence scores of each individual amino acid Fa1921 GHPEYAVSVLLR F1 1559 FEDDAAC 57 02 C 57 02 KDVFARGE Fa 1207 TO GHPEYAVSVLLR Eo Sag IA F3 1717 L5H 509 5198 99 99 98 98 96 95979495 AL Mass Tags The low confidence residues can be displayed as mass tags by adjusting the local confidence score threshold using the button af If the score is set at O all of the amino acids in the peptide sequences will be displayed Increasing the threshold will reduce residues below the threshold to mass tags The Tag Length column in the peptid
105. der with 64 bit PeaksStudio and 32 bit MSFileReader with 32 bit PeaksStudio 8 2 Waters Micromass MassLynx Data Two ways can be used to load Waters s raw data Library Level Support wolf exe RAW data from Waters instruments can be imported provided that MassLynx 4 1 software is installed on the same computer as PEAKS MassLynx 4 0 users can download a different version of wolf exe Command line can be used to convert raw files to mzXML with wolf exe The file Peaks installation directory wolf exe can be replaced with the program compatible with MassLynx 4 0 For links to different versions of Wolf visit the link below http www bioinfor com peaks support formats peaks watersmicromass masslynx data html Converter Level Support massWolf exe Note If you want to use mass Wolf in PEAKS please make sure massWolf is installed correctly and works from command line on your computer If you have massWolf installed on your computer and prefer to use massWolf exe to convert the raw data define a the converter in the Preferences dialog Click the Preferences toolbar icon or select Preferences from the Window menu to open the Preferences dialog Clicking the Waters raw option under the Raw file convertor section on the left will show the preferences for mass Wolf Point to the location of massWo1f exe on your computer and enable it Waters raw Enable the MassLynx Converter MassWolf Location
106. dges that Ronald Beavis is the author of the xX Tandem program BSI is grateful to Or Beavis for allowing us to share X Tandem with our users BSI distributes xX Tandem in accordance with the following Artistic License for all X software binaries and documentation BSI is not responsible for the performance of X Tandem and makes no warranty or guarantee for it View end user license agreement a Warning This computer program is protected by copyright law OK e Ss and international treaties Unauthorized reproduction or l gt gt distribution of this program or any portion of it may result in o 7 severe civil and criminal penalties and will be prosecuted to the License Wizard maximum extent under the law l Click the License Wizard button to continue then follow the instructions in Section 4 1 Activate PEAKS with a trial or purchased license key or Section 4 4 Activate PEAKS manually to re register PEAKS 4 6 Common Errors during Registration 1 The license key contains only English letters and numbers It is recommended to copy Ctrl C paste Ctrl V the license key whenever possible 2 The user information can only contain English characters letters digits and symbols Characters from a non English encoding may cause the activation to fail 3 Ifthe computer is behind a firewall or has other Internet connection problems the activation may fail Please follow the on scr
107. doing an edit An undone edit during the manual de novo sequencing can be reapplied easily Right click and select the Redo command from the pop up menu with the peptide candidate still selected in the Result panel This button can be clicked multiple times to return to later stages in the edit Error Tolerance To set the mass error tolerance in the manual de novo sequencing select the peptide candidate in the Result panel right click and select the Config Error Tolerance in Manual De Novo command from the pop up menu This will open a dialog where the error tolerance can be set PTM Configuration To identify post translational modifications PTM while manually de novo sequencing a spectrum start by selecting the peptide candidate in the Result panel Right click and select the Config PTM in Manual De Novo command from the pop up menu to open the PTM Setup window To know more about the PTM configuration refer to Section 2 3 PTM 83 Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 1 Overview PEAKS software provides a complete set of database search tools for in depth protein analysis With PEAKS DB PEAKS PTM and SPIDER proteins in the sample can be identified with high sensitivity and accuracy It is also possible to find potential sites of modifications and mutations PEAKS supports multiple enzyme digestion by which almost full sequence coverage can be achieved for
108. e UUE 12 0 11 0 550 505 miz 1000 2000m2 3 3 LC MS View LC MS View visualizes de novo sequences in an interactive LC MS heat map Check the option De novo tags to show the locations of MS MS spectra with de novo sequences Those MS MS spectra are indicated by amber squares Only the filtered de novo sequences are shown in the heat map Refer to Section 4 LC MS View for a list of supported interactions in the LC MS heat map 70 Peptide De Novo Sequencing ATA F IBSA LRA eii 4 miz 1845 91 om RT L74 i E C Features AT ha F3ESA Trpen 1 P AW AT E 4 Filtering De Novo Sequencing Results PEAKS de novo sequencing result can be filtered by ALC Average Local Confidence score The ALC filtering threshold can be specified in the top panel of the Summary View Click the Apply Filters button to filter the results The result in the Summary View De Novo Peptide View and LC MS View will be updated accordingly Note Whenever the score threshold is changed the Apply Filters button will be highlighted in red to remind you that the change has NOT taken effect yet The purpose of filtering is to remove poor sequences in which residues are incorrectly sequenced By default the ALC threshold is set to 50 Statistical figures are shown to help evaluate the overall quality of the sequencing result They also provide guidelines for determining a proper ALC threshold Peptide ALC Score Scatter
109. e table is automatically updated when the threshold is adjusted Gh 71 show mass tag for confidence less than y O 10 20 30 40 50 60 70 80 90 3 E Color code gt 90 80 90 60 80 lt 60 f i 8 HSTVFONLNP 497 2 10 1141 5 LAMVPSCGV 374 1 Modifications In the Peptide column modifications are shown as brackets in peptide sequences Consider the example SHM 15 99 TNLNGNPEDR 15 99 shows the methionine M residue is modified with delta mass of 15 99 Da In the PTM column modifications in a sequence are shown as icons Hover the mouse cursor over the icons to show the modification names Note N term modifications are shown after the first residue in the sequence and C term modifications are shown after the last residue Peptide Search The peptide table can be searched by entering the value in the search bar located in the top right corner The peptides can be searched by scan number subsequence precursor m z retention time and modification mass The matched peptides are highlighted in the table and can be iterated by clicking the up and down buttons in the search bar wscn searci AA A noresults gt scan seq contains miz RT PTM Am Note Precursor m z retention time and modification mass are searched approximately For example 130 3 will match any value from 130 25 to 130 35 exclusive And 130 will match from 129 5 to 130 5 exclusive 66 Peptide De Novo Sequencing 3 2
110. e Quality filter is preferred over this filter However under some circumstances it is desirable to apply this filter directly to the MS signal The rule of thumb is to choose the average area around a fold change of 8 as shown in the figure b Ratio 10 10 10 10 107 Avg Area e Charge filter Some low charge states such as charge 1 or high charges like 8 or 9 are more prone to error in feature detection They can be removed using the charge filter e Confidently detected sample filter A peptide is more quantifiable when it is detected in more samples The rule of thumb is to set this filter to the half the number of total samples e With peptide ID filter Set this filter to show only the feature vectors with peptide identification It is recom mended to set this filter for quantification results with attached identification results unless it is desirable to in vestigate the feature vectors having significant fold changes with no peptide identification attached to them e Step 2A The following filters can be used to focus on the peptides with significant changes These filters are of most use when the purpose is to study the peptide changes e Peptide feature significance filter The significance of a peptide is denoted by its 10LogP score The rule of thumb is to set the cutoff value at 20 which is equivalent to a P value of 0 01 133 PEAKS Q Label Free e Peptide feature fold change This filter is highly dependen
111. e View The Peptide view displays all the identified peptides and their intensities The interface is similar to the Peptide view in a PEAKS DB result Refer to Section 3 4 Peptide View for details The relative intensities of the quantifiable peptides are displayed in the intensity columns with label names incorporated into the header e g Light Medium or Heavy The reference sample can be changed from the Show ratio to drop down menu in the Summary view The mode for the normalization factor can also be selected from the Normalization factor listin the Summary view StartPage X df QUANTITATION 11 2013 10 25 10 40 x 1 1000 of 1893 v E Y scan search Q O no results E 5 Peptide 10lgP Y Mass ppm m z RT Scan Spec Accession Light Medium Heavy PTM 1 GC 57 02 TATLGNF 4 03 AK 4 03 1146 5942 574 3062 6072 2 P15880 RS2_HUMAN 1 A 2 SAGPQSQLLASVI 6 02 AEK g 3 AIVAGDQNVEY 4 03 K 4 03 1313 7067 657 8657 5097 1 Q13642 FHL1_HUMAN 1 1 591 0 16 Ba 4 LIIVEGC 57 02 QR 55 96 1086 5856 3 5 544 2982 48 10 6093 2 Q13733JAT1A4_HUMAN 1 0 137 0 061 E 3 5 KVVNPLFEK 54 68 10726281 7 5 537 3173 49 42 6300 1 P62424 RL7A_HUMAN 1 0 216 0 181 3 6 SGVSLAALK 4 03 53 67 848 5269 3 2 425 2721 55 14 7216 1 P10412 H14_ HUMAN 1 0 551 0 645 E 7 TK 8 01 DIEDVFY 4 03 K 4 03 52 68 1272 6932 6
112. e by PEAKS Please note that it is not necessary to decompress the taxonomy files Now that the taxonomy files have been downloaded PEAKS must be given access to them by clicking the Browse buttons and selecting the files Note Please do not rename the taxonomy files otherwise PEAKS cannot recognize the files 2 Databases to be Used in PEAKS inChorus Function The database configured here will also be used in the PEAKS inChorus function to call the X Tandem and OMSSA search engines However Mascot search depends on Mascot s databases only When using these third party software tools note the following with care X Tandem At the time of writing X Tandem has difficulty in searching through large databases and may crash It is therefore suggested that X Tandem only be used with small databases If used with a large database a taxon should be specified When using NCBInr or SwissProt databases with X Tandem it is best to use a sub taxonomy OMSSA At the time of writing OMSSA cannot be used with databases that are not in NCBI or SwissProt format in a way that is available to inChorus Also a bug in OMSSA prevents easy use of databases with OMSSA when they are stored in a folder that contains a space or non english characters in its path This creates problems when 53 Adding a Sequence Database PEAKS creates temporary databases on your behalf To avoid this best practices suggest that all databases are put in a folder named
113. e ncaa ene an goeura eam cate ta cde weer es 24 Ze nostrum nt Select tn iia E A tte 24 Database elec li 25 A ADO MAI ad Information a dd E I eentates 26 e Vendor Instrument O Ware Options a AAA AAA 28 6 Cominit the Chan esta raras I ali detiene 28 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow oooococcococconcnconcnnoncnnons 30 O are a A AE cas celeaniaue tenses ie nauted aa aie eee inuecgecued 30 2 Cede NOW PrO OO AA O o mn O ised Siineles wip ered ee ahee eigen 31 gt Data Renne ment Sete Parametels asian acota ted tenet aes a2 4 IDB Search Stine Parameters ada aaa 33 S Ouantiicalon s Selma Parameters id ao delas cardio ts 33 6 Addie Data toan Existi o Proj ect na E AAA A Ss 34 Te puppe Data FONDIS rasta tri IE lar to 35 S Vendor perito Requirements acia 36 0 15 LACImo Dat ia adicto eicictad 36 0 2 Waters Micromass Mass Lyx ata EA A A 37 Sie PA a 1 01 ID ala sacs cans O oO a tenant iarove tient tat a ties Gant aagosetoasl a ee tacanns tant ok ruhs 3 St Applied BIOS ystemis Sclex Dita n a o a A 38 64 LOS TAR Or OTRAP asias rn A E A ico 38 Bit CONVE res or WIPE as a A A sou leaters 38 SES CADA A SOO rta aaa 39 6 5 Drier Dita aaa 40 SO S PP O 41 111 PEAKS 7 User Manual Bel N VS A A aaa 41 0 PEAKS OPIO E e odos 41 9 Changine the Default Project LOCO ar SSA DAA A A A sds 42 Daa ay VAS UAT ALON ca shapes o y TA Mea deme uate lome anata aan actuc O 43 A W eiei a wantaiiamim
114. e of four algorithms maximizes the number of identifications e Built in Result Validation The decoy fusion method is used to validate the peptide identifications automatically The decoy fusion method is an enhanced result validation method that avoids several pitfalls existing in the commonly used target decoy method With an informative result summary view the results can be easily filtered by false discovery rate FDR e Protein Coverage View All the identified peptides are mapped to the identified proteins and displayed in a consolidated protein coverage view All the PTMs and mutations on the protein sequence are highlighted The interactive graphical user interface GUI allows the in depth and effortless examination of every amino acid in the identification results and every peak in the data e Accuracy and Sensitivity The superiority of PEAKS algorithms has been demonstrated by publications and third party evaluations By com bining four complementary algorithms together the sensitivity is further improved e Multiple enzyme Project To maximize a protein s sequence coverage it is advantageous to use different proteolysis enzymes to digest the protein sample PEAKS supports the use of different enzymes in different samples of a single project The software will automatically use the enzyme specified in each sample and combine all samples results together e Label Free Quantification An Expectation Maximization EM base
115. e para meters If you want to run some differently than others then you must set up separate workflows Once the data is selected you can specify parameters for the identification analysis steps one by one by clicking the other buttons in the workflow dialogue Please refer to the chapters on each individual function for details on setting up the function specific parameters Note PEAKS DB PEAKS PTM and SPIDER are now combined together see Chapter 9 Peptide PTM and Mu tation Identification PEAKS DB PEAKS PTM and SPIDER and are optional in the Identification Work flow You can uncheck them if you do not want to perform those functions 2 Quantification Workflow The quantification workflow is similar to the identification workflow with an additional step for quantification where the quantification parameters to perform labeled quantification can be defined Please refer to the chapters on each individual function for details on setting up the function specific parameters 146 Workflow a De Novo Y a 6 PEAKS Quantification Note Only Reporter Ion Quantification eg 1TRAQ or Precursor Ion Quantification eg SILAC workflows can be set in the quantification workflow For Label Free workflow use the Project Wizard 3 inChorus Workflow The inChorus workflow is similar to the identification workflow but offers the ability to specify inChorus parameters and invoke multiple search engines Refer to Chapter 10
116. e preferences for the AB SCIEX instrument raw file converter Note Refer to Section 8 4 1 QSTAR or QTRAP for details on AB SCIEX wiff preferences 1 2 2 Bruker yep baf fid Click the Bruker yep baf fid option under the Raw file converter section from the menu on the left side to display the Bruker instrument raw file converter preferences Note Refer to Section 8 5 Bruker Data for details on Bruker instrument preferences 1 2 3 Shimadzu AXIMA run Click the Shimadzu AXIMA run option under the Raw file converter section from the menu on the left side to show the Shimadzu instrument raw file converter preferences Note Refer to Section 8 6 Shimadzu Data for details on Shimadzu instrument preferences 1 2 4 Varian xms Click the Varian xms option under the Raw file converter section from the menu on the left side to display the Varian instrument preferences 159 Configuration and Preferences Note Refer to Section 8 7 Varian for details on Varian instrument preferences 1 2 5 Waters raw Click the Waters raw option under the Raw file converter section from the menu on the left side to display the Waters instrument preferences Note Refer to Section 8 2 Waters Micromass MassLynx Data for details on Waters instrument preferences 1 3 Search Engine Preferences This section allows users to configure preferences for the foll
117. e shows the filtered de novo only sequences by the current settings in the Summary View De novo only sequences may suggest novel peptides peptides with unknown modifications or other interesting research subjects The interface is identical to the Peptide View in a de novo sequencing result node For detailed instructions please refer to Section 3 2 De Novo Peptide View 3 6 LC MS View The LC MS View visualizes identified peptides and de novo only sequences in an interactive LC MS heat map Check the option Identified peptides to show the locations of MS MS spectra with peptide identifications which are indicated by blue squares Check the option De novo only tags to show the locations of MS MS spectra with de novo only sequences which are indicated by amber squares Only the filtered peptide identifications and de novo only sequences are shown on the heat map Refer to Section 4 LC MS View for a list of supported interactions in the LC MS heat map 102 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Fi BSA GuC LRAW RT 0 59 mn RT F1 BSA Gluc LRAW ea in As m z 1165 43 RT 28 55 4 Features MS MS Spectra Y Identified peptides Y De novo only tags eo _ 2 a m i 1E9 1E8 167 t 1E6 165 miz F3 BSATrypsin 1 RAW RT LC MS De novo only Peptide Protein Summary DD y de 30 0 M Q ES a reer a E Sy 2 2 a e z
118. e target decoy FDR o 1291 0 267 0 lt 9 1 lt 0 1 N A 41 0 lt 0 1 OMSSA 3 Filtering PEAKS inChorus Results The filtration parameters can be specified using the control panel in Summary View Click the Apply Filters button after changing the parameter values E Peptides Target Inchorus FDR 1 Edit filters Proteins 5core 50 and 0 unique peptides a un SSS SS SSS SSS A AA E De novo only ALC 50 Apply Filters Export Notes q Filtering peptides in the inChorus result 1s different to that in the PEAKS Search result Click the Edit filters button in the Summary View to specify the peptide filtering rules Peptides can be filtered by either inChorus FDR or by each individual engine s score If filtering by inChorus FDR PEAKS calculates the score threshold for each search engine to automatically to reach the target inChorus FDR If filtering by each individual engine s score a peptide is kept as long as at least one engine s score is above the specified threshold 116 Combining Multiple Database Search Engines with PEAKS inChorus K seguest Xcorr Mascot Score XTandem e value E Omesa e value K 4 Exporting inChorus Results The inChorus exporting function contains the same exporting options as PEAKS DB Unlike PEAKS DB the exported results will contain inChorus scores and individual search engine scores for the supporting peptides of the
119. ecause more false positives are being kept as well In this figure the vertical line indicates the current score threshold The number of PSMs and the corresponding FDR by the current score threshold are shown in the top left corner 1500 Erie Pepe Spectrum Meth 2908 Y A AA A RA A eee 3 5 A A A o A A A A A ee nn AIR A LL number of peptide spectrum matches The FDR curve is available when FDR estimation is enabled in the PEAKS Search parameters PEAKS Search esti mates FDR using the decoy fusion approach Decoy fusion is an enhanced target decoy approach that makes more conservative FDR estimations For details of the decoy fusion approach please refer to the publication PEAKS DB De Novo sequencing assisted database search for sensitive and accurate peptide identification Mol Cell Pro teomics 2011 Dec 20 It is commonly recommended to set FDR to be lt 1 by adjusting the score threshold If a rapid growth of the FDR around the 1 FDR threshold is noticed some PSMs may be sacrificed to significantly reduce the FDR Note When counting the number of PSMs PEAKS keeps one peptide per spectrum at most Thus the number of PSMs is actually the number of spectra with assigned peptides Note Decoy matches are not counted in the number of PSMs Unless otherwise specified decoy matches are also excluded from the other statistical numbers shown in the Summary View 3 2 2 PSM Score Distribution Figures 2 a and 2 b i
120. ected in atleast 1 of 6 samples and with peptide ID Protein Significance 20 Fold change 2 has atleast 2 unique peptides Normalization Factor Sample Correlation 3 1 4 2 Normalization PEAKS offers several options to define the normalization factors When calculating sample or group ratios the area of each feature will multiply its corresponding normalization factor By default PEAKS uses total ion current TIC of the samples to calculate the normalization factors To change the normalization factors click on the Normalization Factor button on the summary page title bar The table shows the normalization factor that will be used to display the LFQ results To set the normalization factors manually select the Manual input option and enter the factor values in the table To use the original feature areas without normalization select the No normalization option To use internal standard protein list to calculate normalization factors select the Use internal standard proteins option and then select a set of proteins by checking the check box from the list Right click to check all selected protein or clear selections Protein List search Protein POOS49 KPYK1_ YEAST P52489 KPYK2 YEAST C P10S82 HSP72 YEAST PIS8i HSP71_YEAST POD359 63P3_YEAST _ PO0925 ENO2_YEAST C P16521 EF3A_YEAST E P53978 EF38_YEAST C P27466 KCC1_YEAST E POD358 63P2_YEAST C PO7259 PYRI_YEAST C POO560 PGK_YEAST El P15108JH5C82 YEAST
121. eeds to be set up once PEAKS will call the converter to convert the data to a native supported format before loading The actual conversion process 1s invisible to the user e RAW directory Waters QTOF instruments See Section 8 2 Waters Micromass MassLynx Data e WIFF file AB Sciex OSTAR and QTRAP instruments See Section 8 4 1 QSTAR or QTRAP e T2D file AB 4700 4800 series See Section 8 4 3 ABI 4700 4800 e RUN folders from Shimadzu instruments See Section 8 6 Shimadzu Data e XMS files from Varian instruments See Section 8 7 Varian 8 Vendor Specific Requirements Most vendors provide tools for MS analysis software to read their raw data format PEAKS works best with the unprocessed raw data as PEAKS uses the data pre processing tools built into the software designed to maximize identification results Listed below are the requirements to load raw data from each supported vendor 8 1 Thermo Data RAW data from Thermo Fisher Scientific mass spectrometers can be loaded provided that the XCalibur software or the Thermo MSFileReader package is installed on the same computer as PEAKS The PEAKS Configuration Wizard can download and install MSFileReader automatically see Section 2 Instrument Selection MSFileReader is publicly available and can be found at the following link Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow Note Install 64 bit MSFileRea
122. een instructions or refer to Section 4 4 Activate PEAKS manually 5 PEAKS Performance Configuration The PEAKS Performance Configuration tool can be accessed from the Windows Start Menu By default PEAKS automatically determines its performance parameters to take full advantage of the processors and memory available on the computer In most situations the Automatically Configure PEAKS Performance option should be used The 20 Installation and Activation Manually Configure PEAKS Performance option will come in handy in situations where advanced users want to adjust PEAKS memory allocation and threading O Manually Configure PEAKS Performance Main Program JVM Heap Size MB 14076 Number of Computing Nodes to Start 8 Start Client Separately Client JRE Binary Folder fre bin Browse Client JVM Heap Size MB 1024 Start Compute Node Separately Computing Node JRE Binary Folder fre bin Browse Computing Node JVM Heap Size MB 1024 The Number of Computing Nodes to Start determines the number of parallel processing threads processes that will be used by PEAKS A higher number usually lets PEAKS run faster if the computer still has extra processing power The maximum number of computing nodes PEAKS may start with is determined by the license a Desktop License allows up to 8 nodes and a Workstation License allows up to 16 nodes The configuration is very different between the 64 bit version and 32 bit version due to
123. eferences command from the menubar to open the Preferences dialog Select the preferences category from left to view the options available for that category j rs Preferences General General Display Options RMI Connections Default Input File Directory Cr Users peaks Derby Database _ Browse Raw file convertor Default Project Folder ABL wiff C Users peaks PeaksProjects Browse Bruker yep baf fid Temporary File Directory Shimadzu AXIMA run temp Varian xms Default Log File Location Wi te ol Tay ia le SERVER_LOG log CLIENT_LOG log COMPUTENODE_LOG log Search Engine Mascot Settings XTandem Settings Omssa Settings Spectrum Annotation 1 1 General Preferences Default Input File Directory Select the Browse button to change the directory that will appear when adding data to a project 157 Configuration and Preferences Default Project Folder PEAKS uses USER_HOME PeaksProjects as the default folder for project files where USER_HOME 1s the user home directory in your system Select the Browse button to change this location Please make sure this directory 1s readable writable by PEAKS Temporary File Directory PEAKS uses PEAKS_HOME_DIRECTORY temp as the default temporary file output di rectory where PEAKS_HOME_DIRECTORY 1s the location where PEAKS is installed Select the Browse button to change this location Please make sure this directory 1s readable writa
124. eptides for details of other options Select the output location and click the Export button to save the selected result components to the specified location 155 Exporting Data Reports and Printing Export Images The Annotated Spectrum Ion Match table Error Map Spectrum Alignment and the protein cov erage can be exported to an image file Refer to Section 3 2 Exporting Images for details 5 2 Exporting Label Free Quantification Results PEAKS LFQ results can be exported to other supported formats To export the quantification results press the Export button in the title bar of the Summary view panel The following export dialog will appear HTML Report Text Formats HTML Report Text Formats Report proteins in HTML with Proteins proteins csv summary view Supporting peptides protein peptides csv Let Stl _ Proteins fasta proteins fasta BILDI Lelie Feature list feature csv _ best unique PSM _ Normalization factor normalization _factor csv Collect all the above details in a single html Save into D test PeaksExport LFQ sample project LABELFREE_ 39 Browse Save into D test PeaksExport LFQ sample project ABELFREE_39 Browse Export Cancel Export Cancel HTML Report The options are the same as those for labeled quantification See Section 5 1 Exporting Labeled Quantification Results for details Text Formats The detected features will be saved as the file feature cs
125. eptides for the protein This table is similar to the peptide table in the Peptide View except that three additional columns are added e Start Shows the peptide s starting position in the protein e End Shows the peptide s ending position inclusive in the protein e Checkbox Controls whether a peptide is shown in the protein coverage as a blue bar e Unique Shows whether the peptide is a unique supporting peptide to the protein group 100 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER The supporting peptides with peptide 10lgP score below the peptide filtering threshold are listed in the table as well By default they are shown in grey color and are not displayed in the protein coverage 3 3 4 De novo Tags The Denovo Only Tags tab shows a table of de novo only peptides with sequence tags matched to the protein The de novo only peptides shown in the table are controlled by the de novo tags sharing X AAs and de novo peptides fully matched filters in the control panel 3 4 Peptide View The Peptide view displays peptide identifications that have been filtered by the current settings in Summary View The table on the top shows the details of each peptide identification The bottom section provides additional information about the peptide spectrum matches for the peptide selected in the table 3 4 1 Peptide Table Peptide Table shows the filtered peptide identification result Each row
126. erwise the residue is considered incorrect The figure shows the score distributions of correct residues and incorrect residues in two different colors Figure 3 b shows the score distribution of residues in de novo only sequences As these residues cannot be directly validated using database peptides their ratios of correctness is statistically estimated using the distributions in Figure 3 a The figure shows the estimated score distributions of correct residues and incorrect residues in two different colors As a guideline the ALC score threshold should be increased gradually until the score distributions of correct and incorrect residues are similar as in figures 3 a and 3 b In the following example the ALC threshold is gradually increased to 80 This ensures that the filtered de novo only sequences are generated from MS MS spectra that have the same spectral quality as the MS MS spectra confidently matched in the database search Figure 3 b also allows estimation of the proportion of incorrect residues in the filtered de novo only sequences 92 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER number of residues A i AAA T i numberofresidues 40 25 50 75 90 95 98 99 997 999 T5 90 95 98 94 997 99 9 40 25 50 75 90 95 9899 997 999 25 50 90 95 98 94 997 99 9 De novo residue local confidence 9 De novo seine local confidence 9 E incorect E Correct 2 E
127. ery rate FDR with the decoy fusion method Decoy fusion 1s an enhanced target decoy method for result validation with FDR Decoy fusion appends a decoy se quence to each protein as the negative control for the search See BSI s web tutorial http www bioinfor com peaks tutorials fdr htm1 for more details Enable PEAKS PTM and SPIDER algorithms after PEAKS DB database search By default PEAKS PTM performs a blind search for additional PTMs in the data Users can limit the PEAKS PTM search on a large number of PTMs by clicking the Advanced Setting button SPIDER performs homology search based on de novo sequencing tags If selected the SPIDER algorithm will be conducted on every confident de novo tag ALC gt 15 whose spectrum is not identified by PEAKS DB with high confidence 101gP lt 30 SPIDER will construct new peptide sequences by altering amino acids of database peptides For each spectrum the better sequence constructed by SPIDER or found by PEAKS DB will be used as the identified peptide SPIDER is good for cross species searches and for finding point mutations of the protein It makes no difference whether SPIDER is invoked through this workflow or by clicking the SPIDER icon on the toolbar 11 Overview PEAKS Search Predefined parameters Error Tolerance an Precursor mass 10 0 ppm v Using monoisotopic mass w Fragment ion Enzyme Specified by each sample Allow non specific deavage at one e end of the pep
128. es Section 3 2 2 Spectrum Annotation a ir a co Error da l m ir B00 600 200 400 3 2 5 Spectrum Alignment 0 5 0 5 1000 1200 1400 1600 mz The Spectrum Alignment displays how the fragment ions generated from the peptide align with the spectrum N terminal ions are shown in blue and C terminal ions are shown in red 00 3 2 6 Parent Scan The Survey tab displays the precursor ion spectrum explained above in the Spectrum Annotation section i Max yMax miz 1000 1500 The buttons that appear in this section are identical to those 69 Peptide De Novo Sequencing Jon Match Survey Intensity a 100 50 422 701 844 395 300 1000 1500 2000 ele carte eee BSA GluC 1 RAW ms 1 LA ala E12 2 Erta 7 tow intens ls RT 12 213 scan 533 TIC 5 06E7 3 2 7 LC MS Snapshot LC MS Snapshot displays a zoomed in snapshot of the peptide spectrum match in LC MS view It has three modes of viewing that can be toggled using the selection bar in the top right corner In the 2D 3D mode the view focuses on the peptide feature containing the peptide spectrum match showing the area around the spectrum s precursor m z and retention time In the All mode the view shows an overview of the LC MS run The spectrum is shown as a red square in the view at its precursor m z and retention time a 30 Al p Dja RT D 3D Al 20 20 2D i 20 3D ail 14 0 ean i 13 0 RMF e
129. ese samples are not merged into one data set Select Yes to merge all the samples into one data set 40 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow 8 6 Shimadzu Data gt RUN files from Shimadzu mass spectrometers can be loaded provided that the Shimadzu software is installed on the same computer as PEAKS Instrument Preferences for Shimadzu Data To set Shimadzu data related preferences in PEAKS open the Pref ce erences window by clicking the Preferences toolbar icon or by selecting Preferences from the Window menu Click Instrument and then Shimadzu AXIMA run in the menu on the left This will show the Shimadzu instru ment preferences on the right side Click Browse to tell PEAKS the location of the Shimadzu run2xm1 exe file Shimadzu AXIMA run Shimadzu run2xml exe File Location Browse Note Shimadzu run2xml exe can not read data files containing spaces or non english characters in its path 8 7 Varian A conversion tool is embedded into Varian s data acquisition software which allows the conversion of Varian raw data into pk1 files that can be immediately read by PEAKS The trans data files are converted in Varian programs by clicking File Save As and selecting the px1 file format or by clicking File right clicking Export and selecting pkl If you are viewing a chromatogram with the Varian software all the spectral data in the viewed chroma
130. ew project icon on the toolbar The Project Wizard will appear 2 Use the Add Data button to the files you wish to load and click Open All the selected data files will be listed on the left side o Place the selected data from the list into samples use A to place all files in a new sample use gt A to put O them an existing sample or place them in individual samples for each file using ad 4 Click the Add sample Or the Add data files respectively buttons to add a sample to the project or data files to a sample 5 For each sample specify the sample details Instrument type Fragmentation method and Enzyme name Note Each sample can use a different proteolysis enzyme Using multiple enzymes to analyze the same proteins can produce overlapping peptides and therefore increase the protein coverage Note To apply the same sample details to the whole project select the sample with correct settings and click on the Copy to Whole Project button Overview 6 Click the Finish button to create the project wi File Tools Window Help a O ACI Create Project Create Project Project Name BSA 3 Enzyme Project Location D test PeaksStudio Testing CODEN BSA_GluC RAW ra NETE ins E Ll Digest 1 sw Orbitrap Orbi Trap y CID 1 BSA_Trypsin RAW e Add data files Ll Digest 2 ya w Orbitrap Orbi Trap y CID BSAS y ra YN Data Renenent gt Frish A
131. f ferent samples will have the same precursor m z and are fragmented together In the MS MS scans labels from differ ent samples will produce different reporter ions which can then be used to calculate the quantification ratio between samples User defined labels are supported in PEAKS Q as well as commercial labels such as TRAQ and TMT The quantification analysis is based on a PEAKS DB result See Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Ensure that you have specified the isotope labels as PTMs in the database search After database search is complete follow these steps 1 Select a PEAKS DB result node in the Project view Click the Quantification toolbar icon Q Note Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for how to perform PEAKS DB and quantification in a single easy workflow 2 Select the quantification protocol as Reporter Ion Quantification specify the PEAKS quantification parameters in the dialog box on the right and click OK Wait for the analysis to finish A new quantification result node will appear at the Project view Double click the node to open the result file The quantification results with labels are displayed in a format that is similar to the PEAKS DB result A ratio is added to each quantifiable peptide and protein along with standard deviations for protein ratios 2 Setting Parameters The fol
132. f proteins not found in other protein groups A confident protein identification normally requires at least two unique peptides These protein filtering options affect only the Protein View De novo Only De novo only peptides are filtered by ALC score The ALC score is the average of local confidence score of all the residues in a de novo sequence When the highest peptide 10lgP score of all PSMs matched to a spectrum falls below the peptide threshold the de novo sequencing result on that spectrum is considered a de novo only peptide By default the threshold is automatically set and it 1s locked as the same value as the 10lgP score threshold for filtering peptides The ALC score threshold can be determined using Figures 3 a and 3 b in the Summary View For details please refer to Section 3 2 3 De Novo Result Validation 5 Exporting PEAKS Search Results The Export button in the title bar of Summary View allows exporting of the filtered results into various formats It provides the opportunity to share the report in HTML use the result as a supplemental in a publication or further validate and analyze the result in a third party software Please refer to Section 4 1 Exporting Summary Proteins and Peptides for details Note Projects can be shared by copying the PEAKS project directory It can then be opened in the free PEAKS Viewer http www bioinfor com peaks viewer index php that has the same GUI as PEAKS St
133. fied proteins and peptides can be exported to various formats by clicking the Export button at the top of the summary view More details are discussed in Section 5 Exporting PEAKS Search Results In the report several statistical charts can be examined to assess result quality The report is composed of four sections 89 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 1 Notes In the header of the report information such as a description of the experiment can be entered The notes can be edited by clicking the Notes button 2 Result Statistics Key statistics are shown in the figures and tables The figures provide important information of target decoy result validation which can be used to filter the results appropriately 3 Experiment Control Figures show the distribution of precursor mass error revealing how well the instrument is calibrated A table shows the statistics of missed cleavages in peptides revealing enzyme digestion efficiency of the protein sample 4 Other Information Search parameters and MS instrument information are shown Statistical figures and tables in the report are discussed in the rest of this section 3 2 1 False Discovery Rate FDR Curve Figure 1 shows the FDR curve with respect to the number of PSMs being kept after filtering PSMs by peptide 10lgP score By lowering the score threshold more PSMs are kept in the filtered result In the meantime the FDR increases b
134. fold change Feature vectors 1025 Quality 0 Feature vector with ID 1025 Intensity 0E0 Protein groups 50 Charge 1 Charge l0 Confident sample number 1 With peptide ID Table 3 Search Parameters Normlization Use TIC Quantification type Label free quantification Protein sigmificance ee E a m pe E Retention Time Shift Tolerance 6 0 min Protein fold change Dependent on PID 10 FDR 1 samples Controll 1 Controll 2 Control 1 Control 2 Treatm entl 1 Treatmentl2 2 Treatment24 1 Treatment24 2 Top protem groups 30 Reference Sample Auto detect Confident unique supports 1 3 1 3 Exporting Quantification Results PEAKS label free quantification results can be exported to various supported formats The summary page and the images from various views can also be exported to various supported formats Refer to Section 5 2 Exporting La bel Free Quantification Results for details 3 1 4 Additional Functions 3 1 4 1 Base Sample Selection Use the drop down box to choose the base sample The base sample will be used as the base when calculating sample ratios The group in which the base sample belongs to will be used as the base for group ratio calculations The base sample will also influence the retention time shift and M Z shift distribution figures 139 PEAKS Q Label Free RT Range 10 sRTs 154 Peptide feature Significance 20 Fold change 2 Quality 2 Avg Area 0 1 lt Charges 10 det
135. for every peptide spectrum match PSM reported by PEAKS DB PEAKS PTM and SPIDER The score is derived from the p value that indicates the statistical significance of the peptide spectrum match A peptide may be matched to many spectra resulting in multiple PSMs In that case the peptide s score is calculated as the maximum of all the PSMs For details of the scoring algorithm please refer to the publication PEAKS DB De Novo sequencing assisted database search for sensitive and accurate peptide identification Mol Cell Proteomics 2011 Dec 20 PEAKS Protein Score 10lgP The PEAKS protein score 10lgP is calculated as the weighted sum of the 101gP scores of the protein s supporting peptides After removing any redundant peptides the supporting peptides are sorted by 10lgP scores in descending order and the k th ranked peptide contributes to the weighted sum with a weight of 1 k 3 2 Summary View The summary view reports key statistics as an overview of the result It also provides functions to filter and export identified proteins and peptides 1 Result filtering Filtering rules are located at the top of the summary view Low quality peptide and protein iden tifications can be filtered by 10lgP score Peptide sequences identified only by de novo sequencing can be filtered by ALC score More details about how to properly set the filters are discussed in Section 4 Filtering PEAKS Search Results 2 Result exporting Identi
136. g the database chosen to be downloaded along with the corresponding FTP or website 3 Click OK to invoke the default FTP client software and download the database automatically Click Cancel to copy the URL to the system clipboard If Cancel was pressed click OK on the dialog detailing the copy to the clipboard Next open a browser and paste the URL into the address bar When the file download window opens click Save 4 Once the database has been downloaded check to see 1f it is compressed If so extract the file using a program such as WinZip or WinRar The desired result is a FASTA format text file fas or fasta file 52 Adding a Sequence Database Move the database file into a directory that PEAKS can access Click Browse in the Basic Options to inform PEAKS of the location of the database file If the selected database is an EST database check the box labeled EST database If not ensure that it is left unchecked Based on the selected format from the FASTA Format Database list in Step 2 the accession number information and parsing rules for the database headers are automatically entered in the textboxes in the Advanced Options Fasta Title Format section below If Other was selected in Step 2 enter the parsing parameters into the corresponding textboxes Alternatively 1f the database format 1s similar to one of the public databases such as NCBI nr the parsing rules can be filled
137. gP Mass ppm m z RT Scan FSpec Accession 114 115 116 117 PTM 1 1 144 10 EEETAAAAAVGAGGFAFHEHHEK 144 10 2778 3926 927 1393 F23 3748 Q08655 ASR1_SOLLC A Z V 144 10 LVNIEQQSPDIAQGVHGHLTK 144 10 129 70 2670 4656 1 5 891 1638 47 96 F7 3051 20 A7PRI6 METK4_VITVI 3 A 144 10 LVVHELEDDLGK 144 10 GGHELSLTTGNAGGR 126 92 3132 6365 1 8 1045 2213 52 98 F20 3469 28 065199 50DCP_VITVI L 144 10 SGGDHIHAGTVVGK 144 10 122 31 1734 9579 2 7 579 3281 38 65 F38 1909 25 P56648 RBL_VITVI F 144 10 DVYINDEDYSVSRPK 144 10 120 28 2234 1057 2 0 745 7106 46 99 F1 2393 102 P43311 PPO_VITVI T G 144 10 PLQISWNY 144 10 NYGPAGR 2080 0693 694 3660 P29023 CHIB_MAIZE 1 144 10 WFGIATAHDFESHDDITEER 118 95 2632 2295 1 2 878 4182 54 65 F11 3578 14 QOSFWA4JPSAB_PELHO 1 144 10 INEPTAAAIAYGLDK 144 10 K 144 10 117 58 2219 2830 14 740 7693 50 78 F12 2965 20 P22953 MD37E_ARATH 9 1 144 10 LNEPTAAALAYGLDK 4144 10 K 144 10 117 58 2219 2830 14 740 7693 50 78 F12 2965 20 Q9XCB1 DNAK_RHOMR O 1 144 10 INEPTAAALAYGLDK 144 10 K 144 10 117 58 2219 2830 1 4 740 7693 50 78 F12 2965 20 QOVST6 DNAK_ALCBS G 144 10 PAPLSLALAHADIDDGK 144 10 117 18 2048 1104 2 8 683 7126 50 73 F2 3107 4 Q69GY7 UCRIA_SOLTU Lcjms Peptide Protein Summar pa pl ee pot lp lp ee pa pa Note
138. h 7 ALC 94 ppm 0 7 All canddates j y y o y s RT i All Intensity 9 v T rr ffs x 20 30 nn C6 35 0 34 0 73 74 zI El 3 cs 32 ca ca 2 26 v4 b j miz 31 0 Ma adla a ax zr ErTo 080a F preprocess F low intens label lon Match 7 WCID_ETD_MIX 100625W1R51_Jekob1100625V1R intensity 5 V 1 7 1 E s k tion Mode ECD ETD c and z ions high ener Lon 9 5783 1 7564 y2 a Ya Ys Y3 Y6 b3 H20 miz 100 200 300 400 500 600 700 da da 1 1 2x J ErTol 0 8Da Y preprocess V low intens label 7 De Novo Sequencing on Single Spectrum To run PEAKS de novo sequencing on a single spectrum select the spectrum in the MS MS view of the sample click the right button of the mouse and select the PEAKS Auto Denovo command from the pop up menu Note Residue local confidence scores are NOT calculated for de novo sequences generated in this mode 75 Peptide De Novo Sequencing Orbisample mzXML E Ai DENOVO 7 auiii 11 11 pta oo Ja MARIL PEAKS Auto Denowo Correct Precursor Mass and Charge Export As DTA Export As PEL t De T hase bis Alec eat Map M5 M5 8 Manual De Novo Sequencing PEAKS provides a set of tools to help manually sequence a peptide from the spectrum Note Manual de novo sequencing does NOT support ETD spectra 8 1 Manual De Novo Graphical User Interface To cre
139. h along with protein coverage of their identified peptides in the window below The quantification ratios of the quantifiable proteins are displayed in the ratio columns with their label names as the header eg 114 A protein is considered quantifiable when identified by a unique peptide above the peptide score threshold set in the parameters The ratio is calculated from the unique peptides of the protein Proteins with no unique peptides are not considered to be assigned a ratio The denominator sample can be changed from the Show drop down menu in the Summary view The normalization mode can also be selected in the Summary view see Section 3 5 Filtering Quantification Results The SD is the standard deviation of the peptide ratios in the protein The peptides of the selected protein along with their ratios are displayed at the bottom half of the Protein view 125 PEAKS Q MS MS Level 5 Show top w proteins in each group accession contains search Q O noresults E E Fi Accession 10lgP Coverage Peptides Unique Avg Mass Description 114 115 116 117 SD 114 SD 115 SD 116 SD117 Mark c 5 0 Proteins Y la P43311 PPO_VITVI 67347 Polyphenol oxidase chloroplastic OS Vitis vinifera P z P56648 RBL_VITVI 313 07 m MIN Imm I l3s 18 2 52519 Ribulose bisphosphate carboxylase large chain OS 1 0 716
140. he samples then the measured intensity should not change too much even though it may have some random variation Small variation means the peptide can be well quantified Let to be the estimated intensity fold change of the 1 logs peptide across the samples when the peptide abundance does not change The quality is calculated as 20 Thus if a peptide has an expected 2 fold change then its quality will be 1 if a peptide has an expected 4 fold change then its quality will be 0 5 A high quality value implies that the peptide can be well quantified Significance It is assumes that for all the peptides with the same quality their ratios follow a log normal distribu tion Different qualities may have log normal distributions with different variances The 2 tail P value is calculated as the significance of the feature vector using the log normal distribution corresponding to its quality and its maxi 10log P value mum group ratio is finally displayed as the significance in the column m z Each feature in the feature vector has its own precursor mass to charge ratio This column displays the average precursor mass to charge ratio of all the features in the feature vector 141 PEAKS Q Label Free e RT range The retention time range of the feature vector e z Precursor charge e Avg Area Each feature in the feature vector has its own intensity area under the XIC curve This column displays the average area of all
141. he Spectra data in mzXML format for use with Skyline peptides pepXML peptides pep xml Save into D test PeaksExports Sample project_DBSEARCH_10 Browse Export Cancel The export options are grouped into HTML Report Text Formats and For Third Party based on the output format Select the items that you want to export from the available options Select the output location and click the Export button to save the selected result components to the specified location HTML Report This will generate single or multiple HTML reports in the specified location After the completion of result exporting the index file for the reports protein htm1 will be opened in the default browser automatically The following exporting options are available e Summary view The Summary view page will be saved as a summary html file in HTML format in the specified location e Protein coverage The coverage pane will be saved for each protein e Supporting peptides A list of supporting peptides will be saved for each protein e Best unique PSM The best unique PSM will be saved for each protein An individual protein will have its own HTML output file where the corresponding protein coverage supporting peptides and the best unique PSM are gathered Select Collect all the above details in a single html to collect all the protein reports in a single protein html file 153 Exporting Data Reports and Printing Text
142. he figure a significance 10lgP a 50 sass pg EEEE SEEE E E EEE A o nn AS 4c siti i i gl i i i sl Sl inl ii AAA mm i el mee ww i iu irk 30 Ja oer ca tas eer eee an as nl ss a ie as a nado eon a a is Gm 20 jee yt He A pe A ee A A A e 10 al q A A e e e e a e e e e e e e e e 1m e m ae e e e e e aae e 2m ae e e ae e e HE H 1 4 112 1 7 4 a Ratio 134 PEAKS Q Label Free Whenever a filter value is changed the Apply Filters button changes its color as a reminder Click it to apply the filters 3 1 2 Summary Report 3 1 2 1 Heat Map Only those protein groups which passed the filters are displayed in the heat map The relative protein abundance is represented as a heat map of the representative proteins of each protein group The representative proteins are clustered if they exhibit a similar expression trend across the samples The hierarchical clustering is generated using neighbor joining algorithm with a Euclidean distance similarity measurement of the log2 ratios of the abundance of each sample relative to the average abundance An interactive protein profile heat map pops up in a window when the Heat Map button is clicked Right click on the heat map to save a copy of it pu pr pr Eu Gu gu T T T T T T T T T T log ratio EE E E FP51985ups UB2E1_HUMAN_UPS 40 P00441ups SODC HUMAN _UFS P83185ups SUMO1_HUMAN_UP P01127ups PDGFB_HUIMAN UPS PO0187ups CYB5_HUMAN_UPS di Q06820
143. he spectrum In the Spectrum Annotation panel click and drag the mouse horizontally The selected area will be enhanced and shown in the Spectrum Annotation panel Click the 1 1 button to return to the default view Setting removing ions to from a peak Select a peak and right click anywhere in the Spectrum Annotation panel Select Set y ion from the pop up menu to designate the peak as a y ion or Set b ion from the pop up menu to designate the peak as a b ion Click on Remove ion to remove the ion that you have previously set a i ror 1 intercity ed 1585 79 1375 71 Set y ion Set b ion Remove bon Set other ions eee 55 63 pa 1150 64 50 Right tags 1 6 Rightmost y 10n Leftmest y ion 226 12 246 18 Leftmost b 1on 1604 81 Rightmost b ion 1459 76 Dl bel Te a LA re A AA MAN DL A O A LE 00 400 B00 300 1000 100 A AAA A a Tintan hah DrbiSample maXML ms 2 mz 602 90607 Ma yl 1 2 21 ErTo 0 5Da 9 intensity threshold Eo a TIC 1 79E7 1400 1600 Select Set other ions from the pop up menu to view the Ion Editor dialog box The Ion Editor dialogue allows addition or removal of ion designations to from a peak Select either C Term Ion or N Term Ion to see the C and N terminal ions respectively Then select an ion from the ion list and press the Add button to add it to the selected ion 79 Peptide De Novo Sequencing list Remove an ion from the selected ion list b
144. hreshold used for de novo only is locked to be the same as the 10lgP threshold used for filtering peptides To specify a different value first click the lock icon to unlock it After the filtering conditions are changed the Apply Filters will change to red Click it to apply the new criteria Overview StartPage X W PEAKS 44 2013 10 02 13 47 x Peptides 10loP 15 w FOR Proteins 10lgP 20 yl and 0 y unique peptides summary De novo only ALC 50 wy and 10lgP F 15 Apply Filters Export Notes The top control pane has two additional buttons Export and Notes The result can be exported by clicking the Export button The Notes button allows the user to add a text note about the project that will be displayed in the result summary report After applying filters the statistics report page at the bottom of the summary view will be updated accordingly We only explain two statistical charts here see screenshot below Figure 2 a shows the PSM score distribution in a stacked histogram If the search result and the peptide 10lgP score threshold is of high confidence then very few decoy matches brown in the high score region should be observed Additionally if the FDR estimation method decoy fusion worked properly then a similar or larger number of decoy brown matches than target blue matches in the low score region should be observed Figure 2 b plots the precursor mass error in ppm v s 10lgP peptide score fo
145. iew panel and select the Add Data command a from the file menu or use the add data button E from the toolbar The Project Wizard window will open 34 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow Create Project Project Name New Project 1 Project Location D test ES Sample Enzyme Instrument Fragment EJ Sample 2 Trypsin yw Orbitrap Orbi Trap y CID oll Spike4_040806_3 RA i dp Add data files Le Add sample Select All remove Collapse SelectAll Copy to Whole Project Finish 3 Use the Add Data button to browse to the location of the data files select the files you wish to load and click Open All the selected data files will be listed on the left side 4 The selected data from the list on the left can be placed into an appropriate sample in the project structure There are several options to place the selected data files into the project structure All of the selected files can be placed in a new sample In an existing sample E each file g5 5 More files can be added to an existing sample by clicking the Add data files button Additional samples can be created by clicking the Add sample button or they can be placed in individual samples for 6 Select the Instrument type Fragmentation method and Enzyme name that was used to generate the experi mental data from the drop down lists For more information on adding f
146. iles samples or setting up the instrument configuration refer to Section 2 Create New Project Note It is discouraged to change the Enzyme Instrument or Fragmentation setting for an existing Sample It may cause errors during further analysis or inconsistencies in the analysis result 7 Supported Data Formats The following is a list of supported data formats in PEAKS PEAKS supports these formats at three different levels 35 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow e Native Support PEAKS can read the following files directly without any additional tools e mzXML e mzData e mzML e DTA file or a directory of DTA files e MGF e PKL e PEAKS 6 projects See Section 8 8 PEAKS 6 Projects e Library Level Support The instrument vendor s software library is required to be installed on the same computer as PEAKS PEAKS will call the software library to read the data directly e RAW file Thermo Fisher Scientific instruments See Section 8 1 Thermo Data e D directory Agilent instruments See Section 8 3 Agilent Data e LIFT or D directory Bruker instruments See Section 8 5 Bruker Data e RAW directory Waters QTOF instruments See Section 8 2 Waters Micromass MassLynx Data e Converter Level Support Third party converters are required Users need to install the required converters correctly and let PEAKS know their locations This only n
147. in sequence coverage This area visualizes the coverage of the protein sequence Regions in the protein sequence that are covered by supporting peptides are displayed in bold font with a grey background Confident modifications and mutations identified in supporting peptides are displayed as icons above the protein sequence Modifications are represented by colored icons with the initial letter of its modification name If a residue is modified by more than one modification in the same supporting peptide is used instead of a letter Mutations are represented by framed white icons with a letter indicating the post modification residue Placing the cursor Over a mutation icon shows the full name of the modification mutation The number above a mutation icon indicates the position in the protein sequence The header information of the protein is shown on the top of the protein sequence The supporting peptides and matched de novo tags can be shown as colored bars under the protein sequence Placing the cursor over a bar shows detailed information of the peptide Left clicking on a bar shows the annotated spectrum from which the supporting peptide or de novo tag 1s identified Right clicking on a bar shows a pop up menu for quick operations including Remove peptide which hides the peptide from the protein coverage view Hidden peptides can be restored by re selecting them in the supporting peptide table or the de novo tag table gt sp P02769 ALBU_BOVI
148. in the plots To display other features select the corresponding sample using the check box in the table 3 3 3 Sample Features This tab displays a close up LC MS view of the detected features from the selected samples The feature boundary is shown by a bounding polygon The center of the feature is denoted by a solid red dot on the mono isotopic peak cluster Placing the mouse cursor on this center of a feature pops up a tool tip that shows the m z value mean retention time charge state and intensity The centers of the features are used to calculate the retention time alignment The A button at the top right corner of the tab can be used to toggle between the 2D LC MS view and the 3D LC MS view Feature Details Sample Features RT Alignment V m z 812 4778 RT mean 74 00 o z 2 E o al 812 813 814 815 mfz 812 Intensity 2 7E7 gj4 815 m z RT F7 B08 09062 RAW x Show Controli_2 y 142 PEAKS Q Label Free 3 3 4 RT Alignment This tab displays the aligned TIC curves of the selected samples with the highlighted retention time range of the selected feature Use the toggle switch in the top right corner to switch between the before and after RT alignment view Click the lv button to view the pairwise RT alignment in a pop up window Select the sample pair from the drop down list to view the desired pairwise RT alignment Feature Details Sample Features RT Alignment dh Ea 9 x
149. in the table is a peptide identification repre sented by its highest scoring PSM The table can be sorted by any column A search function is also provided When there are more than 1000 peptides the table is split into multiple pages Note If there are multiple PSMs matched to a peptide the table displays the information about the top scoring PSM Peptides with the same sequence but different modifications are considered different and shown in different rows The Spec column shows the number of PSMs matched the peptide Other PSMs can be examined after selecting a peptide in the table See Section 3 4 2 Peptide Spectrum Matches for details J A 1000 of 1263 aa san ah QU noresults Peptide 10lgP Mass ppm mz RT Scan F5pec Accession PTM Found By 1 T 445 0 D VMENFVAFVOKC 57 023 C 101 22 3350 4714 ALA Dero 32 Fiz 4 PO2759 ALBU_BOVIN ca PEAKS PTH 7 2 O 57 02 C 57 02HGDLLEC 57 0 F3 1944 1 POZ76SlALBUBOVIN BEEE PEAKSDB TS ECC S7 02 57 02 H 57 02960 Frigo 1 PO276S AL6U_BOVIN IBIBIS PEAKSPTM C 57 02 FL SHKDDSPDLPKLKPDPNTL F3 1552 8 POZT69IALBU BOVIN BIB PEAKSD 57 02 ADDRADLAKYIC 57 02 31 18 e3 1708 9 PO276S ALEU_EOVIN BE PEAKS DB 5 O 57 02 C 57 02 AADDKEAC 57 F1 1245 10 PO276SIALSU BOVIN BBB _PEAKSDB 7 DYASLIENRLC 57 02MHERTPVSE 78 76 2498 3203 1 5 833 7794 45 55 F3 2611 1 PO2769lALBU BOVIN E PEAKSDS 3_ VH
150. ing The PEAKS de novo sequencing algorithm and scoring functions are constantly tuned for each instrument type to ensure optimal accuracy Fast sequencing speed PEAKS can perform de novo sequencing on over 10 spectra per second on a moderate PC and even faster on servers Easy to use user interface PEAKS generates a comprehensive result summary and provides interactive views of annotated spectra Support of all major instrument types PEAKS supports de novo sequencing of spectra generated by all instrument types Orbitrap FTMS Ion Trap Time of Flight Quadrupole in common fragmentation modes CID HCD ETD PEAKS can also perform de novo se quencing on complementary CID HCD ETD spectral pairs or triplets Automated result validation PEAKS assigns a local confidence score for each amino acid in de novo sequences The local confidence score ranges from 0 to 99 indicating how confident the algorithm considers a particular amino acid is correctly sequenced Moreover the peptide sequence is evaluated by ALC Average of Local Confidence score ALC 1s the average of the local confidence score of all the amino acids in the sequence Note For details about the PEAKS de novo sequencing algorithm please refer to the initial publication PEAKS Powerful Software for Peptide De Novo Sequencing by Tandem Mass Spectrometry Rapid Communication in Mass Spectrometry 17 20 2337 2342 2003 In addition to automated de novo sequencing PEAKS also
151. ing new enzymes 2 3 PTM To select PTMs for de novo sequencing click the Set PTM button to open the PTM Options dialog Name i Mono mass Residue site IEA F Carbamidomethylation 4 hydroxynonenal HNE 156 1150 CHK A Acetylation 0 aoo SKE Acetylation N tern Acetylation Protein N term Amidation Ammoniadoss CGN term Armonia loss Protein N term Beta methylthiolation Biotin ylation 226 0776 Carbamidomethylation 57 0215 C Carboxylation E 43 9898 oH Carboxymethy Selected Variable PTM Gitrulination QU Oxidation 4 Deamidation NO QU Deamidation No Dehydration e Dioxidation M ass 7 FAD gt Famesylstion Form ylation Formylation Protein N term Geranyi gerany Guanidination aos SCS ae aso O Recent Common Uncommon Artificial Customized View New Remove Remove All Switch Type Ok Cancel PEAKS includes modifications in the Unimod database Unimod modifications are categorized into three lists e Common The Common list contains commonly observed modifications including naturally occurring post translational modifications modifications induced in standard sample preparation and common artifacts e Uncommon The Uncommon list contains less commonly observed modifications including rare post translational modifica tions residue substitutions and rare artifacts e Artificial The Artificial lis
152. ion shows a graphical representation of the spectrum the peaks in the spectrum the user selected peaks and assigned ions Pick a peak on the panel with the cursor and assign ions or tags to it in manual de novo e Ton Table Panel The Ion Table shows the proposed ions with their corresponding masses The default Ion Table will display immonium b b H20 b NH3 y y H2O and y NH3 ions e Spectrum Alignment and Error Map Panel The Spectrum Alignment shows how the proposed ions assigned in manual de novo align with the spectrum By default the Spectrum Alignment displays b ions and y ions The b ions are shown right to left in blue while the y ions are shown left to right in red The Error Map displays the confidence assigned to each ion e Tag Panel The Tag Panel will appear when you search tags or ions in the spectrum You can select the tags in the list using the Select button Clicking Apply will add the selected tags to the sequence candidate 8 2 Manual De Novo Operations When the mouse cursor is placed in the Spectrum Annotation panel a green by default triangle follows the move ment of the mouse This is the Position Bar and it is used as a cursor for all manual de novo operations The cursor s position on the m z scale and its relative intensity are shown in a pop up window on top of the Position Bar 77 Peptide De Novo Sequencing intensity 55 10D 1585 79
153. ion to search with unspecified modifications Database Select a protein sequence database for the search Select one from the list of databases that have been configured in PEAKS and set the taxonomy if applicable To configure a new sequence database refer to Chapter 6 Adding a Sequence Database For searching only a few proteins choose Paste sequence and then paste in the protein sequences Database Select database gt P62258 1433E HUMAN 14 3 3 protein MDDREDLVYYQARLAEQAERYDEMVESMEEVAGMDVELTWEE NLLSVAYENVITGARRASNWE ORE ENE GGEDELEM Note The pasted protein sequences should follow the FASTA format Each sequence follows a description line which starts with gt PEAKS omits invalid characters white space and numbers in a sequence A maximum of 1000 protein sequences can be pasted De novo Tag Options PEAKS Search requires a de novo sequencing result to improve its search speed sensitivity and accuracy By default de novo sequencing runs on the fly with the current parameter settings It is also possible to select an existing de novo sequencing result Estimate FDR with decoy fusion Select this option to enable search result validation with decoy fusion an en hanced target decoy approach When this option is selected decoy sequences are automatically generated from the target database and are searched by PEAKS DB PEAKS PTM and SPIDER This enables the estimation of the false 87 Peptide PTM and Mutation Ide
154. ionally the data provides wrong or no charge information for the precursor 10ns This function attempts to correct the charge information e Low quality spectrum removal this function attempts to remove the junk spectra This will save some analysis time Use this function with caution as it may also remove a small portion of identifiable spectra e Centroiding charge deconvolution and isotope deconvolution centroiding the peaks and deconvolution of multiple charge ions to singly charged ions in the MS MS scans If the data is not refined within PEAKS most analysis functions such as de novo sequencing or PEAKS DB will ask you to input the refinement parameters before the analysis is done You can run the data refinement function separately by selecting a fraction sample or project on the project tree All the fraction s under the selected node will be refined The use of this function is outlined in the following L s Select a project sample or a fraction node Click the data refinement button a on the tool bar 4 E BBEJs 14 890 2 Specify the data refinement parameters in the popup dialog and click OK Most of the parameters are self explana tory and the default parameters provide a good starting point for the analysis 3 Wait for the analysis to complete A new Data Refinement node will appear in the project tree Later analysis on this fraction will be based on the refined data ry Project View Lg D test OtherProjects
155. ions All available samples are listed on the left side of the panel All of the selected samples in the left side list can be added to a new group in the group table by clicking gt pan or can be added to an existing group by clicking and selecting the group from the drop down list Selected groups or selected samples in the group table can be removed by clicking for rearrangement of the samples The groups are color coded by the color column in the group table These colors will be used in the heat map of the Summary view to distinguish the groups Click the Advanced button to open the advanced settings dialog where the reference sample and training samples can be selected manually The reference sample will be used as the center to do the retention time alignment The training samples are used to calculate the feature vector quality For training samples biological replicates are preferred In both cases auto detection should fit most cases 129 PEAKS Q Label Free Reference Sample Reference sample will be used as e center to do retebon bime algament Auto wetecton shoud ft most cases Training Samples Auto detect W Auto detect W Feature vector qualty is calouated based on a par of baning samples Goveoicates are preferred to be used as baining samples Auto detechon shoud Fi most cases To attach the identification results to the label free quantification result click on Attach button in the Attach Iden
156. ir uses in detail 4 1 Noise Level Select the appropriate threshold for noise filtering Once selected the Heat Map view will reflect the changes 4 2 Show Hide Features To view the detected LC MS features mark the Features checkbox in the control The features detected during the data refinement steps will be highlighted by small red diamond shaped points on the heat map Placing the cursor on such a feature point will show the boundary of the feature using a red polygon and display the information regarding the monoisotopic m z mean RT charge state of the feature and the total area under the XIC curves of the feature in a pop up window 47 Data Visualization 30 0 m z 656 3986 RT mean 49 51 z 2 Area 8 09E6 49 0 bob bos uo bod 660 661 4 3 Locate M Z and Retention Time La Input M Z and retention time such as 618 88 50 5 in the following text box to locate a M Z retention time position in the LC MS view 4 4 Synchronize LC MS View between LC Runs Use the button to synchronize LC MS view between different different LC runs When the LC MS views are synchronized the M Z retention time position and noise level are locked as the same in each run 4 5 Show Hide MS MS Spectra Select the MS MS Spectra checkbox to highlight the scans with associated tandem scans by marking them with tiny blue squares on the heat map Place the cursor on an MS MS spectra indicator to view the precursor m z
157. is results Figure 3 The distribution of feature vector ratio a By quality b By intensity a Ratio Ratio 112 14 118 1116 0 0 25 5 0 75 10 0 12 5 15 0 17 5 20 0 Quality Avg Area 3 1 2 5 RT and m z Shift Distribution Figure 4 a plots the retention time shift distribution to the base sample The distribution of the m z shift in ppm of the data with respect to the base sample is plotted in Figure 4 b These two figures can be used to refine the parameter settings for label free quantification 138 PEAKS Q Label Free Figure 4 a RT shift distribution b M Z shift distribution Y a b 70 000 A O 8 O O O ees go O 0 cece cecsessesssssssessss A nz Clete eee EESE 15 000 gn sete EOE ee eke eee eek eee 2 2 12500 A BY a a 10 000 O D a Lc u 7 500 4 MUG a Aa E EE A A 18 18 5 0 al 10 15 20 MZ Shift ppm A Before RT alignment W After RT alignment E M Z Shift 3 1 2 6 Summary Tables Tables 1 and 3 list the filtration parameters and the label free quantification analysis parameters respectively Table 2 contains statistics regarding the feature vectors and protein groups in the filtered result Table 1 Result filtration parameters Table 2 Statistics of filtered result Retention time Features 7184 Retention time 140 Features with ID 2714 Feature
158. izard from the menu the configuration wizard will run and display a welcome message Clicking the Next button shows the following instrument selection panel 24 Configuration Wizard Configure Instruments and Public Databases Instruments Thermo data can be loaded using Thermo MSFileReader library The config wizard will download and install the MSFileReader automatically AB SCIEX AB 5600 Instruments AB 4700 4800 Instruments AB SCIEX OTRAP Instruments AB SCIEX OSTAR Instruments Agilent Technologies _ Agilent Ion Trap Instruments Agilent Q TOF Instruments Bruker Daltonics Bruker APEX micrOTOF HCT Ion Trap TOF TOF Instruments Shimadzu Corporation _ Shimadzu AXIMA CFR Instruments Waters Corporation _ Waters Micromass Q TOF Instruments Varian Incorporation _ Varian Mass Spectrometry Instruments lt lt Back Next gt gt Cancel Help The instrument selection panel lists all the supported instruments and their vendor specific software Select the instru ments as necessary A small description about the instrument and possible action to be taken by the configuration wizard is displayed once it is selected Click Next to proceed 3 Database Selection The database selection panel lists the public databases available for automatic download The automatically down loadable databases are NCBI nr SwissProt TrEMBL and IPI human Select the databases to be configured A small description of
159. label This will bring up the Export Images dialog for selecting the result items to export Image Types 5 Error Map Ion Table and Error Map 8 Current Spectrum View 5 Spectrum Alignment View 7 Annotated Spectrum with Alignment Image Options Basic Options Advanced Options Web Smallest images suitable for viewing online O Print Oversampled images suitable for printing Save As D ltestiPeaksExportlimages 516 79297 2 png Select the desired result elements from the Image Types list The Basic Options tab offers choices for the location to save and the image size Web Smallest images suitable for viewing online or Print Oversampled images suitable for printing 151 Exporting Data Reports and Printing Basic Options Advanced Options Scale 100 S Oversample 1 Xx File Format png Width 1 398 Height 344 The Advanced Options panel offers choices for editing the scaling factor file format resolution and oversample factor PEAKS supports BMP GIF JPEG PNG and SVG image formats After setting all parameters click the OK button to export the selected result item to an image 4 Exporting Database Search Results The exporting mechanism for PEAKS DB PEAKS PTM and SPIDER results are the same as that of a de novo result with the exception of the number and type of available exporting options All exporting functions are availa
160. lder 32 bit operating system It is strongly recommended to use PEAKS 64 bit version on 64 bit operating systems Due to the memory limitation for 32 bit Java a typical configuration for a computer with 4GB RAM looks like the following C Automatically Configure Performance recommended Main Program JVM Heap Size MB 1024 Number of Computing Nodes to Start 1 Start Client Separately Client JRE Binary Folder fre bin Client JVM Heap Size MB 1024 Start Compute Node Separately Computing Node JRE Binary Folder fre bin Computing Node JVM Heap Size MB 1024 The total amount of memory used by PEAKS is Computing Node JVM Heap Size MB multiplied by the Number of Computing Nodes to Start plus the Main Program JVM Heap Size MB In this case 1024 1 1024 2048 MB In some systems PEAKS may not start with 1024MB assigned to the main program due to other programs running on the system Lowering the Main Program JVM Heap Size MB to 800 may help solve the issue 22 Installation and Activation 6 What s Next Depending on the data formats and the type of analysis needed two additional configuration steps may still be needed before data analysis can be conducted e To read the instrument s raw data formats installation of specific software libraries to support the instrument may be required See Section 8 Vendor Specific Requirements for this requirement e To conduct a database search for protein ide
161. line overage pna Browse Export Cancel 5 Exporting Quantification Results PEAKS Q labeled and label free quantification exporting functions are also similar to that of de novo or PEAKS DB All export functions are available through the Summary view panel 5 1 Exporting Labeled Quantification Results PEAKS Q results can be exported to other supported formats To export the quantification results press the Export button in the title bar of the Summary view panel The following export dialog will appear HTML Report Text Formats HTML Report Text Formats Report proteins in HTML with Proteins proteins csv summary view Supporting peptides protein peptides csv id E C Proteins fasta proteins fasta supporting peptides _ best unique PSM Collect all the above details in a single html Save into D test PeaksExports Sample project_QUANTITATION_ Browse Save into D test PeaksExports Sample project_QUANTITATION_ Browse Cancel Export Cancel HTML Report The options are the same as those for PEAKS DB For PEAKS Q except that only the quantified proteins are used in the exporting See Section 4 1 Exporting Summary Proteins and Peptides for details Text Formats The text format exporting options are similar to those available for PEAKS DB For PEAKS Q except that only the quantified proteins and peptides are used in the exporting See Section 4 1 Exporting Summary Proteins and P
162. location to which the DTA files will be exported For MGF and PKL exports the dialog will ask for a name and a location for the file Selecting Export MzXML File command will open the Export mzXML File dialog FY Export mzXML File atartRT 0 End RT 120 Save as D test Celegans_FT_d6ITODDT_01 mzxml ox Cancel 148 Exporting Data Reports and Printing Enter the starting and ending retention times in the appropriate boxes Then click the Browse button to select a des tination to save your file 2 Exporting Result From Project View Fraction information and peptide identification results can be exported to Excel or HTML format from the pop up menu in the Project View Right click on a project node a sample node or a fraction node and select the options Export Result to Excel or Export Result to Html The exported result contains fraction information PTM information and a list of peptide identifications of the selected fractions Project view E D test Peaks60 Pec cation E 4 Protein Standard Data Refine Be Fl identificat Replicate Analysis ie DATA RE a y DENOVO e PEAKS 3 Peaks Database Search AR PEAKS Po Spider Search SPIDER E B l JL iPRG2011 ETD in horus Search ria F3 D100930 AW 4 DATA RE Export DTA file dy DENOVO Export PKL file PEAKS 10 Esport MGF file PEAKS P Export Result to Excel men SPIDER 7 El T D test Peaks650 Pea Export Result to
163. lowing condition De novo ALC score greater than 15 recommend 15 105 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 6 2 Running SPIDER on a PEAKS DB or PEAKS PTM Result Launch SPIDER by selecting a PEAKS DB or a PEAKS PTM result and clicking the SPIDER icon on the toolbar AK Running SPIDER on a PEAKS DB or a PEAKS PTM result is functionally equivalent to running both searches together in an integrated search However the parameters panel in this case appears as follows and allows for a few additional options SPIDER Search Predefined parameters F Carbamidometnyiaton T Oxidation RAY A Fl Oxidation MI Qf Deamidation NQ Filter Perform SPIDER search on spectra satisfying the following condition De novo ALC score greater than 15 M recommend 1596 Note For users of previous versions of PEAKS SPIDER in the current version runs in the mode previously called Homology Match PTM Clicking the Set PTM button will bring up a separate window for choosing modifications The PTM config uration is the same as it is in de novo sequencing Refer to Section 2 3 PTM for details Filter The filtering option asks for the minimum de novo tag score ALC for a spectrum to be analyzed in SPIDER If the ALC score of a spectrum is too low the spectrum is unlikely to provide a significant hit 7 Comparison of PEAKS Results It is possible to compare up to three search resu
164. lowing parameter dialogue pops up when clicking the quantification tool bar ee Select Reporter Ion Quantification eg 1TRAQ from the left side under the Quantification heading in the quantification window Enter the relevant MS MS level labeling quantification parameters on the right side of the window 123 PEAKS Q MS MS Level Quantifications Reporter Ion Quantification Predefined parameters he Basic Options Mass Error Tolerance 20 0 ppm w Peptide Score Threshold 10logP 20 Label Free Select Method Quantification Method Detail A TRAQ Aplex y Name TRAQ 4plex Method Type Reporter Ion Quantification a Reporter Ion Quantificatio Modification Target New N Terminal Modification C Terminal Modification nm Precursor Ion Quantification eg SILAC ay aE al lificati Side Chain Modification at Modification Mass 144 10207 Label Options Name Reporter Ion Mass Da 114 114 1107 115 115 1077 116 116 1111 117 117 1144 OK Cancel Help The following parameters are available in the Basic Options section of the quantification window e Mass Error Tolerance This parameter is used to locate the reporter ion peaks in the MS MS spectrum A little wider tolerance than the fragment ion error tolerance set in PEAKS DB is recommended e Peptide Score Threshold 10lgP Peptides with a score above this threshold are used to quantify the identified proteins Peptide level quantification results are still sho
165. lt before closing the project For details on exporting please refer to Section 7 5 Exporting Comparison Result 7 2 Peptide Comparison Peptides identifications from the compared search results are displayed in the table For each peptide the table shows m z retention time peptide 10lgP score charge and whether there are multiple PSM hits The Coverage Map column graphically illustrates which results the peptide is identified from A solid icon indicates the peptide is identified Using the control panel in the bottom each result can be filtered separately by setting the peptide 10lgP score threshold It is possible to to show only the common peptides shared by all results or the unique peptide in one result The following screenshot is the peptide table in a typical comparison result 107 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER cae eC A MS TENI M pr ss ee a weet 495 291 EEEN es rue su 35 0541 i 785 90533 35 4252 513207 16 5345 pa 315186 35 663 1 13 25 2 oe O 115 02 Tae gl 865 2152 43H 2 AVVDUEPGTM 15 95 050R EEE PEE ae 57 27 809 4083 34 5133 a LOOL AWNEENQIR a Sassy i D ee ona 3657 3 0168 a mam oS lL L O le a U ADINMLGTIAS EEM 6214556 3 11 5 2 2 true 621 8543 39 0524 56 05 2 S2L8541 37 9902 55 72 7 3 P
166. lter the results The results will be updated in the Summary view the Protein view and the Peptide view accordingly StartPage X df QUANTITATION 4 2013 10 10 15 01 x Proteins with fold change 1 w E E A A TE A Show ratio to Light w Normalization factor no ha 1 1 1 Apply Filters Export Notes z ratio to Light auto ratio to Medium log2 ratio Sn to Hea Q7LBR1 CHM1 cL Heavy QSPFW 1 wipes ee 40 E intensity QENIOS NCOAT HUMAN O78M98 CCD3 HUMAN E rc PF02851 ROAT_HUMAN 4 Exporting Quantification Results gt PEAKS Q results can be exported to other supported formats All export functions are available through the Summary view panel To export the quantification results click the Export button in the title bar of the Summary view panel Refer to Section 5 1 Exporting Labeled Quantification Results for details 122 Chapter 12 Reporter lon Quantification e g ITRAQ and TMT 1 Overview Reporter ion quantification with isotope labels at MS MS level is one of the three quantification modes that are sup ported by the optional PEAKS Q module of PEAKS Studio This is based on the relative intensities of fragment peaks at fixed m z values within an MS MS spectrum In this mode isotope labels with the same mass are introduced to several samples The samples are then analyzed together in an LC MS MS experiment The same peptides from di
167. lts in the same project To compare results press Ctrl to select two or three result nodes right click and choose Compare Results from the pop up menu PEAKS will then start to compare the results A D temp LFQ_Heatmap_Blank O LABEL FREE 13 27 Apr 11 21 25 E J Sample 1 Ey PanTumorScxLRAW DATA REFINE 2 27 Apr 11 15 50 22 jy DENOVO 6 27 Apr 11 17 05 a J Sample 2 Compare Results Ey PanTumorScx2 RAW DATA REFINE 1 27 Apr 11 Delete Result iA DENOVO 5 27 Apr 11 16 30 SPEAKS 9 27 Apr 11 19 00 106 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 7 1 Comparison Result When comparison is completed a comparison result node is added to the project as shown in the screenshot The comparison result is opened automatically The result consists of three parts peptide comparison protein comparison and statistical charts SGU D temp LFQ_Heatmap_Blank y Compare run 9 10 12 20 May 11 16 45 C LABEL FREE 13 27 Apr 11 21 25 EJ Sample 1 El PanTumorScx RAW DATA REFINE 2 27 Apr 11 15 i DENOVO 6 27 Apr 11 17 05 f PEAKS 10 27 Apr 11 19 14 Jl Sample 2 Er PanTumorScx2 RAW DATA REFINE 1 27 Apr 11 15 i DENOVO 5 27 Apr 11 16 30 F PEAKS 9 27 Apr 11 19 00 Er i Sample 3 Ef PanTumorScx3 RAW DATA REFINE 4 27 Apr 11 15 Note The comparison is done on the fly and the result is not saved with the PEAKS project It is suggested to export the comparison resu
168. mbers of modifications present in the peptide shown in color coded icons e Found by The name of PEAKS search tool that identifies the peptide can be either PEAKS DB PEAKS PTM or SPIDER 3 4 2 Peptide Spectrum Matches This section displays the spectrum annotation and other information about the peptide selected in the Peptide table When multiple spectra are matched to the peptide the information about the top scoring PSM is shown by default Other PSMs can be viewed by choosing from the pop up list shown after clicking the All matches button The list of proteins that contain the selected peptide can be examined by clicking the Protein button Jump to the protein in the Protein view by clicking a protein in the pop up list The interface of this section is identical to the Spectrum Annotation in the PEAKS de novo result For detailed instructions please refer to Section 3 2 De Novo Peptide View Scan F2 1249 m z 631 7892 z 4 RT 25 19 10ig 159 36 ppm 2 2 All matches Protein menos qE pe JEn Je fe fE fE fs Ju k o o 5 po Ja er 630 635 Fraction D Yyang_data aowen pa n AAA Ena a a Rel y Liu BSA ysC 1 RAW y 12 4 16 i 2 200 2400 Sean A i 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 00 Fragmentation Mode CID CAD y and b ions aS 4 1 2X 2Y ErrTol 0 05Da v preprocess Y low intens label TIC 9 725 lon Match Survey b bH20 3 b NH3 b 2 Seq y y 20 y NH3 y 2
169. ment Fragment az AI Ee J Sample 1 Glu C phosphate y Orbitrap Orbi Trap y HC 1 BSA_GluC RAW ede Add data files Add sample Select All l Collapse Select All Copy to Whole Project Data Refinement gt inis Cancel 30 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow 2 Create New Project 1 To add a sample to the project structure click on the ot Add data files To create a new project select New Project from the file menu or click the new project icon Le on the toolbar The Project Wizard will appear Use the Project Name field to name your project Click Browse to select a directory for saving the project The selected directory will appear in the Project Location text box Note Refer to Section 9 Changing the Default Project Location for changing the default save location for projects Use the Add Data button to browse to the directory of the data files select the files you wish to load and click Open All the selected data files will be listed on the left side The selected data from the list on the left can be placed into appropriate samples in the project structure There are several options to place the selected data files into the project structure All of the selected files can be placed in a new sample in an existing sample ME each file eS or they can be placed in individual samples for el ela
170. mzid in mzIdentML format version 1 0 0 Peptides mzidentml version 1 1 0 A list of peptide spectrum matches will be saved to peptides_1_1_0 mzid in mzIdentML format version 1 1 0 Peptides pepxml A list of peptide spectrum matches filtered by the peptide filters in the Summary page will be saved to peptides pep xml in pepXML format The number of the spectrum_query elements will be the same as the number of PSMs mentioned in the Summary page And the number of the search_hit elements will be the same as the number of PSMs counted from peptide table summing up Spec column De novo only peptides pepxml A list of good de novo sequences with no good database matches or no matches at all will be saved in de novo only peptides xml in pepXML format For Third Party PEAKS analysis results can be used in other third party software packages for validation or further analysis of the data In addition to the result files some third party software packages also require spectrum data files in some specified format The For Third Party pane provides the following exporting options to support such usage of PEAKS results e For Scaffold peptides mzIdentML In addition to exported results in supported mzIdentML formats version 1 0 0 and version 1 1 0 PEAKS will export the spectrum data in MGF format The exported mzIdentML file and MGF files can be loaded into Scaffold for further analysis like PTM verification for example
171. n Search a sequence tag Select a peak and right click anywhere in the Spectrum View to trigger the pop up menu From the menu select either Left tags or Right tags PEAKS will select the appropriate terminal tags and show them in the Tag panel see below To test the suitability of a tag highlight it in the Searched Tags list The corresponding information for the tag will be shown in the Spectrum Annotation panel the Ion Table panel and the Spectrum Alignment and Error Map panel One or more tags can be inserted by highlighting the desired tags clicking Select to move them into the Selected Tags list and then clicking the Apply button Press the Cancel button at any time to exit the search and discard any changes 82 Peptide De Novo Sequencing Searched Tags Selected Tags a i L EZA YA WE as FW Y A NM LT YAY Undoing an edit Errors during the manual de novo sequencing can be undone easily With the peptide candidate still selected in the Result panel right click and select the Undo command from the pop up menu to return to the previous peptide sequence This button can be used multiple times to return to previous edits Manual De Nove New Candidate for Manual De Nova Remove the selected Candidate Config Error Tolerance in Manual De Nove Intensity Ys 100 Config PTM in Manual De Novo 34 Undo Redo Add new sequence Can t Save Re
172. n protocol Precursor on Quantification specify the PEAKS quantification parameters in the dialog and click OK at Wait for the analysis to finish A new quantification result node will appear in the Project view Double click the node to open the result The quantification result display is similar to that of the PEAKS DB result However the ratio and standard deviation columns are added to each quantifiable peptide and protein 2 Setting Parameters The following parameter dialogue pops up when the quantification tool bar icon Q is clicked or Tools Quantification command from the menu is clicked Select Precursor Ion Quantification eg SILAC from the left hand side 118 PEAKS Q MS Level Quantifications Precursor Ion Quantification Predefined parameters he Basic Options Mass Error Tolerance 20 0 ppm w Upper Bound of Precursor Charge 35 Retention Time Range 1 0 min w Peptide Score Threshold 10logP 20 C Label Free ee Quantification Method Detail s Reporter Ion Quantification SILAC 3plex R10 K w Name SILAC 3plex R 10 K8 R6 K4 Method Type Precursor Ion Quantification eg TRAQ TMT New Label Options Precursor Ion Quantification Sample Name Modification Modification Detail 7 Ege SLAG i leavy 130 8 15N 2 Silac label 10 0209095 R 13C 6 15N 2 Silac label 8 0141990 K 13C 6 Silac label 6 0201290 R 4 4 5 5 D4 Lysine 4 0251078 K OK Cancel Help The following parame
173. n the Summary page show the peptide score distribution of PSMs When FDR estimation is enabled in the PEAKS Search parameters the target and decoy matches are respectively shown in two different colors 90 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Figure 2 a is a stacked histogram showing the number of target and decoy matches in each score interval There should be a similar number of target and decoy matches with low scores but very few decoy matches with high scores The vertical line indicates the current score threshold lt lt number of FSMS 75 100 125 150 PEAKS peptide score 101gP Decoy BE Target Figure 2 b is a scatterplot showing the peptide score versus the precursor mass error in ppm for all PSMs This figure is the most useful for high resolution instruments The PSMs with high scores should be centered near the mass error of 0 and the PSMs with low scores are usually scattered to larger mass error The vertical dotted line indicates the current score threshold 0 25 50 75 100 125 150 PEAKS peptide score 10IgP o Decoy Target 3 2 3 De Novo Result Validation Figures 3 a and 3 b in the Summary page help determine a proper score threshold to filter de novo only sequences After the database search de novo sequences can be categorized as 1 Verifiable de novo sequences A de novo sequence is verifiable if the associated MS MS spectrum is confidently
174. nformation is updated in the Result panel Ion Table panel and Spectrum Alignment and Error Map panel The selected ions are also annotated and color coded in the Spectrum Annotation panel After setting two ions PEAKS will estimate the residue found between them 1f a residue corresponds closely to the mass difference The peptide sequence candidate name will change to show the residue and the mass remaining to be sequenced on either side of the residue All other panels will also reflect the changes 81 Peptide De Novo Sequencing gt 802 90507 2 Manual De Mowo Intensity 96 344 25 la 1170 51 1375 71 Y 1150 64 yl m oe 1189 531 39 5 912 52 1000 47 FA o 749 4 226 12 246 18 j L486 1604 81 bo 1359 66 1489 76 bi H20 5 bi l 100 200 300 400 Soo 600 700 eno edo 10010 1100 1200 1300 1400 1500 1600 1200 a yi det 2x 21 ErTol 0 5Da F intensity threshold OrbiSample makML ms 2 m2 802 90607 2 2RT 0 0729T1C0 1 79 7 1189 531 39 45 4b bH20 bHHS Seq Y yH2o 06 y HH 0 5 0 5 1 345 24 327 17 328 23 3544 25 3 2 416 29 398 28 399 27 A 1250 56 1242 56 1243 54 2 Eno 3 1170 51 1189 53 1171 51 1172 50 1 0 5 0 5 00 1000 1500 miz 344 25 A L170 51 He l mumsa 44 25 HyNax Intensity 2 mz 500 1000 1500 Searching the left or right side of the spectrum for the first last y or b io
175. nt Configuration From the Configuration window select Instrument from the left side menu to change the instrument configuration Instrument List lt Built In gt Orbitrap Orbi Trap lt Built In gt Orbitrap Orbi Orbi lt Built In gt Triple TOF lt Built In gt Q TOF lt Built In gt TOF TOF lt Built In gt Ion Trap Instrument Details Instrument Name Orbitrap Orbi Trap Basic Options Ion Source ESI nano spray hy MS Precursor Scan FT ICR Orbitrap hl MS MS Product Scan Linear Ion Trap ty Advanced Options Precursor mass search type 8 Monoisotopic Average Parent mass error tolerance 15 0 pom s Fragment mass error tolerance 0 5 Da Built in Instruments The names of the built in instruments are provided in the Instrument List Select an instru ment to view the detailed instrument information in the Instrument details panel below Note The details of a built in instrument cannot be deleted or edited 169 Configuration and Preferences Create a new instrument 1 Za Click the New button and provide a name for the instrument in the Instrument Details panel Use the Ion Source drop down list to select the ion source that was used MALDI SELDI or ESI nano spray This will help the PEAKS Data Refine tool determine the charge of the ions Use the MS Precursor Scan drop down list to select the type of MS scan that was performed This
176. ntification PEAKS DB PEAKS PTM and SPIDER discovery rate FDR in the analysis report It is NOT recommended to de select this option unless manual validation of the result using another approach is preferred Find unspecified PTMs and common mutations with PEAKS PTM Select this option to enable PEAKS PTM search after PEAKS DB search finishes PEAKS PTM analyzes spectra with good de novo sequences that remain unidentified by PEAKS DB The default setting for PEAKS PTM 1s to search with all the built in modifications in the Common and Uncommon lists which include all the natural modifications and mutations in the Unimod database Alternatively PEAKS PTM can search with a list of preferred modifications In Advanced Settings preferred mod ifications from the PTM Options dialog can be chosen PEAKS PTM allows an unlimited number of variable mod ifications to be searched However limiting the number can improve search accuracy Additionally the maximum number of variable PTMs per peptide can be specified and a value of less than 4 is recom mended A threshold on the de novo ALC score can be specified so that only the spectra with good de novo sequences are analyzed by PEAKS PTM When PEAKS PTM search is enabled a PEAKS PTM result node will be automatically generated after the search In the PEAKS PTM report search result from PEAKS DB and PEAKS PTM are merged Note Modifications selected for the PEAKS DB search are automaticall
177. ntification a protein or EST sequence database must be configured See Chapter 6 Adding a Sequence Database If you are eager to try PEAKS now leave these two configuration steps aside for a while and try out the 15 minute walkthrough to get familiar with PEAKS GUl and basic operations See Section 4 Quick Walkthrough 23 Chapter 3 Configuration Wizard Configure Instruments and Public Databases 1 Overview The configuration wizard guides you through some easy to follow steps to configure PEAKS for instrument raw data support and database searching The configuration wizard can be invoked from the menu Window Config Wizard PEAKS supports different instrument vendors raw data formats A list of supported formats can be found in Section 7 Supported Data Formats Some vendors formats may require the vendors specific software to be installed on the same computer that PEAKS is running on The configuration wizard helps you select the proper instrument and install the appropriate vendor software A sequence database must be configured in PEAKS to identify peptides and proteins with the MS MS spectra by database searching The configuration wizard also helps you select the appropriate databases from a list of public sequence databases download and configure them automatically The list of public databases can be found in Section 3 Database Selection 2 Instrument Selection When you select Window Config W
178. oad License Request File Import License Activation Complete Installation and Activation cna Ares cna ene sree Key Important You will receive your license file via email I have already received the license file 2 Save a generated request file to a removable storage device e g a USB memory key Click the button below to save the License Request File to directory e g an USB drive so that it can be later transfered to a computer that has internet access tad 3 From another computer with an Internet connection upload the license request file to BSI s license server as de scribed in the following screen 17 Installation and Activation Gather The following steps should be done on the computer Information which has Internet connections eee eee Go to http www bioinfor com lces20 index jsp Request File Select the option I have the license request file I want to register the software and click next Upload License Request File Click Browse button to select the license request file that has been copied to this computer type in the visual verification code and dick next Import License Now the software is being registered If you get a Operation completed successfully message please check your email The license file will be sent to you shortly Activation Complete 3 BS Product Licensing Center amp ED www bioinfor com cs20 index jsp 7 Ye Google P D
179. ode wma be lirendiew a a a a ra 43 N WCW E E A T O TA 43 SA MS MS VIEW sE AAA SAS NA ASE AAA AAA NNS 44 E O UI II II cena amet atted ncn casattemiaet cet 45 Ala NOE EOV lid N T NEE 47 A SOW H de Pear S roas a N E E AE A E mbt ahaseaneieas 4 43 Locate M Zand Retention TIAS NI AA TA Mantes 48 4 4 Synchronize LC MS View between LC Runs oooocccoccnconcnccncononcononnoncnnoncononnoncnnonnnonannannos 48 A SHOW Hide MS MS SPECII antaras 48 AO lniens iy VIEW Seite A T E is et ETTE 49 A EXPO EC NUS NS Ra 50 Addn lt a Sequence Database olaa 5I kC onnsurmo Scgucnece Databases siii O A AEE 51 2 Databases to be Used in PEAKS inChorus Function oocococccconconononncncnnonnononnoncononnononnoncnnonnonannns 53 Te Datt REMESAS AS AS AAA AA ANA A AS 55 PSO CEVICW AR E 55 2 Data Refinement Paramete iS is E E AE A mau icsawand 55 Zo Savine the Parameters Tor Future Usa dl iii 57 5 Peptide De Noro SECUENCIA 5 teach SS A A et antacid ohana 58 PI AA NA 58 2 De Noyo SeQuencin Pardee eje ii is 59 2 E m0 101 TOEIC E a Aan UU E E A O ENS 60 Dd ENS A AIN ES ESE aaa EA ES 60 A a TT A at coat cana cet agete eae 61 2A OMer Parametros 63 Zo Savino tne Parameters tor Future Use ua ii ibn 63 3 Understanding PEAKS De Novo Sequencing Results o oooooccoconcononcononcononnoncononconcnnonconcnnononnns 63 Sl SU A NEW is O 63 Ie De NOVO EPS VIEW a abor 64 SA lo A E 65 di PECULIAR a a ala Weekends 67 AS A AI Dn o II A E 68 A
180. of the user s datasets The two main factors affecting PEAKS performance are CPU and RAM For the default 64 bit version of PEAKS 7 the recommended and ideal requirements are e Recommended Intel Core processor and 8GB RAM e Ideal Intel Core 17 or Xeon processors and 16GB RAM or more or 2GB per core For the 32 bit version of PEAKS 7 4GB RAM is required 3 Installation on a Windows Computer Important Please uninstall any older version of PEAKS currently installed on the system before proceeding Important Avoid installing PEAKS in any directory that contains a white space for example the Program Files direc tory as some features may not function correctly in such situations Please make sure that the user account has full access permissions read write execute on the selected directory Important To open an instrument s raw data using PEAKS it is necessary to install PEAKS on the same computer where the instrument vendors own software is installed Refer to Section 8 Vendor Specific Requirements for the vendor specific requirements for raw data loading 13 Installation and Activation 1 Close all programs that are currently running 2 Insert the PEAKS installation disc into the DVD drive If an installation file was obtained elsewhere double click on the PEAKS installation file and move ahead to step 4 3 The installation window should automatically appear after the DVD is inserted If it does not fin
181. oing downloads will be cancelled if the wizard is cancelled Click Next to set preferences for some vendor instrument software A warning message box will pop up in case any downloaded item is not installed 2d Configuration Wizard Configure Instruments and Public Databases 5 Vendor Instrument Software Options This panel allows users to set the raw file converter preferences of some vendor specific software Refer to Section 1 2 Raw File Converter Preferences for details on raw file converter preferences After making appropriate changes to the raw file converter preferences click the Next button to finalize the changes Vendor Instrument Software Options Bruker yeap baf fid No further configuration of PEAKS is needed But you must ensure that Bruker CompassXtract Runtime is installed on this computer Download CompassXtract Runtime from Bruker website Raw File Convertor Options Bruker file reader will export 8 Raw data Line spectra Bruker fid file may contain several files do you want to merge them 5 yes 6 no Shimadzu AXIMA run Shimadzu MS data run can be converted to mzXML format using Shimadzu Launchpad The mzXML data can be loaded into PEAKS readily Please contact your Shimadzu representative to obtain Shimadzu Launchpad Shimadzu run2xml exe File Location C Program Files x86 Programs yun2xml exe Browse Varian xms Varian MS data xms or sms can be converted to PKL format using Vari
182. omy options may be specified for certain databases To add a sequence database click the icon in the main toolbar and select Database from the left hand side This will open the database configuration dialog Click the New button to create a new sequence database entry or select a database from the Database List The database can be configured in the Database Details area below Click the Add Update button to save any changes 51 Adding a Sequence Database Enzyme Database List nl puau t_SwissProt NCBI_nr Labeled Q Method Database Database Details FASTA format database NCBI nr r Basic Options Database name Validated Path Browse or EST database Advanced Options Fasta Title Format Rule to parse accessionjid from FASTA title Maid Rule to parse description from FASTA title ELY Accession id URL http fer ncbi nlm nih govw fentrez viewer fogi db proteinaval lt Accession ID gt Delimiter s Taxonomy Optons moni Eza Follow these steps to configure a database 1 Select the database format from the FASTA Format Database drop down list or select Other if the desired format is not present If Other is selected you must enter custom parse rules 2 In the Basic Options section enter a name for the database If the database FASTA file is already on the local system skip to step 6 Otherwise click Download A window will appear confirmin
183. on PEAKS DB PEAKS PTM and SPIDER e modification table The modification table shows the modifications identified in supporting peptides on the protein For each modification the number of supporting PSMs with this particular modification is shown The checkbox on the left controls whether to indicate the modification in the protein coverage Double clicking on the name of a modification shows detailed information about the modification Right clicking a modification shows a pop up menu for quick operations Tools bar The tools bar is at the upper right corner of the protein sequence coverage It has two icons eae outline coverage TA a ry io FORT 8835 de LVNEL TEFAKTCVAD Show modifications minimal ion intensity 5 AM PIM 57 02 Carbamidom 1584 a e Full screen Left clicking this icon shows the protein coverage in full screen mode Click to return from full screen mode e Tool box S The tool box contains the following tools Copy template protein Copies the original database protein into the system clipboard Copy mutated protein Copies the protein with detected mutations into the system clipboard Save protein coverage as image Coverage statistics NCBI BLAST search NCBI Entrez search Multiple sequence alignment Performs multiple sequence alignment for the selected proteins 3 3 3 Supporting Peptides The Peptides tab shows a table of supporting p
184. ons heading in the quantification window to view the label free quantification parameters on the right side 128 PEAKS Q Label Free Quantifications Label Free Quantification Basic Options Mass Error Tolerance Retention Time Shift Tolerance 6 0 8 Label Free Sample Groups Attach Identification searo sample isi Select All Collapse Reporter lon Quantification in eq 7TRAQ TMT Treatment12_1 j j d Treatmenti2_2 EG z iroun 7 Treatment24_1 T ou 0 Contra J Treatment24_2 o Controli_1 Precursor Ion Quantification Controli_2 eg SILAC PEAKS 10 PEAKS 23 r PEAKS 27 PEAKS 31 p PEAKS 35 Group Color a ril Group 2 l Control2_1 Control2_2 Advanced Move Up Move Down Cancel The following parameters are available in the Basic Options section of the quantification window e Mass Error Tolerance Mass shift between different runs Initially this should be set as twice as the mass error tolerance in database search Later by checking the M Z shift distribution figure in the result you can narrow the tolerance e Retention Time Shift Tolerance The retention time shift between different runs Initially this should be set as the highest possible shift between runs Later by checking the RT shift distribution figure in the result you can narrow the tolerance The Sample Groups section allows you to arrange the samples into groups according to different experiment condi t
185. osing Displayed exports the log2 ratio of the sample average 135 PEAKS Q Label Free Variation Trend Chart log2 ratio Sample 7 Sample 8 Sample Y Sample 10 Sample 11 Sample 12 Samples 3 1 2 2 Sample Correlation To view the intensity correlation plot for the detected features of the two selected samples click the Sample Correlation button from the Summary view title bar The correlation plot will be displayed in a pop up window Select the samples from the drop down lists beside Sample correlation to plot the intensity correlation graph of different sample pairs The Pearson s correlation score indicates the reproducibility of the experiment based on a selected pair of samples Click the Save As button to export the correlation plot to an image file 136 PEAKS Q Label Free Sample correlation Controli_1 tl Controli_2 O a ene Pearson s 0 9962 i o E a a Control1_1 Control1_1 Y S Controli_2 3 1 2 3 Volcano Plot A volcano plot combines a statistical test with the magnitude of the change enabling quick visual identification of those data points that display large magnitude changes that are also statistically significant The volcano plots in Figures 2 a and b plot significance versus fold change for proteins and peptides respectively These plots help quickly identify the most meaningful changes There are one horizontal and two vertical threshold which divide the pl
186. ot area into six quadrants Plotting points in this way results in two regions of interest in the plot those points that are found towards the top of the plot that either to the far left or the far right These represent values that display large magnitude fold changes hence being left or right of center as well as high statistical significance hence being towards the top In both figures the ratio is the group ratio to the base group If more than two groups are present then the maximum ratio is used in the figures 137 PEAKS Q Label Free Figure 2 The volcano plots a For proteins b For peptides a b ao O Epersreccicsescion e F Pa Bp TE O E EEEE EE E H a 55 A WOO 4 nnn ne nnn ne nn nnn nee nee eee ne een m e e m e e ees A A of pe Mp rn rd H 60 AA aL aL 40 a a G70 Pa El El BOQ 4 m 30 4 J 5 525 an an an 20 40 4 15 20 10 O 10 5 14 3 1 2 4 Feature Vector Ratio Distribution Figures 3 a and 3 b plot the distribution of feature vector ratios by quality and average area respectively In both figures the ratio 1s the group ratio to the base group If more than two groups are present then each ratio value depicts a data point in the figures These plots help to determine the threshold values for the quality and intensity filters to obtain reliable information for the analys
187. owing search engines Mascot X Tandem and OMSSA 1 3 1 Mascot Settings Click Mascot Settings on the left side to display the Mascot preferences Mascot Settings Hostname or IP address myMascot mySite com Port 80 Virtual Directory mascot Version M SCOt Server 2eBeK k ur User name myMascotAccount Password seses Email testibioinfor com Test Connection These parameters specify how PEAKS accesses the Mascot server if applicable Enter the hostname or an IP ad dress port virtual directory and Mascot server version As well enter the username password and email address To make sure that everything is entered correctly and that the server is working click the Test Connection button The port and virtual directory match the above settings for most servers 1 3 2 X Tandem Settings Click X Tandem Settings on the left side to display the X Tandem preferences XTandem Settings X Tandem Local Path _ tandem Browse As PEAKS provides a local copy of X Tandem upon installation a default path will appear in the Local Settings section To use another license location for X Tandem click the Browse button to tell PEAKS where to find the search engine 160 Configuration and Preferences 1 3 3 OMSSA Settings Click OMSSA Settings on the left side to display the OMSSA preferences Omssa Settings Default Omssa Path omssa Erowse As PEAKS provides a local copy of OMSSA upon installa
188. pm 2 7 by PEAKS PTM v intensity K v xT s s a R oR 100 8 H20 Y8 NH3 901 48 1 spectrum 10lgP 24 06 1127 66 Seq KVLT sub A SSARQR A T 30 01 bo 310 Y7 H20 971 56 V7 NHB QDTISSKLKE CCDKPLLEKS HCIAEVEKDA 788 40 a YO NH3 RLAKEYEATL EECCAKDDPH ACYSTVFDKL 1000 55 Y6 NH3 47172 474 687 35 be 1085 64 A BA A 843 50 303 18 b4 H20 617 34 b2 H20 424 29 210 16 200 400 600 800 1000 1200 wij 121 2x 2 ErrTol 0 02Da Y preprocess Y low intens label 111 101 116 144 154 d a a ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK 00 AAs per line and 10AA gap Specify the number of amino acids per line and whether to show a gap for every 10 amino acids de novo tags sharing X AAs Show de novo tags with at least X consecutive amino acids matched in the protein sequence de novo peptides fully matched Show de novo tags with its full sequence matched in the protein sequence This control is especially useful for showing short but fully matched de novo peptides modification minimal ion intensity A modification or mutation is considered confident if there are fragment 10ns supporting the modified residue with the minimal peak intensity above the specified threshold Only confi dent modifications and mutations are displayed as icons above the protein sequence 99 Peptide PTM and Mutation Identificati
189. protein identifications See Section 4 Exporting Database Search Results for details 117 Chapter 11 Precursor lon Quantification e g SILAC and ICAT 1 Overview Precursor ion quantification with isotope labels at the MS level is one of the three quantification modes that are supported by the optional PEAKS Q module for PEAKS Studio In this mode the isotope labels with different mass values are introduced as two or more samples The samples are then analyzed together in an LC MS MS experiment The same peptide from different samples is recognized by a set of precursor ion peaks with similar retention time and mass differences within the retention time window and mass error tolerance set by the user The ratio is calculated from the intensities of those peaks PEAKS Q supports both user defined labels and commercial quantification labels The quantification analysis is based on a PEAKS DB result See Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Ensure that the specified isotopes are labeled as PTMs when performing the database search After database search is complete follow these steps 1 Select a PEAKS DB result node in the Project view Click the PEAKS Quantification toolbar icon Q Note Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for how to perform PEAKS DB and quantification in a single easy workflow 2 Select the quantificatio
190. provides a manual de novo sequencing tool to assist in the manual interpretation of individual spectrum Note Refer to Section 8 Manual De Novo Sequencing Other PEAKS tools including PEAKS DB for database search PEAKS PTM for unspecified PTM search and SPIDER for homology search depend on the de novo sequencing result to achieve superior sensitivity and accuracy Note Refer to Section 1 Overview 58 Peptide De Novo Sequencing The use of automated de novo sequencing 1s outlined below For detailed information please refer to later sections 1 Select a data node in Project View The selected node can be a fraction a sample or a project PEAKS will analyze all fractions under the selected node Launch PEAKS de novo by clicking the Auto De Novo button in the toolbar 66806 FN Project View E as A de 1 El 4 F1 BSA GluC 1 RAW o 8 DATA REFINE 1 2013 10 22 11 06 J Sample 2 Ely F2 BSA LysC LRAW 4 DATA REFINE 2 2013 10 22 11 06 J Sample 3 Edy F3 BSA Trypsin 1 RAW i DATA REFINE 3 2013 10 22 11 06 Note Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for how to create a project 2 Specify the de novo sequencing parameters in the pop up dialog Most of the parameters are self explanatory and the default parameters provide a good starting point Click OK to start automated de novo sequencing Note If data 1s not yet refined yo
191. r all the PSMs This figure is the most useful for high resolution instruments Generally the high scoring points should be centered around the mass error 0 Notice that the data points start to scatter to larger mass errors when they are below a certain score threshold Figure 2 PSM score distribution a Distribution of PEAKS peptide score b Scatterplot of PEAKS peptide score versus precursor mass error a o A 15 F AS a A EA i O S A AAA E E A AS eeee tee aaa ase SNe eE EEE eae Seo Ree eke eae eae g E A AAA A J 17 E 400 mra ae hal a 10 oD eee ee ee ee ee 15 AAN lima 40 20 30 40 50 60 70 80 90 60 10 20 30 40 50 60 FO ag 90 A Berra score aa PEARS peptide score 10lgP A Decoy M Target o Decoy Target 4 4 Result Visualization Besides the summary view there are four other views for visualizing the data and the results Protein Peptide De novo only and LC MS e The Protein view contains a list of proteins passing the filters The proteins identified with the same set or a subset of peptides are grouped together e The Peptide view shows all the peptide identifications passing the filters The multiple spectra that identified the same peptide sequence are grouped together e The De novo only view shows all the peptides identified exclusively by de novo sequencing The LC MS view displays the LC MS data as a heat map with highlighted MS MS scans and detected feat
192. r support for Thermo and Agilent raw files on 64bit OS e Collaborate with Skyline group The identification results from PEAKS exported as pepXML can be imported into Skyline e Proteome Discoverer 1 4 support Now PEAKS can load the pepXML result file generated from Thermo s Proteome Discoverer software versions 1 3 and 1 4 e Supports mzIdentML result format Now PEAKS can export results to mzIdentML formats version 1 0 0 and version 1 1 0 for downstream analysis such as Scaffold PTM 4 Quick Walkthrough This section presents a quick walkthrough of a typical data analysis PEAKS user interface 1s introduced by using the sample project included in PEAKS installation filtration and visualization of the analysis result are showcased This will help understand what can be accomplished with PEAKS After that a PEAKS project creation from raw data and data analysis on the project is demonstrated 4 1 Opening an Existing Project The installation instructions for PEAKS can be found at Chapter 2 Installation and Activation After launching PEAKS the sample project can be opened by one of the following two ways see screenshot below 1 If this is a fresh installation click the Sample Project in the Recent Projects list of the Start Page 2 Click the open project button and browse to the directory where PEAKS 7 was installed select SampleProject and click the Open button in the file browser Overview FY PEAKS Studio m
193. reating new PTMs and viewing PTM information 2 4 Other Parameters Report up to peptides candidates per spectrum Displays how many peptide de novo sequences PEAKS will report per spectrum Implicit Parameters Instrument Type and Fragmentation Mode were specified for each analyzed sample during project creation 2 5 Saving the Parameters for Future Use After setting up the parameters you can save them for future use Click the Predefined parameters drop down list select Save as and enter a name for the current set of parameters to save it Saved parameters are available for future analysis You can load a set of saved parameters by selecting from the Predefined parameters drop down list 3 Understanding PEAKS De Novo Sequencing Results When de novo sequencing is done PEAKS attaches a result node to the analyzed data in Project View Double click the node to load the analysis report The analysis report is presented in three pages e Summary Outline of PEAKS de novo sequencing result with key statistics It also provides functions to filter and export de novo sequences e De novo De novo peptide sequences listed in the table Spectrum annotation and other detailed information are provided e LC MS De novo peptide sequences visualized in an interactive LC MS heat map 3 1 Summary View The summary view reports key statistics as an overview of the result It also provides functions to filter and export de novo
194. rogram Note The use of variable modifications increases the size of the computational search space for the de novo se quencing algorithm It is recommend not to select too many variable modifications in PEAKS de novo Note Leucine L and Isoleucine 1 cannot be directly distinguished in mass spectrometry since the two residues have exactly the same mass For that reason PEAKS uses L in de novo sequencing to represent both and L If a user defined modification modifies it should be defined on L instead When is defined as a modification site the modification site will be ignored by the de novo sequencing algorithm PTM name Carbamidomethylation PTM abbreviation Carbamidomethyl Mass Monoisotopic 57 021465 Residues that can be modified Anywhere peptide protein N term C term Formula H 3 CC N 1 O0 1 Rule Help Double clicking any of the PTMs will display the PTM details in the PTM Info popup dialog The same information can be viewed by selecting a PTM from a list and clicking the View button If a desired modification is not listed or 62 Peptide De Novo Sequencing 1s different to the listed modification 1t can be defined as a customized modification Click the New button to show the New PTM dialog and enter the information of the particular modification The newly defined modification will be added to the Customized list Refer to Section 2 2 PTM Configuration for details on c
195. rotein Comparison The top protein identifications from the compared search results are displayed in the table The layout of the protein table and control panel at the bottom is similar to the Peptide Comparison pane The following information is also displayed for each protein e Score PEAKS protein 10lgP score e Spec the number of PSMs matched to supporting peptides for the protein e Pep the number of supporting peptides for the protein e Uniq the number of unique supporting peptides for the protein e Spec the number of peptides detected for the protein over the number of peptides in theoretical digestion e Cov the sequence coverage of the protein The following screenshot is the protein table showing a typical comparison result 108 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 7 Ee A A pasa ae l o H2538 N E e COM SC S IR IR II E E ES E Y TT s a ARO pa es CL O O O a e a A o E 7 4 Statistical Charts PEAKS provides a number of statistical charts which can be easily exported for usage in publications The peptide score distribution protein score distribution peptide number Venn diagram and protein number Venn diagram can help validate the results The charts can be exported from the pop up menu after right clicking the comparison result node Peptide Number Venn Diagram PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 400 600 800 1000 1200 1400 1600 1800 200
196. rter ion mass For precursor ion quantification methods the label options pane will contain the sample name modification and modification details Create a New Method To create a new labeled Q method click the New button to open the New Edit Quantifi cation Method dialog where the quantification method details can be specified 166 Configuration and Preferences Quantification Method Detail Quantification Method Detail Name My RIQ Method Method Type Reporter Ion Quantification w Name My PIQ Method Method Type Precursor Ion Quantification w Modification Target Label Options Terminal Modificati Termi ificati JE haere _ C Terminal Modification Sample Name Modification Modification Detail Heavy SILAC K6 Ey J6 02018K Side Chain Modification at Modification Mass 144 10207 Label Options Name Reporter lon Mass Da 114 114 1107 Add Row Delete Row OK Cancel OK Cancel Specify the modification target modification mass and label options for a Reporter Ion Quantification method Use Add Label and Delete Current Line to add or remove a label Each label is defined by the sample name and reporter ion mass Use Add row and Delete Row to add or remove a label for a Precursor Ion Quantification method Each label is defined by the sample name added mass target residue and labeling efficiency If one sample has multiple labels with different mass shifts a user can add multiple l
197. s Taxonomy Options moni xe Create a new database Click the New button to create a new sequence database entry or select a database from the Database List The database can be configured in the Database Details area below Click the Add Update button to save any changes Refer to Chapter 6 Adding a Sequence Database for details on adding a new database Delete a previously configured database To delete a database file select the database to be deleted from the Database List and click the Delete button at the bottom Moving Updating a database To move a database to another directory the location must be updated in PEAKS Select the database and specify the new location using the Browse button next to the Path field Then click Add Update to save the new settings If the database location is invalid the database name will appear in red in the list of databases Any protein identification using that database will fail If an update 1s made to the database file perhaps by downloading the latest database file and overwriting the old database file PEAKS will show the database information in light gray A light gray color could also mean that the database does not have header information 168 Configuration and Preferences Configure databases for use with other search engines in PEAKS inChorus The databases configured here will also be used in PEAKS inChorus to call the X Tandem and O
198. s chapter provides an overview of PEAKS distinctive features and describes a typical data analysis workflow in PEAKS It is strongly recommended to read this chapter to get a big picture of what PEAKS provides and how PEAKS is used Other parts of this manual are intended for reference and so it does not need to be read from cover to cover Many contents of this manual can be read from the software s inline help The electronic and most up to date version of this manual can be found at http bioinfor com doc peaks7 htmlmanual index html The installation of the software is covered in a separate chapter Chapter 2 Installation and Activation 2 What ls PEAKS PEAKS is a complete software package for proteomic mass spectrometry data analysis Starting from the raw mass spectrometry data PEAKS takes care of every step of data conversion peptide and protein identification PTM and mutation characterization as well as result validation visualization and reporting Overview data analysis result raw data database search visualization gt reporting validation and er filtration homology search quantification PEAKS helps in every step of the proteomics data analysis The following is a partial list of the distinctive features of PEAKS software e Complete Analysis PEAKS is a complete software package for de novo sequencing database search characterizing unspecific PTMs and detection of peptide mutations The combined us
199. selection will tell the PEAKS Data Refine tool whether the survey scan 1s of sufficient resolution to determine the charge and the monoisotopic peak from the examination of the survey scan Use the MS MS Product Scan drop down list to select the type of MS MS scan that was performed This selection will help PEAKS decide which internal parameters for weighing fragments and amount of noise to use during PEAKS auto de novo sequencing and PEAKS DB search Select LIT FT if alternating high res low res modes were used This will allow the algorithm to determine the mass analyzer from the scan header Use the Advanced Options to specify additional parameters Select Monoisotopic or Average as Precursor Mass Search Type For ion trap instruments it is usually ben eficial to allow PEAKS DB search to use an average mass Specify the values for Parent mass error tolerance and Fragment mass error tolerance in Daltons or ppm These will appear on the PEAKS de novo and PEAKS DB option screens when the instrument is selected Click the Add Update button to save the changes The new instrument will appear in the Instrument List where it can be accessed when creating a new project file To delete a customized instrument select the appropriate instrument from the Instrument List and click the Delete button 170
200. single protein studies PEAKS database search tools have built in result validation using an enhanced target decoy approach The False Discovery Rate FDR is estimated to ensure that only valid results are reported PEAKS DB 1s a database search tool assisted by PEAKS de novo sequencing to achieve high sensitivity and accuracy Note For more details refer to the paper PEAKS DB De Novo sequencing assisted database search for sensitive and accurate peptide identification Mol Cell Proteomics 2011 Dec 20 PEAKS PTM is a dedicated search tool for peptides with unspecified modifications and mutations It can search with unlimited number of modifications allowing search with all modifications and mutations in the Unimod database Note For more details refer to the paper PeaksPTM Mass Spectrometry Based Identification of Peptides with Unspecified Modifications Journal of Proteomics Research 2011 10 7 2930 2936 SPIDER is a dedicated search tool for finding novel peptides that are homologous to peptides in a given protein database Note For more details refer to the paper SPIDER Software for Protein Identification from Sequence Tags Containing De Novo Sequencing Error J Bioinform Comput Biol 2005 Jun 3 3 697 716 PEAKS DB PEAKS PTM and SPIDER can be launched together as a workflow in a PEAKS search An analysis report is generated to show the combined database search result The steps to run a PEAKS search are 1 Sele
201. spectrum or click the 1 1 button 3 MS MS View The MS MS View shows the list of tandem scans on the left For each MS MS scan the list of identification results the spectrum the LC MS view zoomed in to the area pointing to the spectrum ion match table and its survey scans are shown on the right Zoom options are the same as described in the MS View section 44 Data Visualization H gt 556 6478 RT 28 1321 Scan 1398 3 MS MS Spectra List b7 2 Ys b 4H20 2 A 755 7366 3 A i 584 8143 RT 28 171 Scan 1400 2 A pa 46 e a A por DVSLLHKPTTQISDFH E an Bda eee PEAKS 44 2013 10 02 13 47 Identification Results G 712 3648 RT 28 2139 Scan 1402 2 yA 2 a E PEAKS PTM 45 2013 10 02 13 47 18 i 3 H gt 868 41614 RT 28 2856 Scan 1405 2 ithaca ana i E iy DENOVO 43 2013 10 02 13 47 G 627 29974 RT 28 3104 Scan 1406 3 DVSLLHKPTTQLSDFHVATR 89 fH 524 919 RT 28 3566 Scan 1408 3 L DVSLLHKPTTQLSN 98 FHVATR 89 Y G 786 8755 RT 28 388 Scan 1409 2 RT i intensity D v s uJuJalxlelr rjolz s plrjalvalr R G 534 2906 RT 28 4219 Scan 1410 2 100 G gt 652 88654 RT 28 4671 Scan 1412 2 Spectrum 30 0 G gt 666 2657 RT 28 5008 Scan 1413 3 G gt 686 3358 RT 28 5344 Scan 1414 2 29 0 fH 877 4204 RT 28 5809 Scan 141
202. st Incorrect M Est Correct a ga 20O O eal ALC260 d d F a 150 Space EEE iii D D a a T a 100 7 mee AI ar i E E 2 Z 50 a a O Last nal His 10 25 50 T5 90 95 98 94 gg7 999 10 25 50 T5 90 95 48 94 39947 99 9 De novo residue local confidence De novo residue local confidence A Incorrect M Correct A Est Incorrect M Est Correct 250 NS AS E MN 100 numberofresidues ala pa PA a a a a So O a D T E peee T _ 2 io E number of residues on Oc So O So het ill In AA Ma g 90 95 98 94 997 99 9 90 95 98 94 997 99 9 um novo tie local confidence 9 En novo E local confidence A incorect MA Correct A Est Incorrect M Est Correct 3 2 4 Statistics of Data and Results Tables 1 4 show the statistical numbers of the data and results 93 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Table 1 Statistics of data Table 4 PTM Profile of MS Scans 4751 Name AMass FPSM Position of MS MS Scans 5152 Carbamidomethyl 57 02 1548 c 57 02 585 DEHK_N term Table 2 Result filtration parameters 98 102 NQ ai ii SEN ii E FENS Dehydration 18 01 57 DSTY Protein 10lgP 20 l Dethiomethyl 48 00 30 M Proteins unique peptides 0 Carbamylation 43 01 29 K N term De novo ALC Score 50 Oxidation 15 99 24 M ds Sodium 21 98 22 DE C term Table 3 Statistics of filtered result Ammonia los
203. sually examined For a typical LC MS MS fraction three views are provided e MS this view shows the TIC total ion chromatogram plot and all the MS scans For each MS scan the corre sponding MS MS scans are also displayed e MS MS this view lists all the MS MS scans For each MS MS scan the corresponding MS scan is also displayed e LC MS this provides a bird s eye view of the whole LC MS dataset with detected features and MS MS scans marked on refined data After opening a data file by double clicking the data node on the project tree the choice of different views can be made by choosing different tabs at the upper left corner of the data view window 0408 441 90 ect View artPage Xx OrbiSample RAW X D ftest OtherProjects Mew Project 12 7 TIC d Intensit s Sample ls Pi 100 SA Aorbisample RAW k DATA REFINE 1 2013 09 04 12 19 y DENOVO 2 2013 09 04 12 22 W PEAKS 3 2013 09 04 12 22 i PEAKS PTM 4 2013 09 04 12 22 SWE SPIDER 5 2013 09 04 12 22 KI XTANDEM 10 2013 09 04 17 28 2 MS View The MS View contains the TIC and all the scans The total ion chromatogram TIC is displayed on the left of the MS view The navigation buttons are circled in the figure To collapse the TIC chart click the left navigation button To navigate the survey scans use the up and down navigation buttons The survey scans can also be navigated by using the up and down arrow of the keyboard Clicking on a specifi
204. sult But you can also choose to attach an existing identification result as the last step of the label free quantification Note For details of the retention alignment algorithm refer to the paper A Combinatorial Approach to the Peptide Feature Matching Problem for Label Free Quantification Bioinformatics 2013 10 1093 The usage of this function is outlined in the following overview L Select a project node in the Project View frame Click the PEAKS Quantification tool bar icon Q or select Tools Quantification command from the menu Note Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for project creation data loading PEAKS DB search and quantification in a single workflow 2 Select the quantification method as Label Free and specify the PEAKS Label Free quantification parameters in the right panel of the window 3 Arrange the samples into groups attach Identification results to the analysis and click OK Note LEQ requires at least 2 groups with at least 1 sample in each group Note Currently only identification results from PEAKS are supported in quantification Inchorus and all other third party engine results are not supported 4 Wait for the analysis to finish A new LFQ result node will appear at the project tree Double click the node to open the result file 2 Setting Parameters Select Label Free from the left side under the Quantificati
205. t contains modifications that can only be artificially induced for a specific purpose including various chemical and isotopic tags for quantification or other experiments In this dialog there are two other lists of modifications 61 Peptide De Novo Sequencing e Recent The Recent list keeps track of previously selected modifications By default modifications are sorted by the last time they were selected You can conveniently select the modifications that are most frequently used for your analysis e Customized The Customized list shows all the user defined modifications User defined modifications can be modified or deleted Modifications can be selected as either fixed or variable modifications e A fixed modification forces all instances of applicable residues to be modified For example when car bamidomethylation C 57 02 is selected as a fixed modification all cysteine residues in the result will be modified e A variable modification gives the option for the residues to be modified If carbamidomethylation C 57 02 is selected as a variable modification only some of cysteine residues in the result will be modified Note For a specific residue only one fixed modification is allowed If more than one fixed modification is selected for the same residue PEAKS will warn about conflicting fixed modifications The warning message can be ignored and only the first fixed modification on that residue will be used in the p
206. t on the data set Usually a 2 fold change is used as a convention e Use volcano plot to choose proper cutoff The volcano plot can be used to choose proper cutoff values for significance and fold change filters The basic principle is to choose the cutoff values to make sure that only the outliers stay above the threshold in the top left or the top right corner of the volcano plot Note These two filters do not influence protein significance and ratio calculation Step 2B The following filters can be used to focus on the quantifiable proteins These filters are of most use when the purpose is to study protein changes or differential gene expression e Protein unique peptide filter A protein can be quantified more confidently when it has more unique peptides The convention is to set this filter to 2 Step 3 The following filters can be used to pick up the proteins groups with significant changes e Protein significance filter The rule of thumb is to set the cutoff value to 20 as in the case of the peptide sig nificance filter e Protein fold change filter Usually a 2 fold change is often used as convention e Use volcano plot to choose proper cutoff Volcano plot can be used to choose proper cutoff values for signifi cance and fold change filters The basic principle is to choose the cutoff values to make sure that only the outliers stay above the threshold in the top left or the top right corner of the volcano plot as shown in t
207. ters Upon clicking the Finish button the project will appear in the Project View panel The outlined G symbol indicates that the file is still loading The solid le symbol indicates that the file has finished loading 31 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow PEAKS supports a variety of instrument vendors raw data formats A list of supported formats can be found in Sec tion 7 Supported Data Formats Some vendors formats may require the vendors specific software to be installed on the same computer that PEAKS is running on Before creating a project with your own data ensure that the ven dor specific requirements discussed in Section 8 Vendor Specific Requirements are met Once the data is loaded it becomes a part of that project so that the original data files can be manipulated or deleted without affecting the analysis in PEAKS To close an open project select the project node in the Project view and choose the Close Current Project command from the file menu or use the close project icon from the tool bar or right click and choose the Close Project command from the pop up menu It is recommended to close the unused projects to preserve computer memory 3 Data Refinement Setting Parameters To run a data refinement on the fractoins immediately after creating a project click the Data Refinement button on the project wizard to open the Data Refinement panel See Sec
208. ters are available in the Basic Options section of the quantification window e Mass Error Tolerance This parameter is used to locate the precursor ion peak group of an identified peptide in the survey scan In a SILAC or ICAT experiment we are usually dealing with non centroided survey scans therefore the mass error tolerance should be set a little wider than the parent ion error tolerance used in the PEAKS DB search Upper Bound Precursor Charge The precursor ion of an identified peptide may produce sibling ions of different charge states Only those sibling ions with a charge less than the upper bound precursor charge will be considered for quantification of the identified peptide Retention Time Range The retention time range is the maximum elution time range that is considered for the quantification of an identified peptide Peptide Score Threshold 10lgP Only peptides with a score above this threshold are used to quantify the iden tified proteins Choose the appropriate quantification method used in experiment from the Select Method drop down list that con tains predefined quantification methods The details of the selected quantification method will appear in the Quan tification Method Detail panel To create a new quantification method click the New button to display the New Edit Quantification Method dialog Refer to Section 2 3 Labeled Q Method Configuration for how to create or edit a quantific
209. that are generated using different fragmentations Mass spectrometers like the LTQ Orbitrap Velos is capable of generating multiple tandem mass spectra using different fragmentations for the same precursor ion Using complementary fragmentations such as CID HCD and ETD the accuracy and confidence of automated de novo sequencing can be significantly improved PEAKS automatically detects spectral pairs and triplets when doing de novo sequencing The fragmentation type of data fractions containing spectral pairs or triplets has to be set to Mixed during the project creation In Mixed mode PEAKS loads the fragmentation type for each tandem spectrum from the meta information encoded in the data file A spectral pair or triplet is detected if the following conditions are satisfied 73 Peptide De Novo Sequencing e The pair or triplet of spectra are under the same precursor scan and are fragmented from the same precursor ion e The pair or triplet of spectra have different fragmentation types For example HCD ETD for spectral pairs CID HCD ETD for spectral triplets Create Project Project Name ETD_HCD Project Project Location C PeaksProjects Browse Project Structure Sample Enzyme z Sample 1 Trypsin SM Add data files Add sample PTI e Select All Remove Up Bown Collapse SelectAl Copy to Whole Project J
210. the Spectrum Annotation view In Place Ion Info Ion information m z value and relative intensity are displayed in a pop up in the Spectrum An notation view when this option is checked and the cursor is placed on a peak Intensity Set the intensity threshold for spectrum annotation to low 2 medium 5 or high 10 To apply this intensity threshold select the intensity threshold checkbox in the Spectrum Annotation view see Section 3 2 2 Spectrum Annotation 2 PEAKS Configuration This section describes the configuration of enzymes PTMs databases instruments and parameters To begin click the Configuration toolbar icon or select Configuration from the Window menu 2 1 Enzyme Configuration PEAKS can use almost any enzyme or combination of enzymes in your analysis Select built in enzymes from the extensive list provided in PEAKS or define a new one From the Configuration window select Enzyme from the left side menu to change the enzyme configuration Enzyme List Built In gt Pepsin pH 1 3 lt Built In gt Pepsin pH gt 2 lt Built In gt Proteinase K lt Built In gt Trypsin lt Built In gt Trypsin with D P lt Built In gt None Enzyme Details Enzyme Name Trypsin Cleave Sites X all amino acids after RK and not before P or after r and before or after and before a or after 7 and before r new AA Delete Heb
211. the amount of memory Java can access In both scenarios if out of memory problems are experienced the number of nodes in PEAKS should be reduced and the amount of memory per node should be increased 5 1 PEAKS 64 bit Version The 64 bit version of PEAKS can access a large amount of memory therefore it is highly recommended to deselect the options Start Client Separately and Start Compute Node Separately The Main Program JVM Heap Size MB is the maximum amount of memory PEAKS can use Although a higher number is better it is recommended to use no more than 80 of the system memory The Number of Computing Nodes to Start can now be calculated For example let X MB memory be assigned to the main program the number of computing node N is the largest integer less than X 2000 As shown in the figure the system has 16000MB RAM therefore 16000 80 12800MB memory is configured to the main program JVM 12800 2000 6 4 therefore 6 computing nodes is configured 21 Installation and Activation C Automatically Configure PEAKS Performance recommended Main Program JVM Heap Size MB 12800 Number of Computing Nodes to Start 6 Start Client Separately Client JRE Binary Folder jre bin Client JVM Heap Size MB 1024 _ Start Compute Node Separately Computing Node JRE Binary Folder jre bin Computing Node JVM Heap Size MB 1024 5 2 PEAKS 32 bit Version PEAKS 32 bit version is produced only for compatibility on o
212. the normalization factors in the textbox to the right a Proteins with fold change 1 w E 5 Show ratio to 114 w Normalization factor auto w 1 1 6 0 96 0 99 Apply Filters Export Notes Note When filtration parameters are changed the Apply Filters button changes color as a reminder to apply the filter by clicking it 4 Exporting Quantification Results PEAKS Q results can be exported to other supported formats All export functions are available through the Summary view panel To export the quantification results click the Export button in the title bar of the Summary view panel Refer to Section 5 1 Exporting Labeled Quantification Results for details gt 127 Chapter 13 Label Free Quantification LFQ 1 Overview Label free quantification is one of the three quantification methods supported by the PEAKS Q module It usually serves as a fast profiling tool for differential studies of large scale proteomics This quantification method is based on the relative intensities of peptide features detected in multiple samples Feature detection is performed separately on each sample By using the EM expectation maximization algorithm more overlapped features can be detected The features of the same peptide from different samples are reliably aligned together using a high performance re tention time alignment algorithm The label free quantification of PEAKS Q module can be performed without any identification re
213. the output format HTML Report This will generate a summary report in the specified location After the completion of exporting the result will be opened in the default browser automatically The following exporting options are available e Result summary The Summary view page will be saved as summary htm1 file in HTML format in the specified location Text Formats The following exporting options are available in various text formats e De novo peptides The peptides identified by de novo sequencing will be saved to de novo peptides csv file in Comma Separated Values CSV format in the specified folder e De novo peptides pepxml In addition to CSV format the peptides can be saved in pepXML format e All de novo candidates All de novo candidates for each spectrum will be saved to all de novo candidates csv file in CSV format 3 2 Exporting Images The annotated Spectrum Ion Match table Error Map and Spectrum Alignment can be exported to image files To do so position the cursor on any of those items in the result panel and click the right mouse button to view the pop up menu and select the Export Image command from the menu 150 Exporting Data Reports and Printing Intensity 2 s Flalc y s t v F p x Export image Print image Copy b2 Set As Anchor Peak cp Remowe Anchor Peak Annotation Settings VI NH3 Y A b3 Y3 H21 100 200 3010 y ji i 11 2X 2Y ErrTol 0 5 Da E preprocess W low intens
214. tide Maximum missed deavages per peptide 3H PIM Maximum allowed variable PTM per peptide 34 Database 3 Select database Database UniProt_SwissProt View Paste sequence Taxa all species Set View taxa De Novo Tag Options Al Available de novo tags de novo with current parameter w 5 Estimate FDR with decoy fusion 6 _ Find unspecified PTMs and common mutations with PEAKS PTM _ Find more mutations with SPIDER 12 Chapter 2 Installation and Activation This section of the manual will guide users through the installation and activation of PEAKS 1 Package Contents The PEAKS package contains e This manual e PEAKS Software e Quick reference sheet for mass spectrometry 2 System Requirements PEAKS 7 is available in both 64 bit and 32 bit versions Installation of the 64 bit version which is the default version that BSI distributes is highly recommended for most of the users For users with a 32 bit operating system the 32 bit version can be downloaded upon request PEAKS 7 supports Windows Operation System Windows XP Windows 7 and Windows 8 PEAKS program files will only use 200MB disk space It requires some extra disk space to store temporary files and intermediate results The amount of disk space required for this purpose depends on the size
215. tion a default path will appear here To use another li cense location for OMSSA click the Browse button to tell PEAKS where to find the desired search engine 1 4 Spectrum Annotation Preferences Click Spectrum Annotation from the left side to open the spectrum annotation preferences Spectrum Annotation Hosoo0e8o8 o 0 A0 0ang HOS EE8g E m E m m E Show Decimal Places 2 m z on fragmentation m z on unannotated sequence fragmentation in place ion info Intensity Low Medium High The spectra in PEAKS results can be annotated by selecting ion types from the thorough collection of ions that PEAKS offers The selected ion types will be displayed in the Ion Match table as well It is possible to annotate the spectrum with various ions for both CID and ETD By default y ion y H20 y NH3 y 2 b ion b H20 b NH3 and b 2 are selected 161 Configuration and Preferences Note To reset the settings to the PEAKS defaults use the Reset default button Show Decimal Places Select the number of decimal places that will appear in the ion table and spectrum view The default 1s set to two decimal places m z on Fragmentation Select this to display the m z value on top of the annotated ions m z on Unannotated Select this to display the m z value on top of the peaks without ion matches sequence Fragmentation Select this to display the sequence fragmentation on the top left corner of
216. tion 2 Data Refinement Parameters for more details on data refinement parameters Data Refinement Predefined parameters default Merge Scans Retention time window for raw files only Precursor m z error tolerance Da Merge CID and HCD scans together Correct Precursor Mass only recommended C Mass and Charge states Min charge 14 Max charge Filter Scans Only keep scans satisfying Precursor mass between and Da Retention time between and min Quality value greater than suggest 0 65 Click the Finish button to exit the Project Wizard without specifying parameters for further analysis Upon clicking the Finish button PEAKS will create the project and run the data refinement on the fractions See Chapter 7 Data Refinement to learn more about data refinement Alternately click the Identification button to proceed to set up para meters for database search 32 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow 4 DB Search Setting Parameters To run de novo sequencing and database search on the project immediately after the project creation and data refine ment click the Identification button on the Project Wizard See Section 2 Setting PEAKS Search Parameters for more details on database search parameters PEAKS Search Predefined parameters Error Tolerance Precursor mass 10 0 ppm using monoisotopicmass w Fragmention 0 5 Da Enzyme Specified by each s
217. tion Workflow Click the workflow icon W on the toolbar and select Identification The identification workflow setup window will appear Click the Select Data button to navigate and locate files to be used for identification analysis Only projects that are open in the Project View panel can be selected for analysis To select the files samples to be analyzed either select the individual file sample or click the All Samples or the All Fractions buttons Then click the Add to Right to transfer the samples files to the Selected Data list on the right hand side Use the Remove and Clear buttons to remove the selected files samples or all files samples from the Selected Data list respectively Click OK to proceed to the next step 145 Workflow j All Data Selected Data FA D te st Peaks60 PeaksProjects Sample Project inChorus d D test Peaks60 PeaksProjects Sample Project Identification E Ll Sample 1 E L Protein Standard HCD 3 fl 0100930_yeast_SCX105_rak_ft8E_pc_O1 RAW e i identification RAW E UD test Peaks60 PeaksProjects Sample Project Identification er Jk I PRG2011 ETD BA I Protein Standard HCD 3 D100930_yeast_5CX105_rak_8BE_pc_01 RAW E m D test Peaks60 PeaksProjects Sample Project PTM E dll PRG_2012 E PRG2012_m2ML mzML E m Ta e Project Labelling Q Note All files loaded in a single workflow will be processed in exactly the same way using the exact sam
218. tion trend chart for that cluster Other information displayed on the summary page is self explanatory 1 Heatmap View A a Q4PD88 CCPR2_USTMA P00489 PYGM_RABIT QOU5905 7L301_YEAST PO0331 ADH2 YEAST PO0330 ADH1_YEAST QE0WC3 TNCTS_MOUSE Q7MTV8 ENO_PORGI P23254 TKT1_YEAST POOS24 ENO1_ YEAST Q6D5V3 PDXH_ERWCT POOT81 TRYP_PIG QSKIV1 HGBE_HAEIN PO27E9 ALBU_BOVIN Q3SZR3 A1AG_BOVIN P12783 FETUA_BOVIM 029443 TRFE_BOVIN P49085 ALBU_RABIT Q3MHN5 VTDB_BOVIN Cell colour represents the log ratio to the Control Sample Control Sample is marked with loge ratio 5 0 0 0 5 0 Default group 1 2 Notes 3 Result Statistics Table 1 Statistics of data and unfiltered result Table 2 Result filtration parameters of MS Scans 11982 Protem fold change of MS MS Scans 22175 4 Other Information Table 4 Search parameters Table 5 Instrument parameters Quantification Type ITRAQ Fractions injl RAW inj RAW inj3 RAW injt RAW inji RAW Quantification Mass Tolerance 0 2Da Ton Source ESI nano spray Quantification RT Range 0 0min Fragmentation Mode CID CAD IRMPD y and b ions Upper Bound Charge 0 MS Scan Mode FT ICR Orbitrap Peptide Score Threshold 20 0 MS MS Scan Mode Linear Ion Trap Label 0 114 11 100 0 Label 1 115 11 100 0 Label 2 116 11 100 0 Label 3 117 11 100 0 3 2 Protein View The Protein view shows a list of proteins that are identified in the database searc
219. togram are converted to pk1 format If you are viewing a single spectrum and choose to convert the data only the viewed spectra will be converted Importing raw data that has not been preprocessed will produce better results when using the preprocessing options native to PEAKS Instrument Preferences for Varian Data To set Varian data related preferences in PEAKS open the Preferences window by clicking the Preferences toolbar icon or by selecting Preferences from the Window menu Click Instrument and then Varian xms on the left This will display the Varian instrument preferences on the right side Click Browse to tell PEAKS the location of the xm1rai exe file Varian xms Default xmlrai exe Location Browse Note Varian xmlrai exe can not read data filse containing spaces or non english characters in its path 8 8 PEAKS 6 Projects Projects created in PEAKS 6 software can be opened in PEAKS 7 To convert the project to a PEAKS 7 project open the project in the same way you would open any existing PEAKS project The project will be recognized as a PEAKS project from an older version The following confirmation dialog box will pop up 41 Project Wizard Create a PEAKS Project Load Da ta and Set up a Workflow A This project was created by an older version of PEAKS Do you want to convert it now Mo thanks Choose Yes to convert the project and proceed The following Project Converter
220. tons or ppm Fragment ion m z tolerance PEAKS de novo uses this value when scoring de novo peptide sequences PEAKS considers a fragment ion to be matched if the calculated m z is within the tolerance Error tolerance parameters need to be set consistently with the mass accuracy of the instrument Default parameters for the instrument generating the data can be loaded by choosing default in Predefined parameters Note When the selected data node is a project of multiple samples generated by different instruments default parameters of the first sample is loaded when choosing default It is recommended to set the tolerance parameters according to the least accurate instrument 2 2 Enzyme Select the enzyme used for protein digestion from a drop down list PEAKS de novo respects the enzyme specificity at both ends when generating peptides Note When the selected data node is a project of multiple samples Specified by each sample allows samples to be analyzed separately using their respective enzyme specified during project creation 60 Peptide De Novo Sequencing Note None is a special enzyme allowing non specific cleavage at both ends of the peptide It is recommended when the digestion enzyme exhibits a high degree of non specificity If the enzyme used for protein digestion is not in the list a new enzyme can be added by clicking the New button Refer to Section 2 1 Enzyme Configuration for details on creat
221. try 532146 04 liil i L Phn i i FDR EXjmati Enabled 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 4 PEAKS peptide score 10lgP PEAKS peptide score 10lgP I Decoy E Target 2 Decoy Target y v lt gt lt gt 4 3 Result Summary and Filtering After opening a result node by double clicking it 1 e the PEAKS DB node in the sample project the summary view is shown by default The summary view provides mainly three functions 1 Specify score thresholds to filter the results 2 Examine the result statistics 3 Export results The top region of the summary view is a control pane and the bottom region is a statistics report page The result filters are controlled at the top control pane see screenshot below e The peptide identification is filtered by the peptide spectrum match s 10lgP score Or by the desired FDR false discovery rate specified by clicking the FDR button e The protein identification is filtered by the protein s 10lgP score and the number of unique peptides the protein contains e The de novo only peptides are the confident de novo sequence tags that remain unidentified by the database search algorithms To report a de novo only peptide the ALC average local confidence scores must be better than or equal to the specified threshold Meanwhile the spectrum s best database search result s score should be no greater than the specified 10lgP threshold By default the 10lgP t
222. ty Significance m z RT range z Avg Area Sample Profile Group Profile RT mean Start End Used Y R IINEPTAAALAYGFDK K 11 14 60 00 847 4564 65 39 69 41 2 4 466 mD IS A 67 02 168 183 Y K FETSAGEFTPQEISAR V 6 67 60 00 885 4336 40 05 45 68 2 3 866 MENT 43 11 103 118 3 W K NQAITNAVNTIR S 11 88 60 00 657 8650 19 87 25 86 2 2 766 i A 23 21 ss 69 4 K NELDTLTYSLEK T 9 50 60 00 713 3655 57 26 61 82 2 2 466 A 59 51 533 544 5 W K AVITVPAYFNDEOR Q 9 47 60 00 811 9212 54 10 60 31 2 1 666 gt MENTE A 56 66 138 151 5 W K HLDMTLTR A 7 89 60 00 493 7610 8 54 11 26 2 825 i 9 60 291 298 7 Y R IPAVQELVK Q iz 6 37 60 00 498 8108 30 12 37 13 2 6 7cS IMM 4033 85 342 350 3 R FDLIGIPPAPR G 6 55 59 23 598 3488 64 29 71 27 2 65m MOMO MOM 67 53 453 463 lv K GETLIGQFAK N lt 5 06 60 00 532 2953 32 36 38 39 2 35 sss 57 0 W R TTPSIVAFTAK G 2 83 11 42 568 3251 30 94 40 80 2 2 0853 E E MA A 35 53 37 47 This table 1s similar to the feature table in the feature view The extra columns are e Checkbox Select the peptides to appear within the Coverage Tab as blue bars e Unique whether this peptide is unique to the current protein group e Start the start position of the peptide in the protein e End the end position inclusive of the peptide in the protein e Used
223. u will be prompted to specify the data refinement parameters first Refer to Chapter 7 Data Refinement 3 Wait for the analysis to finish A new result node will appear in Project View Double click the node to examine the de novo sequencing result ry o view ly DENOVO 4 2013 10 22 11 14 x da 1 10000f 176 E gt Eh 0 m Do LRAW E td E i DATA REFINE 1 2013 10 22 11 06 E 1 F1 535 LLKHKPKATE A Sample 2 gt 2 F3 893 C 57 02 C 57 02 TESLVNR S i F2 BSA4 ysC 1 RAW 3 Fis1490 KKFWGKYLYE wo DATA REFINE 2 2013 10 22 11 06 KVTKC 57 02 C 57 02 TE Ey LL Sample 3 E F1 441 LTKVHKE AL F3 BSA Trypsin 1 RAW g F1 574 LLKHKPKATEE DATA REFINE 3 2013 10 22 11 06 F1 2142 VSVLLRLAKE 2 De Novo Sequencing Parameters After selecting a data node in the Project View click the PEAKS de novo toolbar icon fia The PEAKS de novo parameters dialog will appear 59 Peptide De Novo Sequencing De Novo Predefined parameters Error Tolerance Precursor mass 15 0 ppm a Fragmention 0 05 D Enzyme Specified by each sample PTH Fl Carbamidomethylation Vi Oxidation M QT Deamidation NQ Maximum allowed variable FTM per peptide 35 General Options Report up to 5 candidates per spectrum co o 2 1 Error Tolerance Precursor mass tolerance PEAKS de novo generates peptides within the precursor mass tolerance Precursor mass tolerance can be specified in either Dal
224. udio 104 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER 6 Running PEAKS PTM and SPIDER Separately PEAKS PTM and SPIDER can be launched independently from a PEAKS DB result by selecting a PEAKS DB result node and clicking the a button or the AK button on the toolbar respectively Note SPIDER can also be launched from a PEAKS PTM result In this case select a PEAKS PTM result node and click the AK button on the toolbar 6 1 Running PEAKS PTM on a PEAKS DB Result Launch PEAKS PTM by selecting a PEAKS DB result and clicking the PEAKS PTM icon on the toolbar A Running PEAKS PTM on a PEAKS DB result is functionally equivalent to running both searches together in an integrated search The parameters used in a PEAKS PTM search are similar to a PEAKS DB search Selecting a protein database 1s not necessary as PEAKS PTM automatically uses the database searched in PEAKS DB PEAKS PTM Predefined parameters Error Tolerance Precursor mass 15 0 ppm v using monoisotopic mass a Fragment ion Enzyme Specified by each sample 7 Allow non specific deavage at one end of the peptide Maximum missed deavages per peptide 3 PTH Search with 485 built n modifications 5 Search with preferred modifications Fl Carbamidomethylation Oxidation m Vi Deamidation NQ Maximum allowed variable PTM per peptide 3H Filter Options Perform PTM search on spectra satisfying the fol
225. uence coverage for identified proteins The view has four components Protein table Lists protein identifications that have been filtered by the current settings in Summary view Coverage Visualizes the protein sequence coverage of the selected protein showing the mapping of supporting peptides and de novo tags to the protein sequence Peptides Lists supporting peptides mapped to the selected protein De novo Tags Lists de novo only peptides mapped to the selected protein 95 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER Show top x proteins in each group accession contains search Q amp no results Accession 10lgP Coverage Peptides Unique PTM Avg Mass Description Mark 2 0 Proteins iv a ii Po2769 ALBU_BOVIN Bache Serum albumin OS Bos taurus GN ALB PE 1 SV 4 Aa i Q3SZR3 A1AG_BOVIN Y mm 34 8 8 23182 Alpha 1 acid glycoprotein OS Bos taurus GN ORM1PE 2 Y P00760 TRY1_BOVIN 157 56 mam 6 A MM 37 6 6 edcaa 25785 Cationic trypsin OS Bos taurus PE 1 SV 3 7 gt Q5XQN5 K2C5_BOVIN 152 90 A I 13 8 6 Enarrel 62937 Keratin type II cytoskeletal 5 OS Bos taurus GN KRT5 P Y Q3MHN5 VTDB_BOVIN 128 14 I E MM 12 5 5 2686 53342 Vitamin D binding protein OS Bos taurus GN GC PE 2SV 1 Y QOBD91 K2C75_BOVIN 105 47 I E oe 8 4 2 anar
226. ups PRDX1_HUMAN_UPS _ P08283ups GSTA1_HUMAN_UPS P02753ups RETBP_ HUMAN UPS P81769ups B2MG_HUMAN_UPS P01031ups C05_HUMAN_UPS P10826 8ups TAU_ HUMAN UPS 4 0 P08732ups KCRM_HUMAN_ UPS 000782ups UBE2C_HUMAN_ UPS MO Group 1 P10145ups 1L8_HUMAN_ UPS P00918ups CAH2_ HUMAN UPS P04040ups CATA _HUMAN_ UPS PO02787ups TRFE_HUMAN UPS P38075 PDX3_YEAST P0541 3ups FABPH_HUMAN_UPS P62279ups UBC9_HUMAN_ UPS P07229ups CATD_ HUMAN UPS PO1344ups hGF2_HUMAN_UPS PO1375ups TNFA_HUMAN_UPS P12081ups SYHC_HUMAN UPS P02144ups MyYG_HUMAN_UPS PO27688ups ALBU HUMAN UPS P00915ups CAH1_HUMAN_ UPS Ps5957ups BID_HUMAN_UPS P082311ups CATG_HUMAN_ UPS P01112ups RASH_ HUMAN UPS P168083ups MQ02 HUMAN UPS P10599ups THIO_HUMAN_UPS P0G296ups GELS HUMAN _UPS POOTOSups LALBA HUMAN UPS P15559ups MQ01 HUMAN UPS P02741ups CRP_HUMAN_UPS Posso9ups CYC_HUMAN_UPS P0921 1ups GSTP1 HUMAN UPS PO1008ups ANT3_HUMAN_UPS P6887 1ups HBB HUMAN _UPS Peoo05ups HBA HUMAN UPS P62937ups PPIA_HUMAN_UPS 0158423ups MEDDS_HUMAN_UPS In the interactive heat map move the mouse to the tree to select a cluster and left click to show the variation trend chart for that cluster Place the cursor on any point on the variation trend chart to show the protein accession sample name and the corresponding ratio information in a pop up window Click the Export Data button to export the variation trend in a comma separated text file Original option exports the original abundance of proteins Cho
227. ure vector 130 PEAKS Q Label Free e Proteins The quantified proteins with a list of supporting peptide features for each protein 3 1 Summary View The summary view provides three main functions Result Filtration This is achieved by specifying the filtration rules in the area at the top of the Summary View The filtration function is discussed in Section 3 1 1 Result Filtration Summary Report Several statistical charts assist the user to get an overall picture of the results assess the result quality and examine the reliability and reproducibility of the experiment This function is the focus of this section Result Exporting This is achieved by clicking the Export button at the top of the summary view The exporting function is discussed in Section 3 1 3 Exporting Quantification Results The summary report includes an expression profile with candidate proteins assorted in a heat map result statistics tables and a list of filtration parameters To add a summary note click on the Notes button to open a Notes Entry editor where you can edit the notes to be displayed on summary page Proteins Peptide feature Significance O LEMS Summary Significance 30 Fold change 2 has at least 1 unique peptide 2 Result Statistics P33775 PMT1_YEAST F38787 PANE_YEAST FO7267 CARP_YEAST P4034s8 RFC2 YEAST F61626ups LYEC_HUMAN_UPS PE63279ups UBCO HUMAN UPS P1ss5oups NOO4 HUMAN UPS POA24
228. ures Here the focus will be on the protein coverage view only Click the Protein view tab and select one protein The corresponding protein coverage will be displayed at the bottom of the Protein view The protein coverage view maps Overview all peptide identifications of the selected protein onto the protein sequence It enables the effortless examination of every PTM and mutation on each amino acid Some most commonly used operations on the protein coverage view are listed see screenshot below 1 Each blue bar indicates an identified peptide sequence A gray bar indicates a de novo only tag match Peptide identifications with the same amino acid sequence and the same PTMs are grouped together and displayed as a single bar 2 PTMs and mutations are highlighted with colored icons and white letter boxes Highly confident PTMs and muta tions are displayed on top of the protein sequence A PTM or mutation is regarded as confident if the two fragment ions at both sides of the modified residue have relative intensity higher than the user specified threshold in the display option see item 5 3 Click a peptide to show the spectrum annotation 4 Mouse over an amino acid to show the supporting fragment ion peaks 5 Options to control the coverage view display e The coverage outline choice turns on off the peptide bars e The de novo tags sharing specifies the minimum number of consecutive amino acid matches between a de novo onl
229. v file in CSV format The normalization factor used to calculate the quantification ratios can be exported to the file normalization_factor csv file in CSV format The other options are the same as for Labeled Quantification See Section 4 1 Exporting Summary Proteins and Peptides for details Select the output location and click the Export button to save the selected result components to the specified location Export Images The XIC intensity curve LC MS view of the sample features and RT alignment figure of the samples can be exported to image files from the Features view Click the gt button of the corresponding tabs on the Features view Select the resolution and the location to save the file 6 Export inChorus Result The inChorus exporting function contains the same exporting options as PEAKS DB Unlike PEAKS DB the exported results will contain inChorus scores and individual search engine scores for the supporting peptides of the protein identifications See Section 4 Exporting Database Search Results for details 156 Chapter 16 Advanced Configuration and Environment Preferences 1 PEAKS Environment Preferences This section describes the settings of the environment preferences These settings include general preferences raw file converter preferences search engine preferences and spectrum annotation preferences ae a To begin click the Preferences toolbar icon or select Window Pr
230. whether this peptide is used to calculate the protein profile To view the actual abundance of the peptide features among the samples or groups move the cursor on the corre sponding profile A tool tip will display the actual peptide feature intensity along with the ratio Double click on the peptide feature to jump to the feature view Right click on the peptide feature to show the pop up menu similar to the one in the PEAKS DB supporting peptides tab 144 Chapter 14 Creating a High Throughput Workflow PEAKS provides a simple mechanism to define an easy workflow while creating a new project using the Project Wizard The workflow will be applied on the whole project and the results will be generated at the project level Refer to Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow for details on setting up an easy workflow For convenience PEAKS also provides another mechanism to define workflows for protein identification quantifica tion and inChorus search multi engine protein ID This workflow can be applied on multiple projects that can per form PEAKS analyses at the fraction level or sample level or project level This tool is specially helpful for analyses on multiple projects using the same parameters QWI Identification Quantification InChorus Once a specific workflow 1s selected a dialog pops up to specify the analysis steps and the parameters to use in each step 1 Identifica
231. wn for peptides with a score below this threshold Choose the appropriate quantification method used in the experiment from the Select Method drop down list that contains predefined quantification methods The details of the selected quantification method will appear in Quan tification Method Detail panel To create a new quantification method click the New button to display the New Edit Quantification Method dialog Refer to Section 2 3 Labeled Q Method Configuration for how to create or edit a quantification method Selecting the Save as command from the Predefined parameters drop down list allows the users save parameters for ease of use when regularly performing quantification with the same parameters 3 Understanding the Results rs Once completed the protein quantification result will be displayed in a quantification node in the Project View panel Double click on this node to open the result that contains three views Summary view Protein view and Peptide view The Summary view tab will appear by default 3 1 Summary View The MS MS level quantification result summary is presented in the Summary view The quantified proteins are clustered into a tree structure in the Heatmap Proteins are clustered according to their exhibition of a similar expression 124 PEAKS Q MS MS Level trend across samples Move the cursor to the tree to select a cluster and left click to show the varia
232. y added as preferred modifications to the PEAKS PTM search and cannot be removed However any number of additional preferred modifications can be added PTH e Search with 485 builtin modifications Search with preferred modifications A Carbamidomethylation g Deamidation NQ Q Oxidation 1 Maximum allowed variable FTM per peptide 3 Filter Options Perform PTM search on spectra satisfying the following condition De novo ALC score greater than 15 recommend 15 ES Find more mutations with SPIDER Select this option to enable a SPIDER search SPIDER performs homolo gy search on spectra with good de novo sequences that remain unidentified by either PEAKS DB or PEAKS PTM SPIDER identifies novel peptides that are homologous to peptides in the searched protein database When SPIDER is enabled a SPIDER result node will be automatically generated after the search In the SPIDER report search results from PEAKS DB PEAKS PTM and SPIDER are merged 2 1 Saving the Parameters for Future Use After setting up the search parameters they can be saved for future use Click the Predefined parameters drop down list select Save as command and enter a name for the current set of parameters Saved parameters are available for future analyses A set of saved parameters can be loaded by selecting from the Predefined parameters drop down list 88 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM and SPIDER
233. y safe After a computer hardware upgrade it might be required to re activate the software 4 1 Activate PEAKS with a trial or purchased license key The software activation process is very simple If the computer is connected to the Internet PEAKS can be activated by clicking on the first option in the wizard In the Enter the License Key dialog paste or type in the license key and click the Activate button If the activation is successful PEAKS will start normally Manual activation is necessary when the activation failed with the message An error occurred while communicating with BSI licensing server Refer to Section 4 4 Activate PEAKS manually for manual activation The license key is a 20 character long case insensitive alphanumeric string You may find the key in the software package or from your BSI sales representative Your License Key Activate 15 Installation and Activation 4 2 Register to get a free 30 day trial license key This option allows new PEAKS users to evaluate the software before purchase If the computer is connected to the Internet clicking on this option will bring up a web form in the default web browser Please provide your full name institution email address and phone number in the form After the form is submitted an email from lt support bioinfor com gt will be sent with the trial license key This key can then be used to fully activate PEAKS for evaluation purposes Important
234. y selecting it and pressing the Remove button Click the Apply button to apply the changes to the selected peak WN Ton Editor Please choose ion type Selected peak information N Term Don intensity 31245952 0 x wH AHS Y Ha tHI z z H20 Zs y THID After setting an ion the Spectrum Annotation panel the Spectrum Alignment and Error Map panel and the Ion Table panel will reflect the changes The peptide sequence candidate name as displayed in the Result panel and on the top of the Spectrum Annotation panel will also change to reflect the mass remaining to be sequenced on either side of the ion In the example below the selected peak at 1260 5649 m z was designated as a y ion 80 Peptide De Novo Sequencing 502 90607 2 E Manual De Neve Enbensity i 100 344 25 1241 55 1375 71 I 1150 64 5 1045 6 en ie 1090 47 226 12 246 18 1031 49 160481 1359 66 1489 76 bi H20 bi Pe i m miz 100 200 300 400 500 600 200 d 900 1000 1100 1200 1300 1400 1500 1600 1700 a dh 1 1 2 2 ErrTol 0 502 F intensity treshold OrtiSample mzxML ms 2 mz 802 90607 z 2AT 0 0729 TIC 1 79 7 1260 5649 62 7 Seg H3 ry 70 5 0 5 1 2 D 0 5 i 0 5 sog 1000 1500 ma pesa s je a S aas te 7 1241 551 344 25 Nytta Intensity 35 mia 307 1000 1500 Note The manual de novo candidate i
235. y sequence and the protein before it can be displayed as a gray bar e The de novo peptides fully matched checkbox allows a de novo peptide to be displayed if the sequence re gardless of its length is fully matched in the protein e The minimum ion intensity specifies the minimum fragment ion relative intensity in one of the MS MS spectra before a PTM location is regarded as confident and displayed on top of the protein sequence e The checkboxes in the PTM list specifies which PTMs are interesting Click the color boxes to change a color Double click a PTM name to see the PTM detail 6 The full screen button and tool box button Full screen provides a larger view of the coverage The tool box provides common tools such as exporting the coverage pane as a high resolution image file Overview gt sp P02769 ALBU_BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 foutline 8 coverage 63 a i 25 31 33 35 37 41 58 2 g 6839 73 757 80 eee a C ae ee E 8 E de A E Show de novo only sequences 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE TAHRFKDLGE EHFKGLVLIA FSQYDQOCPF AEHVKLVNEL TEFAKTCVAD Y de novo tags sharing 6 3 AAs aa de novo peptides fully matched pa E E E PA Mia d eo N a d nl E B 43 01 Carbamylation 22 17 03 Ammonia oss N 18 15 99 Oxidation M 21 98 Sodium adduct i 17 03 Pyro alu from Q 13 a
236. zard is going to make will be displayed Click the Finish button to commit to the changes to the configuration file The changes will be immediately available to PEAKS 29 Chapter 4 Project Wizard Create a PEAKS Project Load Data and Set up a Workflow 1 Overview Mass spectrometry data needs to be loaded into a PEAKS project before any analysis can be done After creation a PEAKS project is shown as a project node in the top left corner of the PEAKS user interface On the computer s file system a project 1s saved as a directory that contains multiple files including the compressed spectral data and the analysis results To share a PEAKS project you can copy the whole project directory The project can be opened with the same version of PEAKS Studio or the free PEAKS Viewer The Project Wizard guides you through easy to follow steps to create a Peaks project set up the project structure load data into the project and set up the workflow for data refinement identification and quantification To run the Project Wizard simply click the new project button io on the toolbar or select the File New Project command The rest of this chapter discusses the details of project wizard project creation data loading and setting up a simple workflow Create Project Project Name New Project 1 Project Location C Users zrahman PeaksProjects Browse Project Structure Add Data search a BSA _LysC RAW Sample Enzyme Instru

PEAKS 7 User Manual - Bioinformatics Solutions Inc.

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