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PEAKS Studio User Manual (v5.3)

1. 55 Peptide De Novo Sequencing Clicking the header of a column in the Ion Table highlights the corresponding points on the error map and peaks in the spectrum annotation 3 2 4 Error Map The Error Map shows the mass errors of the annotated ions and it is displayed on the right hand side of the Ion Table The m z ratio is displayed on the x axis and the error is listed on the y axis in Daltons The most confident results lie on the centerline C i S z i i i 200 400 600 B00 1000 1700 i400 1600 miz 3 2 5 Spectrum Alignment The Spectrum Alignment is displayed under the Error Map and it shows the entire spectrum and is used as a tool to help us navigate the Spectrum Annotation The blue bar along the horizontal m z axis of the alignment indicates the range of the spectrum in the Spectrum Annotation This alignment displays how the proposed ions 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 reser Soe ices aane sianet Intensity 9 5 miz S00 1000 1500 3 2 6 Parent Scan The Survey tab displays the precursor ion spectrum The buttons that appear in this section are identical to those explained above in the Spectrum Annotation section Info Ion Match Survey Intensity 55 100
2. f peaks Studio 4 Et File Tools Window Help de TI ATN wi Boe BH sBKXROQW DX FN Project view StartPage X 9 PEAKS 4 04 Apr 11 14 07 X RA rz test Pesks Peake Projects PEAKS D8 Tutorial m 2 AAA AAA asinis M M f Peptides 10kP 15 FOR Proteins 10P2 29 and z 0 wiquepeptdes Demovo iCr 3 v amd AC W z 50 Apply Export EdtNotes DATA REFINE 1 04 Apr 11 13 42 53 T estende pei 2 Result Statistics C Data Result View 7 ens rae 1407 Hn XI XTANDEM 5 04 Apr 11 15 50 E Figure 1 The false discovery rate FDR curves X axis is the number of peptide spectrum matches being kept and the y axis is the FDR O INCHORUS 6 04 Apr 11 15 50 E s e gt 5 identified Peptide Spectrum Matches 394 B Project Tree io FOR 1 0 c 80 100 120 140 160 180 number of paptide spectrum matches Tasks Running Info Properties PEAKS Search details Semi Trypsn f Carboxymetiyt v Oxidati D Information Panel Search Engne Name PEAKS 5 3 Parent Mass Error Tolerance 0 10 a Pragment Mass Error Tolerance 0 508 E i Precursor Mass Search Type morosotopic E a Enzyme Sem Trypan a Max Missed Cleavages 2 9 Fixed Modficatons 3 Carboxymethyt 58 01 Variable Modifications e OxdatonM 15 99 Max variable PTM per peptide 3 5 10 15 20 25 30 35 40 45 50 55 5 10 Database swiss 1 PEAKS peptide score 10IgP Taxon nau des att
3. 1585 79 1375 71 E 1048 6 226 12 245 18 749 96 1604 81 1459 76 1031 49 200 400 600 800 1000 Ak y 1400 ell zal es ey Fl chen Urnmsample mzXML mss mz 802 90607 a wy 1 1 2X 2 ErrTol 0 5Da F intensity threshold A SaaS 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 arrow above another peak A pop up window displays the difference between the two peaks in the example below the difference is 109 92297 Fam n Inbensity 7o 100 1585 79 1375 71 PRA 109 92297 s 1049 5 912 52 226 12 246 18 749 45 021 40 1604 81 i 1489 76 l l LEES EE miz 200 400 600 ai 1000 200 1400 m E i a li EUNT OrbiSampie mzxML ms 24m2 802 90607 a y 1 zx zv Ere 0 5 Da 7 intensity threshold Cerpi 29 Tied POET Deselect a peak Double click anywhere in the Spectrum Annotation panel to deselect a peak Zoom in on part of the 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 on the 1 1 button to return to the default view Setting removing ions to from a peak Select a peak and then right click the mouse anywhere in the Spectrum Annotation panel Select Set y 10n from the pop up menu to designate the peak as a y ion or Set b ion
4. File Tools Window Help Note Refer to Chapter 4 Loading Data to a PEAKS Project for how to create a project 2 Specify the PEAKS de novo parameters in the de novo parameters dialog and click OK If your data is not refined yet you also need to specify the data refinement parameters first and click next Most of the parameters are self explanatory and the default parameters provide a good starting point for the analysis Note Refer to Chapter 7 Data Refinement for PEAKS data refinement function 3 Wait for the analysis to finish A new de novo result node will appear in the project tree Double click the node to open the result file F Project View Mg D test Peaks Peaks Projects denovoProject3 Jl Sample 1 EI Orbis pela jy DENOVO 2 VATA KE LL 14 27 4 The result contains two different views Summary and De novo The summary view allows you to specify rules to filter the results and provides statistics of the results The de novo view shows the de novo sequencing results in greater details 47 Peptide De Novo Sequencing i DENOVO 3 04 Apr 11 14 01 X A De novo TLE 5 and ALC 3 2 4 Apply Export Edit Notes L be nowo Sum 20 25 30 35 4D 45 50 55 60 65 FO 75 80 B5 De novo ALC scores 5 You can optionally export the results to other formats by using the summary view 2 De Novo Sequencing Parameters In th
5. This will create a collection of files in the target directory Refer to Section 2 Export De Novo Result for details 6 Run Auto De Novo Sequencing on a Single Spec trum To perform auto de novo sequencing on a single spectrum select the spectrum in the MS MS view of the sample and click the right button to display a pop up menu Select the PEAKS Auto Denovo command from the pop up menu OrbiSample mzXML jy DENOVO 7 08 Mar 11 11 00 la OR oe BAL 434 47 53 PEAKS Auto Denowo Correct Precursor Mass and Charge Export As DTA d Export As PEL Ei RA mera al P 05 Fac 7 Manual De Novo Sequencing PEAKS 5 3 provides a set of tools to help you manually sequence a peptide using graphic cues from the spectrum Note ETD manual de novo is not supported 7 1 Manual De Novo Graphical User Interface The figure below shows the main panels related to manual de novo The five main panels are indicated in the figure below 57 Peptide De Novo Sequencing StartPage x Orbisample mzxM x An OrbiSample mzXML gt 802 90507 2 Tag P Searched Tags a ane Selected Tags 2 HUIR eio novo Result Panel 6 Dal 902 910 6 4 OMSSA 8 08 Mar 11 11 00 A E gt gt 695 34015 RT i Xf XITANDEM 9 08 Mar 11 11 00 pr 2 H gt 507 30252 RT 7 jg DENOVO 16 09 Mar 11 13 52 E mm gt 547 5924
6. 1 Heatmap View Q4PD66 CCPR2 USTMA P00489 PYGM_RABIT Q05905 YL301 YEAST P002231 ADH2_YEAST P00330 ADH1 YEAST Q80WC3 TNC18 MOUSE 7MTV8 ENO_PORGI P23254 TKT1 YEAST P00924 ENO1 YEAST Q6eD5V3 PDXH ERWCT P00761 TRYP PIG Q9KIV1 HGBB HAEIN P02769 ALBU BOVIN Q3SZR3 A1AG BOVIN P12763 FETUA_BOVIN Q29443 TRFE BOVIN P49065 ALBU RABIT Q2MHNS VTDB_BOVIN Cell colour represents the log ratio to the Control Sample Control Sample is marked with C Ls log2 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 Protein fold change 22 of MS MS Scans 22175 4 Other Information Table 4 Search parameters Table 5 Instrument parameters Quantification Type ITRAQ Fractions inj RAW inj2 RAW inj3 RAW inj4 RAW inj5 RAW Quantification Mass Tolerance 0 2Da Ion 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 search together with their identified peptides in the window below The quantification ratios of those quantifia
7. 10536 240 2131 19 1201 12 164 791 2252 6 glisss9s81 GTDLVAVTGVHIEPLGAYSSK mi ausis 00 108756 150635 Uem 3 alasosa De novo only Peptide o ja 10161 1830 9839 0 5 916 50 111 680 14300 7 glisssos3s E O EE TR saer EC 1 AGFLSAIVKGEATSPLIDK 100 27 1916 0618 09 959 04 146 860 20205 6 glisss se4 T ATVTDKQVSYEEAVEKPAEAPOQAA 38 95 2402 1965 L 1202 11 53 018 4678 4 gi l15536997 The table provides the following controls e Sorting by the column Table can be sorted by clicking the headers Going to a different page Use the combo box or the left right arrows located at the left upper corner of the table Searching for a specific peptide First select the search criterion by clicking the triangle beside the search box and then type in the value in the search box Search criteria include scan id partial sequence m z retention time RT and PTM delta mass Once a search is done click the circled up and down arrows to navigate in the matched peptides For each peptide sequence in the table several columns are given 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 Note If multiple PSMs have the same sequence then only the top scoring one is displayed The Spec column shows how many spectra are assigned to the same peptide
8. DB search peptide spectrum matches DB search psm csv More Peptides pepxml peptdes xml Save into OD test Peaks Export FlesTRAOproject QUANTITATION 14 Browse The following exporting options are available Result summary The Summary view page will be saved in summary html file in HTML format in the specified location e Proteins A list of protein identifications will be saved in proteins csv file in Comma Separated Values CSV format in the specified location e Supporting peptides A list of supporting peptides for each protein identification will be saved in pro tein peptides csv file in CSV format in the specified folder DB search peptide spectrum matches A list of peptide spectrum matches will be saved in db search psm csv file in CSV format in the specified location Peptides pepxml A list of peptide spectrum matches will be saved in peptides xml file in pepXML format in the specified location Click the Export button to save the selected result components to the specified location From the Peptide view the Annotated Spectrum Ion 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 2 2 Export Images for details 4 2 Export Label Free Quantification Results The label fre
9. 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 60 Peptide De Novo Sequencing intensity 55 100 1585 79 1375 71 Set y ion Set b ion Remove ion Set other ions lius 355 639 Tue 1150 64 SD Right tags 3 6 Rightmost y ion Leftmast y ion 226 12 246 15 Leftmast b ion 1604 81 Rightmast b ion 1485 76 AH zm e 2 H a 00 400 eno 1000 L200 1400 1600 allzu nena Fintensity trohad Abisample maxML ms 2 mz B02 90607 Ma yl 5 2x 21 ErTo 0 5Da 9 intensity threshold ECO 9 TIC 1 79E7 Select Set other ions from the pop up menu to view the Ion Editor dialog box The Ion Editor dialogue allows you to add or remove 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 list Remove an ion from the selected ion list by selecting it and pressing the Remove button Click Apply to apply the changes to the selected peak FY Ton Editor Please choose ion type Selected peak information mjz 1260 565 i N Term Ion intensty 3245952 0 x x H2O x H3 Y Hz z z H20 z MH3 y 7 HO After setting an ion the Spectrum Annotation panel the Spectrum Alignment and Error Map panel and the Ion Table
10. CIE Validated Path Browse or Download Database EST database Advanced Options Fasta Title Format Rule to parse accession id from FASTA title Mandy Rule to parse description from FASTA title ELA Accessionfid URL http www ncbi nlm nih gov fentrez viewer fogi db 2protein amp val lt Accession ID gt Delimiter s Taxonomy Options taxonid Browse Download taxdmp Browse Download Follow these steps to fill up or update the required fields to configure a database l Select the database from the FASTA Format Database drop down list or select Other if the desired format is not present and a custom one is to be defined If the database FASTA file is already on the local system skip to step 6 In the Basic Options panel enter a name for the database and select Download Database A window will appear confirming the database chosen to be downloaded from the appropriate FTP or website Click Yes to invoke the default FTP client software and download the database automatically Click No to copy the URL to the system clipboard If No 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 Once the database has been downloaded check to see if it is compressed If so extract the file using a program such as WinZip o
11. In particular the summary view allows you to specify rules to filter iD T 2 EL iD cL De novo only 65 PEAKS DB the results and provides information for you to judge the quality of the experiment The de novo only view shows the high confident de novo sequence tags that do not have any high confidence database matches These may be novel peptides in your sample 5 You can optionally export the results to other formats by using the summary view 2 Set PEAKS DB Parameters After selecting a data node in the project tree click the PEAKS DB toolbar icon ti The protein identification parameters dialogue window will appear PEAKS DB Predefined parameters default Error Tolerance Parent ion 0 1 Da T using manoisotapic mass Fragment ion 0 2 Da Enzyme Trypsin View Enzyme New Enzyme Maximum missed cleavages per peptide 1 PTM Remove Maximum allowed variable PTM per peptide 3 Database Select database Database swiss sprot Paste sequence Taxa all species De Novo Tag Options Available de novo tags de novo with current parameter General Options Estimate FDR with decoy sequences Gd Oe Note If your data is not refined in PEAKS yet you will be prompted to specify the data refinement parameters Refer to Chapter 7 Data Refinement for data refinement parameters Error Tolerance The mass error tolerance of the parent precursor and fragm
12. Zi TESTS 221106 CT OTnew HCD 02 RAW 4 amp DATA REFINE 1 D4 Apr 11 13 42 53 Ar E XI XTANDEM 5 04 Apr 11 15 50 f INCHORUS 6 04 Apr 11 15 50 5 Double click the node to examine the de novo sequencing results 5 What s Next Now you ve learnt some of the very basic operations of PEAKS with the sample project To start using PEAKS for analyzing your own data and explore the full functions of PEAKS the following additional work is highly recommended Read Section 4 Creating a New Project for creating a PEAKS project with your own data This chapter also discusses some additional configuration that may be needed for PEAKS to read the instruments raw data f you need to identify proteins from a sequence database read Chapter 6 Adding a Sequence Database for configuring a protein or EST sequence database Attend a free training webinar to go through PEAKS basic functions The webinar information can be found at BSI s website http www bioinfor com products peaks support webinar php Read the corresponding chapters for the analytical tasks you want to conduct It is recommended to become familiar with the de novo sequencing and PEAKS DB analyses before moving on to the more advanced 22 Chapter 4 Loading Data to a PEAKS Project 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 nod
13. panel and the Spectrum Alignment and Error Map panel You can insert one or more tags 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 Searched Tags Selected Tags EN wl r 1 D is L N Apply T YA WF Mr MM LT YAN v Undoing an edit If you have made an error in your sequencing it is possible to undo the change With the peptide candidate still selected in the Result panel right click the mouse and select the Undo command from 63 Peptide De Novo Sequencing the popup menu to return to the previous peptide sequence You can use this button multiple times to return to earlier stages in your edit New Candidate for Manual De Novo Remove the selected Candidate Config Error Tolerance in Manual De Nove Intensity Ye 100 Config PTM in Manual De Novo 34 Undo Redo Add new sequence Can t Save Redoing an edit With the peptide candidate still selected in the Result panel right click the mouse and select the Redo command from the popup menu if you have undone one too many changes You can click this button multiple times to return to later stages in your edit Error Tolerance To set the mass error tolerance in manual de novo sequencing with the peptide candidate selected in the Result panel rig
14. 11 09 36 X Peptides with fold changez 2 y Export EditMotes SelectSamples Summary 5 Export Quantification Result to Other Formats PEAKS quantification results can be exported to other supported formats To export the result press the Export button in the title bar of the Summary view panel Refer to Section 4 2 Export Label Free Quantification Results for details 102 PEAKS Q Label Free 6 Replicate Analysis in LFQ In liquid chromatography_mass spectrometry LC MS based proteomics multiple samples from different groups are often analyzed in parallel Tools that access the quality of proteomics data based on sound statistical principles are needed in this field In PEAKS 5 3 comparison functions are provided in three levels e Assess the reproducibility of MS data from technical replicates e Perform compare analysis of peptides and proteins e Assess the reproducibility of protein quantification from biological technical replicates This section is organized to first introduce how to assign replicate numbers to samples in the project The replicate analysis of MS data comparisons and label free quantification are done together and so each function will be introduced together in one section 6 1 Assign Replicate Number to a Sample A sample can be assigned with a replicate number in two ways in the New Project window when adding a sample to a project and in the quantification win
15. 2076 of MS MS scans 900 a o al e de o o bo 20 25 30 35 40 45 50 55 PEAKS peptide score 10lgP E Decoy E Target ppm 5 10 15 20 25 30 35 40 45 50 55 PEAKS peptide score 101gP a Decoy Target Table 2 Result filtration parameters Peptide 101gP 220 Denovo TLC 23 Protein 10igP 220 De novo ALC 250 Proteins unique peptides gt 0 3 Click the peptide tab to view the identified peptides Click a peptide to check the spectrum annotation below the table 19 A 15 Minute Walkthrough StartPage x H PEAKS 4 04 Apr 11 14 07 X de 1 251o0f251 gt T gan seach ale no results RT Sem Spec Accession PTM 1 1 FOR 27 2 2 PO2769 ALBLI BOVIN 3 D 4 965 83 Fi PO2769 ALBU BOVIM E 2264 19314 L5 755 74 LL 2 PO0722 BGAL ECOLI 1995 8190 0 2 998 92 31 036 1 OE BOVIN Sid sao ae 332 61 35 357 1665 1i PO2769 ALBU_BOVIN mes me 1 BTaBGALRCQ 8 PA TAVEAGEEANOQU 48 5 5212251 LO 841 42 E 1 a ECOLI J E 706 28 22811 3 En ru Lm mecencmuew mecs os moo oom wn 1 pos Yao Y14 Yo 1554 58 1726 64 1237 48 yii 237 1423 55 Ys 193 72 628 32 136 08 n Ya 530 26 V6 E Es 786 41 Ya es 1890 71 PL SSS mlz ten 2000 Aa y ast 20 zr Errtol 0 508 V TIERS 4 Try the following ways to zoom and navigate the annotated spectrum using the mouse e Cli
16. 5 shows the number of peptides by number of miss cleavages which indicates the efficiency of the enzyme a b number af PSMs mass error ppm 10 5 0 H 10 15 mass error ppm Table 5 number of peptides by number of missed clear i Missed Cleavages 2 Peptides 173 26 3 3 Peptide View The Peptide View shows identified peptides The interface contains a peptide table that supports sorting and the search for a peptide Selecting any peptide in the table will display the peptide spectrum matching details at the bottom half of the peptide view 70 PEAKS DB 3 3 1 Peptide Table All peptides above the user specified peptide score threshold are listed in the table If there are more than 1000 peptides the list is broken into multiple pages da 1 1000 of 1934 E san sexc QM amp noresults Peptide 10lgP Mass ppm m z RT Scan FS5pec Accession PTM 1 IA e CUI NR 110 53 E 18 8611 1 3 I 142051 19458 20 gi 15599442 O 2 wiwrGwrGGcYGvck 10599 1726868 12 86444 111836 14530 3 gliseoss 3 VAAAGPLLAAEIDALGK 1089 15 5 8981 0 3 790 46 160 238 21983 1i liss9s789 4 LNAVSAGGNDGLQARPAAEPK 10463 1949 9806 1 4 976 00 48 773 3296 4 glissoesss S _ EAPLHVSNVATFNTETSK 10451 i955 9952 16 979 01 96 308 11504 6 glisso944 BEN GIFPONQWEGGRMATAEAAK 1058 2029 0480 16 1015 53 130 443 17557 5 gliS5S896S ANDAAGDGTTTATVLAQATVNEGLK
17. 85 or PEAKS 225 or Mascot z 45 or X Tandem x 1E 4 or Omssa 1E 4 or SPIDER 35 Edit filters Proteins Score gt 50 and 0 unique peptides edhe filters At least one of the following conditions is true InChorus Score Ex Ex Mascot Score X Tandem p value Omssa e value S a SPIDER SPIDER Score 85 Part Ill PEAKS Q Protein Quantification Table of Contents I PEAKS Oi IS Se Vell mum 88 Nis OVC VAC Mr 88 Ze Sets ParameetS id E E aero enacted entries 88 SM Onderstancdins thie RESU O 90 Sl SUMMA UNCERT t EP 90 Si SU MEW TR TD IE RS 90 SSMUS SAI T erTT 91 TZ PEAKS ORO MYMS Dove Ls A S 92 INEO cu TETUER 92 2 56 LI E al AMICUS cranes RA PI tds A nua aien ssiaa AE 92 S dg mderstandibe the AAA on O ees 93 Su SUMMA NEW r E A EN 94 SM uM i MEER 94 Doe CDBG A Kec 94 155 PEAIKSSO Label BIG iiciso daa ias islas ic aleae Lipi Eee 96 IO vo C A 96 2 Sep DotatielefS accedi rentis ibat a iet foie Tila 96 Sy Understandino DEO Resto ale pit 98 SL SUMMA VIEW sd id a acid editada contada 98 SUM LOC VIS WA Ea 99 3 2 le Bxtracted Ton C bronmidto Mar ia ti iaa 100 S MA A E 100 Did Sa A 101 9 274 VS 2 AMOO exe scat a cine Secu vues et neds eat ovum Pes Saw utasea headed nee sedan UE Pod dant nado dd 101 DRE LT 102 d lA bidder Prts euo D IN ML NI LS DU 102 5 E
18. Axis is Logarithmic scale base 2 Feature Venn Diagram The feature Venn diagram is a standard Venn diagram showing the number of common peptide features and unique peptide features of the two data files Peptide Scatter Plot The peptide scatter plot compares the peptides quantified in two label free quantification results The x axis is the ratio of the peptide of label free quantification result 1 and the y axis is the ratio of the same peptide relative intensity ratios in corresponding samples of label free quantification result B Ratio LABEL FREE 18 sample sample 1 118 1n 5 Ratio LABEL FREE 20 sample sample 1 Peptide Venn Diagram The peptide Venn diagram is a standard Venn diagram comparing the number of quantified common peptides and unique peptides of label free quantification results Protein Q Q Plot The protein Q Q plot is a standard quantile plot comparing the protein ratios from selected samples of label free quantification results The ratios of the proteins in the first sample is plotted against the ratios of the proteins in the second ratio both in ascending order of size and scaled from O to 100 In the ideal case both replicates should result in the same ratios for the proteins and thus the expected result is represented by the diagonal line in red 107 PEAKS Q Label Free 80 au FO 50 d 30 Protein ratio of LABEL FREE 86 sample sample 1 0 20 1 1 9 T
19. Frachons Spiked 040806 2 RAW SpikeO 040806 2 RAW Spikel 04080 Mass error tolerance 0 2 Da 6 2b RAW Spike 040800 2 RAW Spikes 040806 2 RAW Spiked 04080 Retention time Range 3 0 min 6 2RAW Spike6 040806 2 RAW Spike7 040806 2 RAW Spike amp 040806 Upper bound charge 3 2RAW Spikes 040806 1 RAW Spike0 040806 1 RAW Spikel 040806 Minimum peptide score 20 0 RAW Spike2 040806 1 RAW Spiked 040806 1 RAW Spiked 040806 1 The summary includes an expression profile with candidate proteins assorted in a heat map result statistics tables a list of instrument parameters and a list of search parameters To add a summary note click on the Edit Notes button to open a Notes Entry editor where you can edit the notes to be displayed on summary page The summary page can be exported to other formats by clicking the Export button For more details refer to Section 4 2 2 Export Summary Page Heat map The hierarchical clustering of proteins is represented as a heat map depicting relative protein abun dance normalized SC values logged to base 2 of the protein list with filters The hierarchical clustering is mea sured with a euclidean distance similarity measurement of the log2 ratios of the samples relative to a canonical sample 3 2 Protein View Click the Protein View tab The quantified proteins supporting peptides of each protein and peptide features in the survey spectra from each sample will be displayed in the result panel The qu
20. P23396 RS3 HUMAN 40S ribosomal protein ETIITCTITT ma 922 lapa e 2 QS 730717 77787773 NN T RN NN NN RN NN RN RN are prozac 991 UAE jb ame M 1 286822 Histone Hi s0S Ho 0 6 os owo oo m LC PNSEKCE HUMAN 332 1308 os M 3 65065328 Keratn tpe rotos 83 os 99 posoan 92 1803 m 1 1 191613 201 Gattvnheavy chan o7 335 on w m e erore 92 153 a B 13 27259903 pesacoroten Froon 9m on ow on O P38527K1C9 HUMAN MEC AECI E ACI E 98 9 oem 92 mam ex s zmesmpemhmeshs or am os os amp Gvbecopoco HU 91 1932 ne 0 7 33178 9WAFUSSNRL 03 88 owm 90 O PIASKICIO HUMAN So ee REEL SH oran 98 2 398 rm B Hu jerome o 8x om S rpommem m so mu e e a orses omaras no es om owo PIXSSNW HUMAN 992 10537 iow M 8 imemaoSNewemethemy 035 oa ow 99 m porszem 99 1 1492 xw 10 9 35076738 auennenuceotdets 05 os os or MES IT 91 ma anl 7 E Qi es os m QrswopsH mn o1 32m a 9 7 31324452 srta om osn ow 95 an l sn i ean Lenna ee bimbi i lo asa lame 1 Ix s keessa j oe j xm sme mo ars 20m im 3 9 95 35 jj 4 R GLC 57 O2 AIAQAESIRY e 4289 12876605 28 2 64486 77 926 10808 7 090 077 95 106 e 5
21. Project View El iy D test Peaks Peaks Projects PEAKS DB Tutorial GJ Sample 1 E TEST6_221106_CT_OTnew_HCD_02 RAW Be DATA REFINE 1 04 Apr 11 13 42 53 fi DENOVO 3 04 Apr 11 14 01 MI MASCOT 2 04 Apr 11 14 01 p PEAKS 4 04 Apr 11 14 07 XI XTANDEM 5 04 Apr 11 15 50 f INCHORUS 6 04 Apr 11 15 50 There are already several analysis results in the sample projects Open a database search result node PEAKS 4 as an example 17 A 15 Minute Walkthrough 1 Double click the PEAKS 4 result node on the project tree The following result view is brought up at the right side of the main GUI The default summary view shows the result statistics StartPage x f PEAKS 4 04 Apr 11 14 07 X Crepes 209 2115 gt D Proteins 10P 20 v and 0 w uniquepeptdes Demovo TIC 3 y and ALC 50 e EdtNotes 2 Result Statistics Figure 1 The false discovery rate FDR curves X axis is the number of peptide spectrum matches being kept and the y axis is the FDR O 5 0 4 5 1 4 096 1 3 5 3 0 2 0 1 596 1 096 0 596 0 096 identified Peptide Spectrum Matches 394 De novo only Peptide Protein Summary 1 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 number of peptide spectrum matches Figure 2 The distribution of PEAKS peptide score 10lgP a histogram of score b The plot of precursor mass error vs score Y wm a d
22. TA O a as 20 A 15 Minute Walkthrough 2 Click the index html file in the target directory to view the exported results with a web browser You can zip the whole directory and email it to your colleague or put the whole directory under your own website 4 Conducting the Data Analysis In this section we illustrate how to conduct data analysis using de novo sequencing as an example Note that PEAKS has many more functions than just de novo sequencing These will be introduced in later chapters of this manual 1 If the sample project is not opened open it as explained in Section 3 Examining the Analysis Results 2 Select the fraction 1 data node Click the de novo sequencing button at the toolbar File Tools Window Ein a la iu Y le View i DENOVO 3 AT 1 14 01 M MASCOT 2 04 Apr 11 14 01 f PEAKS 4 04 Apr 11 14 07 XI XTANDEM 5 04 Apr 11 15 50 d INCHORUS 6 04 Apr 11 15 50 3 Use the default parameters Click OK Error Tolerance Farentian 0 01 Enzyme Maximum alowed variable PTM per peptide 35 General Options Preprocess this data on the fly peak centroxding charge deconvolution and deisotope Report up to 5 candidates per spectrum 4 Wait for a few minutes A new de novo sequencing result node shows up at the project tree 21 A 15 Minute Walkthrough ra Project View D test Peaks Peaks Projects PEAKS DB Tutorial E Jl Sample 1
23. XICs for precursors ions of identified peptides in multiple data sets No chemical label is required Different samples are measured separately in the same instrument The same peptides from different samples are correlated by their m z and elution time Label free quantification relies on the assumption that the changes in analyte signals reflect their concentrations in one sample relative to another This technology employs overall spectral intensity normalization by interpreting signals of molecules that do not change concentration from sample to sample PEAKS Q uses the overall protein concentration in each sample for the normalization making spiking unnecessary Label free quantification is based on the PEAKS DB search results See Chapter 9 Protein Database Search with PEAKS DB The use of this function is outlined in the following overview l Select a M PEAKS DB fraction sample or project node in the Project View frame Click the PEAKS Quantification toolbar icon Q or select Quantification from the Tools menu Important In order to use the label free quantification analysis of PEAKS Q the survey scans in the data have to be in profile un centroided mode Note Refer to Section 2 Quantification Workflow for how to conduct PEAKS DB and quantification in a single workflow 2 Select the quantification protocol as label free specify the PEAKS quantification parameters in the right panel of the window and click
24. a a a o a 2 3 e 20 25 30 35 4D 4 5 0 15 20 25 30 35 40 45 50 55 PEAKS peptide score 10IgP PEAKS peptide score 10IgP E Decoy E Target a Decoy Target Table 1 Statistics of data Table 2 Result filtration parameters ofMS scans 2076 Peptide 10igP z15 Denovo TLC 73 of MS MS scans 900 Protein 101gP 220 De novo ALC 50 Proteins unique peptides gt 0 2 Change the score threshold beside the 10lgP from 20 to 30 Click Apply button This will apply a more stringent filter to the peptide identification results Notice the changes in the result statistics 18 Start Page x Y PEAKS 4 04 Apr 11 14 07 X Peptides 109P 20 FOR Proteins i0kP 20 and z 0 unquepeptdes Denovo CZ 3 y and ALC 50 ro pn some A 15 Minute Walkthrough 2 Result Statistics Figure 1 The false discovery rate FDR curves X axis is the number of peptide spectrum matches being kept and the y axis is the FDR O 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5951 1 0 0 5 0 0 FDR identified Peptide Spectrum Matches 377 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 number of peptide spectrum matches Figure 2 The distribution of PEAKS peptide score 10lgP a histogram of score b The plot of precursor mass error vs score O number of peptides 5 10 15 Table 1 Statistics of data of MS scans
25. analysis result node will appear in project view Double click on the result node to view the result F Project View E ip D test Peaks Peaks Projects replicateAnalysis Te REPLICATE ANALYSIS 25 Mar 11 17 40 REPLICATE ANALYSIS 25 Mar 11 17 13 Q LABEL FREE 19 24 Mar 11 19 20 i C LABEL FREE 20 24 Mar 11 19 23 O LABEL FREE 21 24 Mar 11 19 33 The results consist of a few charts to compare the data and results of the two samples If you selected both the data and result comparisons when setting up replicate analysis the following charts will appear Feature Comparison The feature comparison scatter plot represents each feature vector which consists of two features detected in the two data files you want to analyze and aligned in the label free quantification The x axis is the log intensity of the feature detected in the first data file and the y axis is the log intensity of the feature detected in the second data file The Pearson Correlation Coefficient is calculated and listed under the chart The standard box plot is shown on the right side of the scatter plot 106 PEAKS Q Label Free 20 4 5 Bau mE 3 97 5 256 m um c i S _ 1 25 3 amp 22 5 ma z as 34 gt 54 5H gc xS l l l 16 18 20 22 24 2b 78 30 Log intensity of fraction A Pearson correlation coefficient 0 864 Fraction A 070828 01 02 070824 DMSO 0h_1 Frachon B 070623_01_03 070924_DM50_0h_2
26. as the number of confidently identified peptides and how many contain a particular PTM 3 Instrument Control Two figures plot the precursor ion mass error distribution revealing how well the instrument is calibrated 4 Other Information The search parameters and MS instrument information are given here In the rest of this section we discuss the most important charts in the summary report False Discovery Rate FDR Curve Figure 1 in the summary page is the FDR curve for the identified pep tide spectrum matches PSM PEAKS keeps at most one peptide for each spectrum peptides with only I L iso form difference are counted as one Thus the number of PSMs is the same as the number of spectra with assigned peptides The PSMs are sorted according to their 10lgP scores The curve shows the FDR with respect to the number of PSMs to be kept in the final result If a score threshold has been provided in the result filtering a vertical dashed line indicates the score threshold Identified Peptide Spectrum Matches 6703 FOR 1 095 FDR F ct 0 096 n O 500 1000 1500 2000 2500 3000 3500 4000 4500 45000 number of peptide spectrum matches i500 6000 6500 7000 7500 s000 L Normally a lt 1 FDR is recommended for score filtering If you notice a rapid growth of FDR around the the 1 FDR threshold you may decide to sacrifice several PSMs to significantly reduce the FDR The FDR curve is estimated with the decoy fusion method a
27. au te tiesa cuerdas c ac qiue 132 Pook Mascot SeN eneee E do 132 1 5 2 Al Tandem 9 eU IE tr cio tinea geen tonsa iets irae cine mata Tee tob due C CUm swat cadet 133 1 5 3 VIS SACS CILITIBS e Caos iueps bd Pd In tamene Riu Caras D UE p LENIN Ini snupeE RC arca PUE EDDIE 133 1 9 45 SSGQUESESS CUELTIO A A 133 1 4 Spectrum Annotation Prefetetl 6S e ect utes vean ule toe Rev ova ctu tende vat oudustoc onu oH UDINE 134 2 PEARS CONSUMO ts tt DO aca bitte a eru Erba lee its 135 Za Enzyine CONSTA OI da doi 135 Aa A O e us O a eadadae tes 136 249 Database C omoun OI A A EONA 137 2 4 Instrument Cont Sur ON sir tas dro dic ott eat dades 137 25 sat aime ter COn UOC dados 139 110 Chapter 14 Identifying More PTMs with PTM Finder 1 Summary The PEAKS DB database search function can already identify modified peptides with user selected PIMs How ever specifying very many variable PTMs in PEAKS DB is not the best practice since it will significantly increase the running time PTM Finder is provided to fill the gap PTM Finder uses the same algorithm as the PEAKS DB search engine To reduce the running time when many PTMs are specified PTM Finder limits the search in two ways 1 It only searches using the spectra that have highly confident de novo sequence tags The other spectra typically do not have the required quality to confidently identify a modified peptide 2 It only searches in the proteins that were identified by a previous PEAKS
28. bo 200 400 600 1000 Qe y 1 2X 2Y Errtol 0 5Da V intensity threshold 800 1200 TIC 1 25E6 Info Ion Match Survey y12 1400 Y13 Y14 b13 TESTe 221106 CT OTnew HCD 02 RAW ms 2 mz 965 8784 z 2 RT 24 5746 y15 b14 miz 1600 1800 2000 Selected MS MS TEST6_221106_CT_OTnew_HCD_02 RAW 965 8784 RT 24 5746 Scan 1249 2 Retention Time 24 575 TIC 1 25F6 Number of Peaks 202 Fragmentation Type CID CAD IRMPD y and b ions 653 36176 RT 25 0268 gt 523 23615 RT 25 0419 gt 695 83325 RT 25 0661 875 8321 RT225 1029 St gt 561 79193 RT225 1376 e RT 25 1714 S 1S MS Spectra List gt 872 3772 RT225 2368 S Y Number of Results 5 INCHORUS 6 04 Apr 11 15 50 has 1 matches DENOVO 3 04 Apr 11 14 01 has 5 matches MASCOT 2 04 Apr 11 14 01 has 2 matches PEAKS 4 04 Apr 11 14 07 has 1 matches XITANDEM 5 04 Apr 11 15 50 has 1 matches 20 E B1B 8 B B1 B1B E B BD 9 BD B E E B 9 9 B B 9 9 H E H E Information and Survey Scans gt m 4 Heat Map Heat Map view shows the distribution of LC MS signals features 40 97 35221 29 75 24 65 19 75 1471 9 71 200 900 1000 110 Placing the cursor on the heat map will show the m z value retention time and intensity of that point in a pop up window 34 Data Visualiz
29. ciiin is io ad obi engi ta did oa 24 SPIEL M E a cen waen 24 9 2 Waters Micromass Massi Data tara iii 24 A Dec 25 2d Npphied Diosystems Sciex Data ws usi secretes dieat Sepisuc e aorta cis qubd dies 25 SAL OS TAR Or OTRAP A AA d enda nep aestatem wae senate 25 34 2 Iisttumieht Preferences Lor WIEP indir ai 23 SE NONICTLIE T DERI PE 25 PME Broker Dt 26 3 5 L Instrument Preferences tor Br ker Dala v oiecoctes AD 26 3 6 35 hmad Dalai durae Fe timber re tbt at ta etatem a oie meas teats dome diu eos 21 DM N a ra A A x ETT Ze 25 REASON dond co 28 A Crean E a NEW PLONE 28 2 zxddine Data toan Existing Pro el dit ar as alii 30 Oy Changing the Detrault Project Location ii ERAI E E II I iubar neues 30 gt Data Visa IZ MO addict 32 Tis OO VCR VIC EE 32 2e MSN ON Ac a A A A A ANE 32 SRI Mb DI a D O 33 A Heat WIAD osussto dosi uet int eiu a lae i ees I DI MEME LIDMIFU EL CI R TAR 34 J Blur J Unblut Heat Map A A wae ama cq osi ente ous 35 42 Fiebig ht Beatie Mide Fh Care it tdi de notatae 35 2 5 Mak Teature 7 Unmark Feat axes denegat dadas 36 AA SHOW Mis 2 1 Taide MS Pp mr E 37 AO PI erase buses m bene vudusde Beason ane vu iwc eats Salads MM nnd enna EUER Semana 37 AO SOW A N TEW Pre 38 NOC ECS APN A E O PS CO A A 39 0 Adame a Sequence Database sti dao deta Forme TUE cis 40 Basic Operations 1 Configuring Sequenc
30. convertion process is invisible to the user RAW directory Waters QTOF instruments See Section 3 2 Waters Micromass MassLynx Data e WIFF file AB Sciex QSTAR and QTRAP instruments See Section 3 4 1 QSTAR or QTRAP T2D file AB 4700 4800 series See Section 3 4 3 ABI 4700 4800 LIFT or D directory Bruker instruments See Section 3 5 Bruker Data e RUN folders from Shimadzu instruments See Section 3 6 Shimadzu Data e XMS files from Varian instruments See Section 3 7 Varian 3 Vendor Specific Requirements Most vendors provide tools for MS analysis software to read their raw data format PEAKS works best with raw data because it is unprocessed This allows it to use the data pre processing tools built in to the software designed to maximize identification results Listed below are the requirements to load raw data from each supported vendor 3 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 5 3 MSFileReader is publicly available and can be found at the following link http sjsupport thermofinnigan com public detail asp id 586 Converting with MSFileReader will only work if there are only English characters in the file path 3 2 Waters Micromass MassLynx Data RAW data from Waters instruments can be imported provided that MassLyn
31. d z a E ait Pi tee Fe E pOT 7 a 1 MN ide 2975 yl i ce B ais a a L Es a i L E Es x a B 7 i X du zr 1 A a a f an a 715 x LM T a Ba ts PA k 5 rE Be E a t h cc L S P oe E 3 i os i Ji P i m ae B gt i a E m i i z ad E E a a ue LAT Tg a b a 2465 MB ae ee E sil T M ien P E d a Ga ee A Ha Do 4o Et o h 3 F MA d tg A i SE LE ju Ce be t A eee A rm ho Eas M M AR E 7 LER Ves s el 3 A brand EA E Sp Lt EA 7 i a ree 75 bh a 1 F ig r P riia Eg ams B i E a m 1975 La hn toU Te ee oe a z eur e E eo i EL y ie y hi E z E EA z q LE E IT z E nha int ee AM Ss E a Gee E 1471 4 45 35 i age Roca AT wes 75 gt miz TE Y ee 2 3 E hins c f E ge yg D SS ss 3 E a So Pete ent zo e E A de i E ey O pl 34 0s S00 500 700 300 ana 1000 1100 35 Data Visualization To change the default highlight colour click on the colour icon of the highlight feature button CE os Aa E n C gl Semele Text sample Text a i LI Sample Text Sample Text 4 3 Mark Feature Unmark Feature Mark Feature marks the identified features by circling around them using a selected colour mau f o 0 0 Py a jo NE ENT MET ergo l 9n y jo 0 0 oo an do oc ev 5 e LP T 4097 4 5 o E WE ULM SQ ooa 6 Sp i 0 k fi g 101 m pls 0 n
32. discovery rate FDR curve X axis is the number of PSMs peptide spectrum matches being kept and the y axis is the FDR 5 096 4 4 0 y FDR 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 number of peptide spectrum matches PEAKS DB Mascot Venn Diagram A Venn diagram shows the number of peptides identified by each possible combination of engines Note These numbers do not distinguish high or low confidence for each engine s score For example if a peptide got a high inChorus score but low scores in every individual engine the peptide is still counted 84 3 PEAKS InChorus in the intersection of all engines in the Venn diagram For this reason this Venn diagram is NOT the best way to compare different engines performance Figure 2 Venn diagram for confident PSMs up to three selected search engines Peaks DB Ma scol AlTandem Filter PEAKS inChorus Result The filtration differs from the PEAKS DB filtration mostly by the peptide filters The peptides can be filtered by the inChorus score and each individual engine s score StartPage X M PEAKS 5 19 May 11 10 10 X jjj INCHORUS 6 19 May 11 10 10 x i INCHORUS 15 19 May 11 10 18 X m E E 3i n E T Click the Edit filters button in the summary view to specify the peptide filtration rules A peptide 1s kept as long as one of the specified rules is satisfied Peptides InChorus
33. explained in the following sections B4 1 90f9 gt eb 0 v scan seach Q no results Scan ft Peptide TLC ALC m z z RT ppm o 1 NINE eoe E 2 4 MGNADLINVIMPLK 8 4 70 695 34 2 0 320 48 2 3 5 ANELLLNVK 7 6 84 507 30 2 0 369 2 3 4 7 SHRMTNLNGNPEDR 8 8 63 547 59 3 1 344 3 3 5 8 HSTVFDNLNPPEDR 1L6 83 820 88 2 1 422 3 0 6 10 WAWFFQLYLAMVPSCGVCDR 13 1 65 798 04 3 1 551 3 5 7 12 LVDELTEF AK 8 9 89 582 81 2 1 703 2 5 8 14 SHCELEVEX 6 6 74 537 25 2 1 778 3 1 9 16 ECEADESHAGFDR 9 8 76 733 28 2 1 873 5 4 Sequence DNPQTHYYAVAVVK TLC 11 4 ALC 82 ppm 1 8 v Irkensity 100 y y12 p n p o Tt u v v Aa v a v v x 1375 71 Y11 H20 b11 y10 1260 56 Yo 1150 64 a 1049 6 ye b10 912 52 1189 53 y2 y7 Y9 H20 246 18 Y3 H20 Ya 749 46 1021 49 bi y13 307 17 416 29 Y b7 1359 66 1489 76 A yg 99944 b6 H20 856 36 Y l 675 28 1278 69 D13 H20 147 11 345 24 515 35 1440 2 mj 100 200 300 400 500 600 700 800 500 1000 1100 1200 1300 1400 1500 1600 1700 9e wily 1 31 2X 2r ErrTol 0 5Da intensity threshold info Ton Match Survey Error da b e e in 2 J L o in 500 1000 1500 miz O EEES 500 1000 1500 Ai lt lt p lt p lt lt TI AO vizio Aol plnia v e ojuw pw mui 3 2 1 Peptide Table PEAKS displays the peptide sequence candidates at the top of the screen You can
34. few basic tasks in this chapter users can become reasonably familiar with the PEAKS user interface and some of the important concepts in PEAKS This will prepare them for the more advanced analyses introduced in the following chapters and save tremendous time later on during the use of PEAKS 2 he Main User Interface When you first open PEAKS the main graphical user interface GUI is divided into the following sections as illustrated in the screenshot A The user menu and toolbar B The project tree The data analyses from one experiment are grouped into one project The data are grouped in samples and frac tions of samples Data analysis is done by selecting a data node and clicking a button in the toolbar Analytical results are added as result nodes on the tree after analysis is done C The data result views Double clicking a data node or a result node will bring up a panel in this section to display the corresponding data or results Depending on the types of the data or result nodes the views may be different and are introduced in more detail in their corresponding chapter of this manual Multiple views can be opened at the same time as tabs Note It is recommended to close the unused projects and data result views to preserve memory D The information panel This panel provides information corresponding to the data in the progress tree including parameters and running progress 16 A 15 Minute Walkthrough
35. for noise filtering Once selected the Heat Map view will reflect the changes les le les Noise Level 15848 39 Chapter 6 Adding a Sequence Database 1 Configuring Sequence Databases In addition to de novo sequencing of peptides PEAKS 5 3 has the ability to search through a database to identify proteins 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 from servers Additionally taxonomy may be specified with certain databases Adding or editing a sequence database can be done by clicking the New and Edit buttons when specifying the parameters for a database search analysis A database configuration dialog will pop up for you to configure the database Database Select database Database iPRG E Alternatively clicking the W icon in the main toolbar and selecting Database from the left hand side will open the database configuration dialog The database can be configured in the area below the Database List FY Configuration Database List IPRG New Database Delete Database Instrument Set As Default 1 Parameters In both cases the database configuration parameters appear as following 40 Adding a Sequence Database FASTA format database NCBI nr Validate Database Basic Options
36. get the license file and use the Import License function to activate the software some email servers may treat the license email as spam So please do not forget to check your junk email box 10 Installation and Registration 4 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 from the License Wizard Either save the attached license file or copy the content between gt and lt in the email to Windows clipboard Important If an email 1s not received in several minutes check the junk email box as some email severs treat the license email as spam 5 Click Next in the window of step 3 the following window will appear Either copy paste the license content between gt and lt in the email to the text area or import the license file 1icense 1cs Click Next Paste the license Paste from Clipboard Import the license file the email attachment Browse 6 The following window will open Click Finish 11 Installation and Registration License import completed The license file has been imported The license will be verified the next time the software is launched 7 Restart PEAKS to begin using the software 4 2 Registration without Internet Connection If the computer does not have internet connection or it is behind a proxy firewall or failed using the first
37. kpuPIPHEKG O e 458 1497684 264 78591 3 9037 3 04 04 25 27 6 KK 4 05 PLPOHVSIVERKG4 05 0 e 38 40 195874 360 09 65 95 310 i398 9 032 oan m 24 7 RGCG5202AMQNESR HD0 e 3764 297 6088 27 5 49 86 77932 108098 s 09 om 95 M6 e 8 kcoeeeawQpeTA J e 3297 192738 391 78244 5676 7 amp 3 1 03 035 2X8 29 3 kpePTIPEEQKG80 G e ass 19755 343 72932 66381 909 2 oa o 25 27 10 RGCG5202AMQNERGGODY e 3579 12365 279 647 87 79935 i9 2 om 04 95 mw e ar KeuowswEKD O e 3486 19842 372 43697 9470 15853 2 oa os m2 24 12 KGCGS7028WWSGKAD3I e 3462 937 841 225 45976 20032 1850 2 os 06 139 mi 13 KK9 amp 0DRPDHUSEPKG80 D e 3373 14738525 351 43231 S482 15861 9 03 os m 24 ER e 3o si zs sno ao ws 3 os os f s Mi e 15 RTEMATR H0 0T e 3L87 1038 6312 3 4 8430 iu 1 os os 5 16 kanname o e 29 7 191560 54681 69164 948 21 078 ow z 17 RKG8 01FVADGIFK 48 01 A e 26 mam 343 520 03 102182 i496 10 os 032 i Puma 1 1 3 317 s1281 102387 1449 7 os os a EME is kamea j s 2205 sr ma ses cos wn i on os 2 3 Rawa e 2585 85123 4428 32973 3665
38. menu to open the Preferences window Clicking on Instrument and then ABI wiff on the menu on the left hand side will show the preferences for ABI instruments W preferences b General ABI wiff Instrument ABI wiff Default wiff Raw File Convertor Location Bruker yep baf fid Shimadzu AXIMA nun Variant xmi Search Engine Raw Fie Convertor Options Inn Config ABI raw files may contain several samples do you want to merge all the samples into one data set yes a no Survey Spectrum Centroiding luct Spectrum Cen traiding Default wiff Raw File Convertor Location Click Browse to tell PEAKS the location of the default wiff raw file converter MSX a conmmercial wiff converter developed by Infochromics is available from BSI Raw File Converter Options ABI raw files may contain several samples By default these samples are not merged into one data set Select yes if you would like PEAKS to merge all the samples into one data set Select Survey Spectrum Centroiding if centroiding has been performed before loading the data into PEAKS Select Product Spectrum Centroiding if centroiding has been performed on the product spectrum before loading into PEAKS This is important to insure PEAKS performs optimally 3 4 3 ABI 4700 4800 T2D files can be extracted and imported into PEAKS with a free tool created by BSI If you require this separate free tool contact your sa
39. on the menu on the left hand will show the derby database preferences on the right hand side Derby Settings Derby Host localhost Port 15270 Derby Server Start Memory 512 Derby Jar Location fib 130 Configuration and Preferences 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 settings or the memory utility see Section 5 Adjusting PEAKS Memory Usage 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 Its location cannot be changed 1 1 4 Performance Clicking on Performance on the menu on the left hand side will display the performance preferences Performance Computer Performance LJ Low Mid High Number of Computing Nodes to Start 1 Show 3D View Advanced Options Start Chent Separately Clent JRE Binary Folder yre eabitibn Browse Client JVM Heap Size MB 1024 Start Compute Node Separately Computing Node JRE Binary Folder e 54bitlbin Browse Computing Node JVM Heap Size MB 1024 Computer Performance For 512 M
40. option please follow these instructions 1 Use the registration interface to Request license file without Internet connection and follow the instructions 12 Installation and Registration D Ihave already obtained the license file Description If you have purchased a license have the registration key or want to evaluate the software and this computer does NOT have Internet connections please use this option to request the license file FY License Wizard Click the button below to save the request file to a USB key or a floppy drive Copy the request file license request to another computer PC2 which has Internet The next step will be performed on PEZ 13 Installation and Registration 3 Transfer the license request file from this computer PC1 onto a computer with an Internet connection PC2 using a USB key ora removable storage device On PC2 go to http www bioinfor com lcs20 index jsp and follow the onscreen instructions The license file will be sent to the registered email address 4 After the license email is received on PC2 save the attachment 1icense 1cs as 1s and transfer the file to the computer with PEAKS PC1 If the license email was not found in the inbox please check the junk mail folder 5 Complete the license wizard on PCI to register 4 3 Re registering PEAKS Re registering PEAKS may be necessary when an additional software module was purchased or SPS was renewed BSI will mo
41. possible to annotate the spectrum with various ions for both CID and ETD By default y ion y H2O y NH3 y 2 b ion b H20 b NH3 b 2 are selected Note If you are upgrading from an earlier version of PEAKS or simply wish to reset the settings use the Reset default button to update to the 5 3 defaults 134 Configuration and Preferences Show Decimal places Select the number of decimal places that will appear in the ion table and spectrum view The default is set to two decimal places m z on fragmentation Select this to display m z value on top of the annotated ions m z on unannotated Select this to display m z value on top of the peaks without ions sequence fragmentation Select this to display sequence fragmentation on top left corner of 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 Annotation view when this option is checked and the cursor is placed on a peak Intensity You can set the intensity threshold for spectrum annotation to low 2 medium 5 or high 10 To apply this intensity threshold you have to select the intensity threshold checkbox in the Spectrum Annotation view 2 PEAKS Configuration This step includes the configuration of enzymes PTMs databases instruments and parameters To begin click the Configuration toolbar icon or select Configuration from the Win
42. replicates To assign the replicate number in the quantification window click the Assign replicates button below the Pa rameter Table on the right hand side This will open the Assign Replicate dialogue where the replicates can be defined 104 PEAKS Q Label Free rm replicateAnalysis Replicatel needs 1 samples qh Sample 4 L9 i dl Sample 1 i Sample 5 i Sample 3 J Sample 6 LU Replicate needs 2 samples dh Sample 2 Select the number of replicates from the Number of Replicates drop down list on top of the window All available samples are listed in the unassigned samples list on the left hand side The list of samples in each replicates are displayed on the right hand side To assign a sample to a replicate select a sample from the unassigned sample list and click on the gt button beside the list of samples in the corresponding replicate To remove a sample from a replicate select the sample and click the lt button beside that replicate To remove all assignments click on Clear All button Relative order of a sample in a replicate can be controlled by Up and Down buttons beside the corresponding replicate 6 2 Run Replicate Analysis Select the project from the Project View and right click on the project node Select Replicate Analysis from the pop up menu This will pop up the Replicate Analysis window ds Jl RiS1 Peaks D
43. some of the following functions can be applied to the data in a project A correct data refinement especially the precursor m z correction can often result in significant improvement in the final anal ysis result Merging scans the redundant MS MS scans from the same precursor m z and similar retention time will be merged together Precursor m z correction the precursor m z value given by some instruments is often not of the monoisotopic ion This creates problem in the downstream analysis By examining the isotope shapes in the corresponding MS scans this function can accurately correct the precursor m z to be monoisotopic Precursor charge correction occasionally the data provides wrong or no charge information for the precursor ions This function makes an attempt to correct the charge information Low quality spectrum removal this function attempts to remove the apparently junk spectra This will save some analysis time Use this function with caution as it may also remove a small portion of identifiable spectra Centroiding and charge deconvolution centroiding the peaks and deconvolute the multiple charge 1ons to singly charged 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 However you can run the data refinement function separately by selecting fraction sample
44. ta llas atada 66 3 Dnderstandinie PEAKS JDB ReSsulb id Sut m cietem tetas sese etes 67 Sel The Peptide and Protein SC Ores ar ur Peleo V e cendo de Pallade Desde d 67 LS ANY WIG Weste esate alia oi ade about qtiae ai ovid aeuo a dettes uias ci ute Dno dtt 67 3 9 PODES VIC W dis 70 3 9 oL P pde Tables ooa dd cd ncc ica 71 3 5 2 Peptide Spectrum Maitei a is e b cet als abe usd e fa uova 71 3 5 A IN o ae ata leak tab E oodd udie UE 73 S PLOT VEN 73 at DR aolre L T b cme ee ene ote E a ete oh Suae ener ey do Ue sob nct olus 74 2912 A mT Peu ehe ott Mae utu MS ea ceca s dia uS MR aot 74 3 4 9 ONTI EO Cu Conon site dio d O ta ofpatiA d 75 92 4 LOO BOX A 75 DENOVO Only NIE ia ci E es 75 a IMecPBEARSODB RE SUI 2 nd A AS AEA E wend 75 5 Export PEAKS DB Results for Publication sun dav ta eme eldest uias 57 ooComparison or PEAKS DB Results seu oe oe ed bretagne e DU arcte NA T O T cC omiparisonseSulbezoso dd TI 5 2 Peptide COMPA SON tae eed uds es eM novo ed Pale eM ood atus Mou A Poe 78 6 5 Protem OMI AS a 79 GA Statistical Charts eiiiai p i petite ties psu vna bs e Mita heben tiui dvsuM ues Dua boo LA iR 80 6 5 Expor ne Comparison Results ai a id 80 IO BEATES MENOS aa cluster aol cee S Ms curd one t Va cori wc teeta ed AA ant 82 SPEARS InChorus OVEEVIOW ds oA xs 82 2 Jnderstandins PEAKS 1nChor s Result eee Nar teat eL a we eb hialameutedmenehidss 83 3 Bilter PEAKS mC horus Resultados rec
45. telae set Pu aua AS 121 EXPO Dita pus Etet en tatu a Pubs acutis ftedoto Qe ote Past Mob same nected a teat unas Pie came togas 121 Ze Bxport DANOVE Result aida Dua i eas 121 212 Export ummsary and PeplHdes ica taeda tuique t e I es 122 LA S Sore Eius er MEM 122 on Export PEARS DB Result scada S 123 SA EXport Summary Proe mns and Pepides cad a A 123 IN SA O TAR 124 a Export Quanh caton Results A Ai 124 A Expor babeled Quautiticauon Results etii dieto tre tl ra ru beers 125 4 2 Export LabebEree Quantification Results 2 di 125 4 21 Export Result Im Excel or HTML dicio pobre rdunu dai eo cut utpiueid u aUa 125 2 22 POR uiniaty PAGE aessresnescadu mU A A E E 126 Export SPIDER Re c2 tics cconsiacotet aos delito daa 126 O Export anc horis Resto SEEDS 127 15 ontieurationm and Prel ToN ES sd o edo t 129 L PEAKS Environment Frer ONCE Sues TU weder quo sun ences wae sutras As 129 kl General Preletef eS ratita te etes rectis iiem teinte Macaca aa lbs 129 I SSH Opo TNR ID T 130 KLZ As cus iot ERI O A rian sc Heu D vitu iti C 130 LES Derby Database cpm 130 as A A Get dances 131 1 2 Instrument Pre fe nC ES ass 131 KABL CH tddi eroeeer Tere 132 1 2 2 Bruker Vep Dabs Hid at A Es 132 1 2 5 5Biiadzu AIT EU oio rese tact hor Aere AA Ire eL che ero etu dedu I Pouf ene 132 Ir o iu esp RN TTE DNO RNC DX RUP 132 1 3 5eartchiErernme Prel ronces cuelan utate iie MOI QUII Dane
46. the Delete button Viewing a previously saved parameter set Selecting a parameter set will display the details of that set below For an explanation of the parameters please see the references listed in the Creating a new parameter set section above 140
47. the diges tion rule These semi versions are recommended due to some degree of non specificity of the digestion enzymes If your enzyme or combination of enzymes is not in the list click the New Enzymes button to define the enzyme used in the experiment in Enzyme Editor window afta RE and rat before E or after and before ar after ed bene er atm mnd chore Mow up ta one end of a pepade to disobey the clemage rule You can provide the name of the new enzyme and define the custom cleavage rules sites and select if you would like to allow up to one end of a peptide to disobey the cleavage rule 2 3 Fixed and Variable PTMs To select the PTMs for the de novo sequencing click the Set PTM button to open the PTM Setup window y mA All PTM Selected Fixed PTM gt Carboxymethyl Name Mano mass Residue ste Methylation 14 0156 CKRHDENG x EM Myristoylation 210 1584 Kc G amp N N acy diglyceride cysteine 788 7258 C N sopropylcarboxamidom 89 0584 C N Succinimudyl 5 morphali 127 0533 K X an O18 label 12 0042 str hac O GleNAcylation 203 0784 ST Oxidation M 15 9945 M A Oxidation Hi 15 9945 rw Selected Variable PTM Palmitoylatian 238 2297 CSTK IS Deamidation gt Phosphopantethene 1340 0858 5 Oxidation M Phosphorylation 79 9663 STYHCDR Phosphor ylation STY 79 9563 sTY Propionamide 171 03
48. the fol lowing steps And the detailed instructions of registration with an Internet connection can be found in Section 4 1 Registration with Internet Connection 1 Provide the registration key to the license wizard or request for the 30 day evaluation without a key 2 Receive an email containing the license file 3 Copy the content of the license file into the license wizard However when the computer does not have an Internet connection or is behind a firewall that blocks the registra tion the registration process requires the assistance of another computer with an Internet connection or outside the firewall and consists of the following main steps The detailed instructions can also be found in Section 4 2 Registration without Internet Connection Provide the registration key to the license wizard or request the 30 day evaluation without a key on the computer you want to run PEAKS Installation and Registration 2 Save a generated request file to a removable storage device e g a USB memory key 3 From another computer with an Internet connection upload the request file to BSI s license server 4 Receive an email containing the license file 5 Transfer the license file to the computer running PEAKS and import the license file into the license wizard Important Keep your registration key safe After a computer hardware upgrade it might be required to re register to get a new license file 4 1 Re
49. the residue and the mass remaining to be sequenced on either side of the residue All other panels will also reflect the changes 62 Peptide De Novo Sequencing 802 90507 2 j Manual De Novo Intensity 5653 344 25 A 1170 51 1375 71 y2 1150 64 yl an 1189 531 39 5 912 52 1090 47 M 743 46 226 12 246 18 ii 12 48 1604 81 b2 1359 66 1489 76 b1 H20 bi IN E ai L miz 100 200 300 400 500 600 ri B00 200 1000 1100 1200 1300 1400 1500 1600 1700 Ba lh ii zx zv Brie 0 5 0a internaty threshold Orbisample mzXML ms 2 mz 802 90607 2 2 AT 0 0729 TIC 1 79E7 1185 531 39 45 b H20 b MH3 Seq y Hi yMH3 O 327 17 328 23 344 25 3 338 28 399 27 A 1260 56 1242 56 1243 54 2 1170 51 1189 53 1171 51 1172 50 1 Error da 500 tao 1500 344 33 A 1170 al Hax l d 1130 51 a 44 25 MW ax Searching the left or right side of the spectrum for the first last y or b ion Search a sequence tag Select a peak and then right click the mouse anywhere in the Spectrum View Frame to trigger the popup 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 You can test the suitability of a tag by highlighting it in the Searched Tags list the corresponding information for the tag will be shown in the Spectrum Annotation panel the Ion Table
50. the top right corner of the title bar of the Peptide Candidates Frame The available options for a search are scan number m z value and retention time RT value The resultant peptide candidates can be iterated by clicking the circled up and down arrow buttons in the search tool mjz 733 oO 8 1 1 scan ppm lb mza 95 3 1 3 72 RT 8 6 Note To search with an approximate mass value type only the necessary number of digits after the decimal points 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 3 2 2 Spectrum Annotation The spectrum annotation displays a graphical representation of the peptide spectrum spectrum 03 Peptide De Novo Sequencing Sequence DVGGLPK TLC 6 1 ALC 87 ppm 38 7 All candidates Intensity 95 5 we Ie jr P x is bs 442 13 YG 570 33 bd gt 329 04 yy 414 24 y2 H20 y 3 Ys TA 226 16 357 23 471 19 e b2 NH3 b4 H20 b2 H20 311 08 197 39 m z 100 200 300 400 EI G00 Foo get S00 1000 1100 da silly 1 1 2X 2 ErrTol 0 8 Da V intensity threshold The title bar shows the peptide sequence of the spectrum that is being displayed Press the All candidates button in the title bar to open a pop up window to display all alternative peptides Click on a peptide sequence in the pop up window to select and display the annotation sequence DWGGLPK TLC 6 1 ALC 28
51. vou EE ISA Sa 65 2 DEUCE EAK SDB Parimet I ess dado deta lilas 66 gt Understanding PEAKS DB Result diia 67 Sx The Peptide and Protein COTOS dadas oda tet an E eue Ivi d edad 67 A A s TT c 67 3 9 Pepe VION erinin Tin en nat ceed ut a tt 70 95 Peptide Tablet load 71 23 5 2 Peptide 5pectrum Match ti das erae qu tud icu gti Iu iei eed 71 Dee MSN TTE LU LIU D 73 S Miu mA P 73 SN Protein Tables aaa aan 74 34 Ze Beptides si de dos 74 SO CONCA A AE T 75 Sx NIETO C E 75 30 DE NOVO ONI V EWN Me rr 75 4 ier TEARS DD Restle A di tias E 3cBxport PEAKS DB Results tor PubliCauOl sume tUe rue REP EE 57 0 CoOmpan sorol PEAKS DB Resol ciii cta is di sia TI 61 Comparisom Result 2 6e p aloe T 02 PepEIde C Omparis OM satellite 78 6 9 Proen C OT pabiSODu ud ereaep ena AAA AA AAN 79 oA Statistical CNRS lt td tn oss eden desea tinas 80 6 5 Exporting Comparison Results risu ii li iis Ur eek 80 TO PEAKS ECHOS tara i s dad ii id iii eddie cio eids 82 L PEARSUBChOPDUS OYE WAS 82 2 Understandime PEAKS inchorus Result sata int Reiten dd umi HeR caidas 83 5 Filter PEAKS 1m COrus Result dto pute 85 44 Chapter 7 Data Refinement 1 Overview The raw LC MS MS data often contains noise redundancy as well as errors due to sample preparation and in strument approximation PEAKS Data Refinement tool can be used to improve the overall quality of the data Upon users choice all or
52. 1 T 1 0 10 20 20 40 50 60 ri BO 90 100 Protein ratio ol LABEL FREE 85 sample 4sample 1 95 6 4 Export Replicate Analysis Result You can export the replicate analysis plots and diagrams to an image file To export to an image file position the cursor on any of the plots or diagram 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 Refer to Section 2 2 Export Images for details 108 Part IV Advanced Data Analyses Table of Contents 12 TENIS EBGGE nica fos o A a 111 SEP MEE Pe O Pu E 111 2 setting Up PT NM Binder Patatlietets i ete Eod opidi IH P e I Etude iP I UP taxes 111 3 Understand P TM EIndeEReSUlta retur eu EE 112 5 Homolosy seareh with SPIDER cascara ne ps ties etd ORTI aia A at decade lanes dp tada 113 le DEt SPIDER Parametros UMPesML PUDE da Nds 113 LT1 Run SPIDER on PEAKS DB Results do Eie 113 1 2 Iun SPIDER Tigdependentlby a nevada HResuebenev bene sUe vacent va sna 115 2 Understand SPIDER Results 22 97 tt ti Mat tudax Tos Feet IUe Lans 115 Zas SPIDER Peptide Vic asiatica 116 ASES Pro VIEW PR emm 116 T6 WV ORT W SS 118 T Iden tication Worki OW 5 os etasute ro theta de Pure tints Mardin epe ties teed tates sagt Marsinteue Piece D D Tees 118 2 9 uantrbteattob WOoEKEIONW 2 e CREDE EP Aia C dissert ita Riu Sue Canalo eae eatin UA EE 119 2 Anc hors MWOPDSHON a da tds 120 LT Exportins Data Reports and Pinan usos ern A ues
53. 2 2 Export Images for details 128 Chapter 18 Advanced Configuration and Environment Preferences 1 PEAKS Environment Preferences This section will describe the settings of the environmental preferences including general instrument search engine and ion editor configurations EN To begin click the Preferences toolbar icon amp or select Preferences from the Window menu to open the Preferences window Use the and boxes to expand and collapse the nodes FY Preferences General General gt Display Options RMI Connections Default Input File Directory C Users farahman 13 Derby Database i Performance iiS Project Folder Search Engine C Users zrahman Spectrum Annotation Temporary File Directory d PeaksStudio5 3f temp Default Configuration File Directory C Users fzrahman Default Log File Location SERVER LOG log CLIENT LOG log COMPUTENODE LOG log 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 Project folder PEAKS uses UsER HOME as the default output folder for project files where USER_HOME is the user home directory in your system Select the Browse button to change this location 129 Configuration and Preferences Temporary File Directory PEAKS uses PEAKS_HOME_DIRECTORY temp as the default temporary file outp
54. 3 os os 5 Ca RBADGSOERGEE A 287 amen 7 537 7284 19 1 os 05 4 rre 247s sos oe 2 as 19389 1999 3 3 im 3 3 Peptide View The peptide view displays all the identifiable peptides and their quantification ratios The interface is similar to the peptide table in a PEAKS DB result The quantification ratios for quantifiable peptides are displayed in the ratio columns with sample names incorporated into the header e g Heavy No Label gt search Q Q no results 1 1000 of 1670 BEME o gas as 253 51938 19399 196 3 rasca pow MEC mme MEE 290 ENE 3 erases HAT Di A ETE 5 GENLVSM 15 99 TVEGPPPK 2 00 569 7709 31 6 78 tr Q6 CECI um E AE eat gas ome E TC EC NN NN NUN RN 7 GAM HSSSVTAVAR srar masa 37 5 563 31 39978 472 2 wiBeDce2eaDGo2 HUMAN 030 ow gt s orsscsawsccsr sie 1253600 425 627 83 36 825 4x2 4 PSSOSKZEHUMN 9 swwswweGeEK S506 15537759 318 777 92 68331 9337 8 VIQ MGIIQ GGIHUMAN ow oa 10 GQrsTHYAEWER G60 30 88 171289 6 37 9 85247 108 08 15364 3 QU6IJQHLHMN o Mijn 0 me mum me me sem sm 5 vbeesbomerses e se EDIYSGGGGGGSR Pentide Snectrum Match Dentain Peptide 9 Chapter 12 Quan
55. 6 6 4 Export Replicate Analysis Result ocio won tail xee eon chau niue totis Coo ae OR oniwa 108 PV Advanced Data Anal tarea 109 4 PIM EMOS est stati IS ETA ESA A 111 ESA ora 111 2 seme Up FIM Pinder Parametros dois 111 3 Understand P TM Finder Result ass 112 15 Homology Search wath SPIDBR ii A A edebat b dic Qu ddnde Da 113 Pe Sel OP IDE Re Parameters iaa 113 11 Run SPIDER on PEAKS DB Resul 46 uie hebes om eoo Nea e Doa et ots 113 1 2 eum SPIDER Independently sacd 115 Ze Understand SEIDER ReSUlts 5r NN 115 2 1 SPIDER PEpude V 16 Wi at o si n 116 22 SPIDER Protect Vie Wai sion 116 TO VV OURO ceased et cca aerate aes ete cla Mol bos diet Me clics dedos Ronee Ae comes 118 T Ident fication W OF KINO wW cca beer SAN AN it aa en b ES 118 2 A lt OUAa MCA Work IOWA 119 SMELL OD WY Eod n ia iii 120 PE xporune Dat Reports and Prine a I abeo E S Ene oa 121 Esport DIU A aor e uem l eau Quot aee aper wae es 121 Ze EXPO De NOVO RES ido ts 121 2A Bxport Summaty and Peptides ula 122 2 27 eX DOU MMA CCS o oto uto Oc qot imet Mate cous ues co atau sea b ecole dto ced ire 122 S Bxport PEAKS5 DDB ReS5SUlUa2254 a AAA SAA A Ve fu ous 123 3 I Export Summary Proteins and Peptides iiic eio t bio Rad altis ba Rea etus 123 SA EX DOLE TIM AS ES MT ai 124 4 Export Qu ntification Results 222 ot dis 124 PEAKS Studio User Manual v5 3 4 1 Export Labeled Quantification Results ooooocoooconcnnonconon
56. 7 RT Edi DENOVO 7 08 Mar 11 11 00 YY E DENOVO 17 09 Mar 11 13 53 YD mM iaa B P PEAKS 10 08 Mar 11 11 00 YL c H gt 798 0384 RT i 9 PEAKS 3 07 Mar 11 14 24 YM Came H gt 582 81006 RT i d INCHORUS 11 08 Mar 11 11 00 YA f 537 2491 RT 2 PEAKS 5 07 Mar 11 17 00 ul 3 gt 733 2819 RT Z DENOVO 2 07 Mar 11 14 11 Intensity 95 100 H 692 29 1893 51 Spectrum Annotation Panel 1375 71 1260 56 1150 64 SO 1049 6 Y1 1090 47 1189 53 226 12 246 18 om 1031 49 1604 81 1359 66 1489 76 b1 H20 my 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Aa y 1 2X 2Y ErrTol 0 5Da V intensity threshold OrbiSample mzXML ms 2 mz 802 90607 z 2 RT 0 0729 TIC 1 797 Info Ion Match Survey b b H20 DNH3 Seg y y H20 y NH3 5 30 5 0 5 1 693 30 675 28 676 27 692 29 2 a Error Map 2 693 51 912 52 894 51 895 50 1 Foo 0 5 e 1 0 5 lon Table S00 1000 1500 mz 1222350 02 050 a 3 863 51 652 26 y Mao Eb miz Tan 90 Spectrum Alignment 1500 The panels are briefly described below Result Panel The Result Panel shows all the sequencing results The results of manual de novo are listed in the sub tree with root Manual De Novo Spectrum Annotation Panel The Spectrum Annotation shows a graphical representation of the spectrum the peaks in the spectrum the user selected peaks an
57. 71 C Pyridexal phosphate 229 0140 w Pyro glu from E 18 0106 EJEN Pyro gu from Q 17 0255 Q i amp N E Show unmod New Remove Remove all Switch Type mm The PTM Options list displays all available PTMs To view additional modifications select the Show unimod checkbox To select a PTM as Fixed or Variable click the PTM from the All PTM list and click the arrow beside the Selected Fixed PTM box or the Selected Variable PTM box respectively To remove a wrongly selected 49 Peptide De Novo Sequencing PTM click the PTM from the Selected Fixed PTM or Selected Variable PTM lists and press the Remove button The Switch Type button can switch a selected PTM between fixed and variable If a desired PTM does not appear on the list or is different than what is listed select the New button and the PTM Editing window will open where you can fill in the information pertaining to your particular PTM The newly edited PTM will be displayed in the All PTM list FY PTM Editing PTM name Mass Monoisotopic Neutral loss mass Monoisotopic Residues that can be modified Maximum Number of Variable PTMs per Peptide To reduce uncertainty limit PEAKS de novo sequencing vocabulary by restricting the number of variable PTMs found on a peptide Specify a number by typing it in the textbox beside Maximum allowed variable PTM per peptide 2 4 Other Parameters
58. 795 ppm 2 38 7 Al candidates Intensity 56 ii 5 peptide sequences x mu Peptide TLC ALC amp ppm D V GIG L E E DVGCGLPK 5 1 87 38 7 DRGLPK DVNLPK 75 38 7 VDGGLPRk 64 38 7 mear as 64 18 2 H20 226 16 b2 NH3 b2 H20 197 39 100 00 300 400 enn Sa yl Li A 2 ErrTol 0 8Da JJ intensity threshold Moving the mouse over the spectrum will display a tooltip to show the annotation the m z ratio and the relative height intensity as a percentage of 100 of that particular peak Both the m z ratio and the height of the peak can also be found at the right hand side of the bottom bar of the spectrum annotation panel The annotation provides a few convenient ways to zoom and navigate in the spectrum Zoom to a m z region click the desired start m z and drag horizontally to the desired end m z release the mouse button 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 ncrease the peak intensity place the mouse pointer in the spectrum scroll the mouse wheel button e See the whole spectrum double click in the spectrum or click the 1 1 button 54 Peptide De Novo Sequencing The ErrTol is used to adjust the error tolerance to view the display of matched ions You can use the profile m and peak Il buttons to switch the spectrum view from profile mode to peak mode and vice v
59. 800 Rank PEAKS 12 27 Apr 11 19 54 Protein Number Venn Diagram PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 0 500 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 PEAKS 12 27 Apr 11 19 54 6 5 Exporting Comparison Results To export the comparison results of PEAKS DB searches please right click on the comparison run node and choose to export to Excel file Here you can choose image quality and filter the content you want to export 80 PEAKS DB Export statistics graph se deomes mne e watts o aoc weg sole 81 Chapter 10 Combining Multiple Database Search Engines with PEAKS inChorus 1 PEAKS inChorus Overview It is well recognized that properly combining the results from different database search engines can enhance the accuracy and sensitivity of peptide identifications PEAKS inChorus is such a tool to invoke or import the results of the database search engines SEQUEST v27 rev12 Mascot v2 3 X Tandem v2010 12 01 1 and OMSSA v2 1 8 PEAKS inChorus uses a probabilistic model to combine multiple engines results We assume that a reader is familiar with PEAKS DB Chapter 9 Protein Database Search with PEAKS DB before reading this chapter The use of this function is outlined in the following overview Details of each step can be found in later sections of this chapter 1 el Select a project node
60. 802 91 0 200 400 600 eno 1000 1200 1400 1600 MY yl 2 zx zv Erro ntensity threshold Orb Sample mzXML ms 1 RT 0 0106 scan 1 TIC 1 27E8 1607 9911 4 Filtering De Novo Sequencing Result PEAKS de novo sequencing result can be filtered based on TLC Total local confidence and ALC Average local confidence score filters You can set the appropriate 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 button The result will be updated in the Summary view and the De novo view accordingly DENOVO 2 24 Mar 11 14 27 X Denovo LC z 3 and ACz 30 i Apply Export Edit Notes Summary 56 Peptide De Novo Sequencing Note Whenever you changed a score threshold the Apply button changes color to remind you to apply the filter by clicking it 5 Export De Novo Results The Export button at the top of the summary view allows exporting of the filtered results into a list of html text file readable in a web browser and csv text file readable in Excel files This provides the opportunity to supplement the results in a publication or put up the results on your website To export the filtered results 1 Click the Export button at the top of the summary view A file chooser appears 2 Choose the location and directory name where you want to put the exported files Click OK
61. 9 97 O CRE 52 23 81 33 2E 5 1 1289 35 65 e 3 DLLFKDSAHGFLKVPPR F12 685 970 5 19 1939 0 das 3 100 00 Bl BOR 55 53 55 79 8 7E 6 1 22E 4 52 93 3 LVDSKNFDDYMK F5 521 737 8 13 1473 6 2 5 99 98 a HEBE 48 61 65 03 5 2bE 3 2 546 49 28 10 VKDSDDVPMVLVGNK F5 552 808 4 14 1614 8 3 E 99 98 SL EDU e A 53 98 81 27 1 6E 3 1 19E 8 51 08 11 LADFALLC 57 021LDGK F2 810 668 5 123 1334 6 28 7 99 97 Fa a 52 30 8131 L7E 3 1 2 69E 10 44 22 e The blue background means the engine identified the peptide with high confidence above the engine s own filtration score threshold See Section 3 Filter PEAKS inChorus Result The white background means the engine identified the peptide with low confidence below the engine s own filtration score threshold A dash symbol means the engine did not identify the peptide Different engines are coded by different letters as follows e P PEAKS DB M Mascot S Sequest X X Tandem O OMSSA e R SPIDER Individual Engine s Score In the peptide view each engine s own score is displayed A dash symbol means the peptide is not found by the engine FDR curves If Mascot is part of the inChorus result and decoy validation was chosen in the Mascot search parameters then Mascot s FDR curve is displayed together with PEAKS DB in the summary view Figure 1 The false
62. As PEAKS provides a local copy upon installation a default path will appear here To use another license location for OMSSA click the Browse button to tell PEAKS where to find the desired search engine 1 3 4 Sequest Settings Clicking on Search Engine and then Sequest Settings on the left hand will display Sequest preferences 133 Configuration and Preferences Sequest Settings Sequest Locaton Browse Default Sequest Parameter File params Browse Sequest Result Output Folder Browse To use Sequest click the Browse button to tell PEAKS where to find the search engine Make sure to specify the location of the Default Sequest Parameter File params and the Sequest Result Output Folder 1 4 Spectrum Annotation Preferences Clicking on Spectrum Annotation on the left hand side will open the following window Spectrum Annotation Settings pa H20 NH3 2 ig m s 3 s O e a Em HO o x E PS C H internal n Show Decimal Places i m z on fragmentati m z on unannotated J sequence fragmentation J in place ion info Intensity Low Medium High Reset default The annotated spectrums in PEAKS results can be annotated by the selected ion types from a big collection of ions that PEAKS offers The selected ion types will be displayed in the Ion Match table as well It is
63. B database search Peptide Score Threshold 10lgP Only those peptides with a score above this threshold are used to quantify the identified proteins The labels used in the experiment are configured in the Label Options section To add label click on the Add Label button To delete a label from the list select the label and press the Delete Label button Each label is defined by the following parameters sample name reporter ion mass and the labeling efficiency The labeling efficiency defines rate at which of the chemical reaction add labels to the peptides It is used to adjust the ratios calculated from the MS data Clicking the Save As button at the top right allows the user to save parameters for ease of use when regularly performing quantification with the same parameters 3 Understanding the Result L 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 93 PEAKS Q MS MS Level 3 1 Summary View The MS MS labeled quantification results are summarized in one page in the Summary view In the heatmap proteins are clustered into a tree structure Move the mouse onto the tree in order to select a cluster and left click to show the variation trend chart for that cluster
64. B to 1 GB of RAM select low Select medium for 1 to 2 GB of RAM Select high for 2 GB or more 3D View PEAKS can display a 3D view for quantification results Check the Show 3D View box to enable this function PEAKS comes with the Java3D program to support the viewing of 3D images If this feature 1s not required deselect it to increase performance Advanced Options These options are present for users who want to take full advantage of their 64 bit Windows operating system Start Client Separately Select this option to load the client JRE separately Select Browse to choose the location of the Client JRE Binary folder It is recommended that the client JVM be a 32 bit JRE The default setting is the 32 bit JRE which comes with PEAKS Start Compute Node Separately Select this option to load the compute node separately It is possible to choose a 64 bit JRE downloaded from Java Sun by clicking the Browse button Select the location of the JRE binary folder With a 64 bit JRE the Heap Space can be set to any amount within the maximum physical memory of the computer 1 2 Instrument Preferences This section allows users to change preferences for the following instruments ABI Bruker Shimadzu and Varian 131 Configuration and Preferences 1 2 1 ABI wiff Clicking on Instrument and then ABI wiff on the menu on the left hand side will show the preferences for ABI instrument Note Refer t
65. DB search Thus the purpose of PTM Finder is to identify more modified peptides and increase the protein coverage It will also not search scans that have already been well identified The use of this function is outlined in the following steps Details can be found in later sections of this chapter 1 Select a PEAKS DB results node Click on the PTM Finder x button on the toolbar Note You cannot execute PTM Finder on a raw file or de novo results 2 Specify parameters for PTM Finder in the popup dialog and click OK 3 After the analysis complete a new result node will appear in the project tree 4 Results are viewed in the same user interface as PEAKS DB 2 Setting Up PTM Finder Parameters The parameters of used in a PTM Finder search are very similar to a PEAKS DB search The differences are The database selection pane disappears since the search will be on the identified proteins of the selected PEAKS DB search result e The filter option asks for the minimum de novo tag score ALC and maximum database search score 10lgP in order to determine which spectra are searched If the ALC of the de novo result is low then the spectrum is unlikely to provide a significant hit If the database search score is high then the spectrum was already assigned confidently in the PEAKS DB search step 111 PTM Finder PTM Finder Predefined parameters Instrument_default Mass Options Parent Mass Error Tolerance 0 1 Da Pr
66. Data Refinement Query Type Tag Match Replicate Analysis Mass Error Tolerance De Novo Fragmention tolerance 0 2 Leucine Isoleucine W Lysine Glutamine PEAKS Search PTM Y SPIDER Search PTM Finder Maximum allowed variable PTM per peptide 35 Database Database Swiss sprot Taxa all species De Novo Tag Options Available de novo tags de novo with existing parameter Jnstrument default The majority of options are the same as the previous section The differences are Database select the sequence database for the search If a taxa 1s specified then the search is limited only to the proteins that belong to that taxa De Novo Tag you can either run SPIDER based on an existing de novo sequencing result or create a new one 2 Understand SPIDER Results Clicking on the Peptide View tab will display results that look very much like the results for PEAKS DB 115 Homology Search with SPIDER 2 1 SPIDER Peptide View Click on the SPIDER Peptide then the Protein tab to see the SPIDER matches shown in red Note that only SPIDER homology search results will display a reconstructed or real sequence If the search was done against a PEAKS DB result the PEAKS DB Peptide tab will display hits from the PEAKS DB search gt SPIDER 36 11 May 11 16 13 X da i 1000o0f7255 Mm v scan se
67. EAKS DB result With SPIDER there are two options for searching Search in an existing PEAKS DB result The highly confident de novo sequence tags of the unassigned spectra are searched against the proteins iden tified in the PEAKS DB result This way we can potentially increase coverage get additional evidence for a particular peptide match or identify new mutations in an existing protein using reconstruction 2 Search in a protein sequence database The highly confident de novo sequence tags are searched against a protein sequence database This allows you to find protein matches from a homologous database even if the studied organism does not have a protein database The use of this function is outlined in the following steps Details can be found in later sections of this chapter Select a PEAKS DB result node search in an existing PEAKS DB result or select a data node search in a protein sequence database Click on the SPIDER button X in the toolbar 2 Specify parameters for SPIDER in the popup window and click OK 3 After the analysis complete a new result node will appear in the project tree 4 Results are viewed in almost the same user interface as PEAKS DB The main change is for the search in an existing PEAKS DB result we will display three views protein view SPIDER peptide view and PEAKS DB peptide view In the protein view coverage column distinguishes between SPIDER hits PEAKS hits and overlapping hits by c
68. FAEDK 144 10 DVC 90 81 2889 4795 4 0 1445 75 50 602 F2 3172 191 P02769 ALBU BOVIN 6 48 0 53 4 00 3 L 144 10 ITAIGDVVNHDPVVGDR 87 96 2033 1027 4 9 1017 56 55 036 F2 3664 144 P00489 PYGM RABIT 1 22 0 32 0 74 2 4 1 144 10 EEELGDNAVFAGENFHHGDK 144 10 L E z 2728 3335 2 6 910 45 49 599 F2 3061 2 P00924 ENO1 YEAST 1 95 0 28 1 10 amp 5 S 144 10 GETEDTFIADLVVGLR 1965 0176 3 5 983 52 69 426 F1 4939 P00924 ENO1 YEAST 1 81 0 32 1 10 P 6 144 10 PIVSIEDPFAEDOWEAWSH 86 18 2549 1509 19 1275 59 61163 F2 4297 5 PUS24ENOLYENST 16 on 102 e BURGOS uc COST RO E EEES IET NM GNE NN A s Y 144 10 NGVFQEC 57 02 C 57 02 QAEDK 2034 9019 0 2 1018 46 43 745 F5 2174 P02769 ALBU_BOVIN 5 92 E 59 gi 60 a y 1 i P00489 PYGM RABIT 0 75 0 37 1 1 P00924 ENO1 YEAST 1 33 11 A 144 10 VDDFLLSLDGTANK 144 10 P00925 ENO2_YEAST 1 33 12 G 144 10 AAGGLGSLAVQYAK 144 10 PO0330 ADH1 YEAST Ac a Ts mmm mmu nan amis mane 1 Lm If there is no special view for reporter ions then you can select a peptide and zoom to the reporter ion region of the MS MS to examine the reporter ions 95 Chapter 13 Label Free Quantification LFQ 1 Overview Label free quantification is one of the three quantification modes supported by the optional PEAKS Q module This quantification type is based on the relative intensities of extracted ion chromatograms
69. G Real KMFNVGGYLQAVLDR LH dtd DatabaseKDFNVGGY LQAVLDR RSD h 0 1 2 3 0 0667 0 0667 0 0667 0 0667 SPIDER matches shown in red MSRPLSDOEK RKOISVRGLA GVENVTELKK NFNRHLHFTL VKDRNVATPR DYYFALAHTV 1 RDHLVGRWIR TOOHYYEKDP KRIYYLSLEF YMGRTLONTM VNLALENACD EATYOLGLDM EELEEIEEDA GLGNGGLGRL AACFLDSMAT LGLAAYGYGI RYEFGIFNOR ICGGWOMEEA 61 DDWLRYGNPW EKARPEFTLP VHFYGRVEHT SOGAKWVDTO VVLAMPYDTP VPGYRNNVVN TMRLWSAKAP NDFNLEKDFNV GGYIQAVLDR NLAENISRVL YPNDNFFEGK ELRLKQEYFV 121 VAATLODIIR RFKSSKFGCR DPVRTNFDAF PDKVAIOLND THPSLAIPEL MRVLVDLERL DWDKAWEVIV KTCAYTNHTV LPEALERWPV HLLETLLPRH LOIIYEINOR FLNRVAAAFP 181 GDVDRLRRMS LVEEGAVKRI NMAHLCIAGS HAVNGVARIH SEILKKTIFK DFYELEPHKF ONKTNGITPR RWLVLCNPGL AEIIAERIGE EYISDLDOLR KLLSYVDDEA FIRDVAKVKO 241 ENKLKFAAYL EREYKVHINP NSLFDVOVKR IHEYKROLLN CLHVITLYNR IKKEPNKFVV PRTVMIGGKA APGYHMAKMI IKLITAIGDV VNHDPVVGDR LRVIFLENYR VSLAERVIPA 301 ADLSEOISTA GTEASGTGNM KFMLNGALTI GTMDGANVEM AEEAGEENFF IFGMRVEDVD RLDORGYNAO EYYDRIPELR OIIEOLSSGF FSPKOPDLFK DIVNMLMHHD RFKVFADYEE 361 YVKCOERVSA LYKNPREWTR MVIRNIATSG KFSSDRTIAQ YAREIWGVEP SRORLPAPDE KIP For Homology Match results the above display contains additional information in the form of an alignment Let ters on a green background with vertical bars indicate agreement Letters on a red background indicate sequencing error Colour codes on the de novo sequence letters still indicate positional confidence Letters on a blue back ground indicate unce
70. H3 y 245 amp 08r 1 7204 5403 5502 36 52 A als ys om 169 10 151 09 152 07 85 05 1942 98 1943 96 980 99 A mrasa 3 282 18 264 17 255 15 141 59 eT 1845 92 1846 91 932 47 ES 383 23 365 22 366 20 192 11 1750 67 1732 84 1783 82 875 92 0 8 pa J 470 25 452 35 15 16495 64 567 25 63 1562 61 1544 64 1447 55 1429 58 7a 724 37 10 7 44 11 1 J 500 1000 aol APITSD ALY sl ErS E Big avi V ii PHP Sh zh E de 1350 45 1332 68 1333 66 675 84 E 1245 51 1231 63 A 112 52 1102 59 1049 49 1031 55 1032 54 anal D i i i i 4 i 4 i i i I 4 i i f Inbersi The default display is divided in four areas The spectrum information When multiple spectra match the same peptide the top scoring spectrum is chosen by default The spectrum information including the peptide spectrum matching score and mass error are displayed in this area The other spectra can be examined by clicking the All matches button 2 Thespectrum annotation The annotation provides a few convenient ways to zoom and navigate in the spectrum Zoom to a 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 scrol
71. NES HE Elsie 20 20 68403 273 gi 15599955 ref NP_253449 1 Lm ranis Mil 35x 1 19 93628 242 15596984 ref NP 250478 1 27451 ER HOM 2 25 25 150837 jglISSSO4GGlreflN 252960 1 25310 IM FI lao U 17 55553250 gi5600749 efiNP 25424311 O gilt55967 260 29 I INI VH 43 18 18 71668 055 g15596793 eFINP_250287 1 GG ghsseei 25535 MMA HW E Mss 20 20 49369 684lgi15590461 refINP_252955 1 gi559635 Lzem X LIII a s 0 os E 3947 11 B CHNEN ITE TONS GENE GNE 5 HL IULIAE DIU 5 n 2n V Mass ppm ze RT Scan Start End 1 KWANDAAGDGTTTATVLAQATVNEGLE A 103 36 2400 2 CE UCAWANAQXAEASCGSLODADW AR e S877 2281 21i mes us i987 7 38 a4 8 3 RqwawGpEPSWWOKV e oL 163298 0 2 820 54 49 10 453 8 3 R QCT OS IVANAGDEPSWVDRW O e 91 07 i8499 13 92 97 iumo 7 48 49 e S KRWINKENTTIIDGAGVQADIEAR e 83 50 2581 3 16 861 36 11558 3 322 38 6 R GIDKATVAIVAQUKELAKPC S7 0ADTKA 827i 2590 4 14 847 156 21561 14 ii 12 8 RGIANAGDEPSWYDQKQ e 79 9 H670 10 334 86 6177 15 43 am ENS e Bos 1988 1 m9 m Smp EE KOT 05 ETTSDYOREKL fam 3 pope Fre Sn om as m re mame lo 0m 1 ma O D 3 4 1 Protein Table Each row of the table is a group of proteins that share the same set or a subset of identified peptides A dark blue
72. New Candidate for Manual De Novo from the pop up menu A new candidate will be created under the Manual De Novo heading The new candidate will not have been sequenced so it will be represented by the mass of the peptide less the mass of water see an example below 802 90607 2 El Manual De Novo AC am m uc omo so Pas ma Note The pop up menu will not be accessible if you have highlighted any of the results in the Result Panel 7 2 Manual De Novo Operations When the mouse cursor is placed in the Spectrum Annotation panel a green by default arrow follows the movement 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 intenzb 951 100 1565 79 126 56 1150 64 50 1049 6 917 52 225 12 246 18 732 56 1031 49 ee 1489 75 HIL 1 mM Al LLL L l miz 200 400 eo 200 1000 1200 1400 1600 1 3 9 n LL OrbiSample mzXML msz2 mz2802 590607 Ma y 11 2x 2r Erro 0 5 Da 9 intensity threshold ron aao 11001 T9E7 260 5649 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 59 Peptide De Novo Sequencing Fe 1 Inbensity 7o
73. OK 3 Wait for the analysis to finish A new quantification 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 hand side under the Tools heading in the quantification window to view the label free quantification parameters on the right hand side 96 PEAKS Q Label Free Quantification Tools Label Free default Basic Options Mass Error Tolerance 0 2 Da Peptide Score Threshold i0logP 20 Retention Time Range 3 0 min Protein Score Threshold i0lpgP 20 t M5 leve Upper Bound of Precursor Charge 3 5 A Parameter Table Label Free Project Name Sample Name E Protein ID HL Number quantification LI NN RN MM TT 532 26 Bees el ate Ra PEAGS32 mete LLL k E The following parameters are available in the Basic Options section of the quantification window Mass Error Tolerance Used to locate the precursor ion peak group of an identified peptide in the survey scans We deal with non centroided survey scans in a LFQ experiment So set the mass error tolerance a little wider than the parent ion error tolerance in the PEAKS DB database search Retention Time Range The retention time range is the maximum elution time range that is considered for the quantification of an identified peptide This also defines the search range for peptide feature pairing across samples Upper Bound
74. PA 2152 43 74 40 80743 18 2455 FL als 812 9081 410055 59 45 aim mape 46 8385 38 74 555 85176 33 1193 i FONETIK mum ise xii 0843 2 gue a E ODS ROSCA ZA Do me E 6 3 Protein Comparison The top proteins identified in the PEAKS DB results are displayed in the table The display setting score filter and cover map function the same as in the peptide comparison frame The following information 1s also displayed for each protein Score PEAKS protein score Spec the number of spectrum on which this protein has been detected Pep the number of supporting peptides of the protein Uniq the number of unique peptides of the protein Spec the ratio of detected peptides to the theoretical numbers Cov the peptide coverage of the protein The following screenshot is a typical results tab for protein comparisons 79 PEAKS DB 3517 BLU p 5 1 239 1 Rm 6 9 1 de 1 Br 1 pn x 1 pus o O Pa 5 ar E Qu a r 5 a ro T M wu 7 5 Q7visziRposc HeH owo Jooo 6 4 Statistical Charts PEAKS 5 3 provides a number of statistical charts which are easily exported for use in publications The peptide score distribution protein score distribution peptide number venn diagram and protein number venn diagram help users to validate their results Peptide Number Venn Diagram PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2
75. PEAKS Studio User Manual v5 3 PEAKS Team PEAKS Studio User Manual v5 3 PEAKS Team Publication date 2011 Table of Contents be Basic Operas atenas a Lodi sue od ke ta due ud tan E ine td eds l 1 Welcome 10 PEAKS O irete a a e a a ust 4 Pe Malia Pic HOS cacra N A 4 Ze IMA Or New Peares ih 9 RIA S 4 So Guidelines tor U sme this Manual coi dis 5 Ah CONN T DT LET 6 AY A D A O 6 Z Installation and REIS TAO A A A sae tu Etudes da AREE UC 7 Le PACK Ae OBITU S ur a a aia thes 7 ES A Requiteiebibs ou ore E oun pause alesse ap ioe uaseetee ea ELO MUS 7 3 Installat h on a Windows Computer dsd acre ptvique ee dai Hd cota J A NES MISA OS A IS A A Pcr 8 4 1 Registration with Internet Connection coto a ii 9 4 2 Registration without Internet Connection aia 12 2 5 WRC VECISICTING PEARS APP o COH ELI PAD CIELO LPS 14 4 4 Common Errors during Registration ccccc ccc ceec ese eceeeee eee ee ence eeeeeeeeeneeeeneenees 14 Ad justo PEAKS Memory Usa sene catets08t ra teta ir aaa 15 O Whats o cT Dido 15 SA IS Minute Walk Brolg Bises io islas da ituiqu ct uerius epu es 16 TE ADVISER aie DM RE 16 2 be Main Ser INCH ACG stas dentate satan Dto ticos 16 2 Exarminmb the Analysis Result id pocas 17 A Conducting the Data ATalvsls s ioriavuce nct tedio tardado Lisa ebd dataset ceat Ute 21 SR dris Mod mC A 22 A Loadine Data toa FEARS Project ue uocem data 23 OVAS Wii ao a 23 A NAAA A 23 J Mendor
76. PTMs on the fly for PEAKS de novo or PEAKS DB refer to section Section 2 3 Fixed and Variable PTMs 2 3 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 or an EST database of DNA sequences Point PEAKS to an existing database on the system or download one Additionally it is possible to specify taxonomy with certain databases From the Configuration window select Database from the left hand side menu to change the database config uration Refer to Chapter 6 Adding a Sequence Database for details on configuring a new database Delete a previously saved database To delete a database file select the database to be deleted from the Database List and click on the Delete Database button to the right of the Database List Setting the default database To set a database as the default select the database from the Database List and click the Set As Default button This database will now be used by PEAKS when PEAKS DB is run Moving Updating a database To move a database to another directory the location must be updated in PEAKS Select the database and then 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 database
77. Precursor Charge The precursor ion of an identified peptide may have sibling ions of different charge states Only those sibling ions with charge less than the upper bound precursor charge will be considered for quantification of the identified peptide Peptide Score Threshold Only identified peptides with a score above this threshold will be used in quantifi cation Protein Score Threshold Only identified proteins with a score above this threshold will be used in quantifi cation do normalization If selected normalization of protein ratios based on total ion intensity will be done auto matically The Parameter Table includes the following information Project Name name of the project selected for quantification gi PEAKS Q Label Free e Sample Name names of samples in the project Note You need to have at least 2 samples with at least 1 file fraction in each sample Fraction Number the number of the fractions in the sample File Name name of the data file Protein ID PEAKS DB result that will be used in quantification Select the PEAKS DB result to be used from the drop down list containing all available results Add to quantification Check uncheck to add the sample to the quantification There must be at least two samples in label free quantification and the number of fractions within each sample must be the same Clicking the Save As button at the top right allows the user to save parameters for e
78. Report up to peptides Set how many peptide sequences PEAKS will report from its de novo sequencing analysis per spectrum Preprocess this data on the fly PEAKS has its own built in preprocessor for removing noise centroiding and deconvolution Check this box to turn preprocessing on only if you have not done the Data Refinement Step Note If you have already pre processed your data in the data refinement step you do not need to do this again 2 5 Saving the Parameters for Future Use After setting up the desired parameters you can save them for future use Click the Save as button at the top of the window 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 updated and displayed 3 Understanding PEAKS De Novo Sequencing Result Once de novo sequencing is completed a new de novo result node will appear at the project tree Double click the node to open the result file The following results will be available to view 50 Peptide De Novo Sequencing 3 1 Summary View The summary view provides three main functions 1 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 a separate section Se
79. Save into D test Peaks Export Files PEAKS DS Tutorial INCHORUS 6 Browse Export 127 Exporting Data Reports and Printing The following exporting options are available Result summary The Summary view page will be saved in summary html file in HTML format in the specified location Proteins html A list of protein identifications will be saved in proteins html file in HTML format in the specified location Supporting peptides A list of supporting peptides for each protein identification will be saved in pro tein peptides csv file in Comma Separated Values CSV format in the specified folder inChorus peptide spectrum matches A list of peptide spectrum matches will be saved in inchorus psm csv file in CSV format in the specified location Proteins fasta A list of protein identifications will be saved in proteins fasta file in FASTA format in the specified location Peptides pepxml A list of peptide spectrum matches will be saved in peptides xml file in pepXML format in the specified location Click the Export button to save the selected result components to the specified location From the Peptide view the Annotated Spectrum Ion 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
80. Specie Requite Fels AAA AS 24 AA O E tte tte ru patet tanda tees fta o E TES EPIS bios 24 3 2 Waters Micromass MassLynx Data s iere Rer EP aras 24 A A O ose quud 25 2 4 Applied Biosystems 5Sclex Data ende tere gue tunedin IS 25 OL OSTAR OP OLRAP cda ra tasca 25 2 42 Instrument Preferences for WIPE 2 ois cana da clas 22 205 3 ABU E200 4800 nd omnendent A 25 SON LSU qo Da aasa a EA TT E A N T ses 26 3 5 1 Instrument Preferences tor Bruker Data zie iat e s 26 O OM ZU A P c p 27 Dida V A ERE a LL ELE 2 3 5 FEARS 2x PrO OCIS rr en E ded etu Donde ue sede doris acsnmar dian bende uui ddr 28 AC reais d New PEIolecE oux cues e bc iios rd att atest gona ties Oe 28 Addis Data toan Existme Pro cobol 30 6 Changmie the Derault Project Locator ds ia IAS 30 de Data Vial o NEL PU toute eB oveas aes 32 NOV Weiss a A ias 32 DIVAS O 32 SIISE nee 33 As eae MAD Mp Y wee E 34 A Blur Unb lie Mle at Wap aec tte it ase 35 2 2 Hirnet Peairt 7 Hide Peatonal ovid aide 35 4 5 Mark Feature 7 Unmark Pedtulfe ducc ca ida Dem QNM 36 AA Show IMS2 4 EideaVES2 Aa 37 25 OW PEU taa aid 37 ANI O O O II ibo quud 38 NS M EET 39 iii PEAKS Studio User Manual v5 3 6 AdGing a Sequence Database tois e oni Maso duci tica 40 Le Contistans Sequence Databases us dis ett bitum Umm tetur este um unte ieee 40 2 Databases to be Used in PEAKS inChorus Function ooocoocc
81. StartPage X TEST6_221106_CT_OTnew_HCD_02 RAW X 4 PEAKS 4 04 Apr 11 14 07 x y DATA REFINE 1 X 1000 9505 RT224 2213 a gt 535 60583 RT 24 2678 526 7623 RT224 2933 St gt 532 2498 RT 24 3271 S 802 90607 RT 24 3729 gt 634 9555 RT 24 3856 St gt 608 8044 RT 24 4126 S gt 476 46844 RT 24 4568 L C 58 01 C 458 01 AAN 98 DKQ 98 AC 458 01 FAVEGPK 0 7 oo 58 01 C 58 01 AADDKQ 98 AC 458 01 FAVQ 98 GPK 0 7 C 58 01 C 58 01 AAN 98 N 98 KQ 98 AC 458 01 FAVEGPK 0 7 MASCOT 2 04 Apr 11 14 01 C 58 01 C 58 0 1 AADDKEAC 58 01 FAVEGPK 107 3 i DGKEVGC 58 01 C 58 01 SIESMN 98 DAR 27 5 PEAKS 4 04 Apr 11 14 07 Heat Map MS MS Ms Identification Results n gt 644 25494 RT 24 4739 Intensity gt 682 84955 RT 24 4976 gt 951 92957 RT 24 5433 cc A JA p p e JAJc F a v E c ex Spectrum 965 8784 RT224 5746 S gt 875 3408 RT 24 6086 St gt 670 3275 RT 24 6479 St gt 495 72058 RT 24 6828 gt 584 8758 RT 24 7176 St gt 619 7951 RT 24 7624 St gt 705 86426 RT 24 7963 gt 729 85693 RT 24 8299 gt 608 80493 RT 24 8746 gt 532 2504 RT 24 8881 St gt 778 32184 RT 24 9109 549 2551 RT 24 9513 St gt 797 8719 RT 24 9726 St 519 2165 RT 24 9959 St Yo Y10 Y3 Y8 Y11 bo be b4 YS yg y7 6 bs Y2 H20 Y2
82. The other PSMs can be examined by selecting the peptide See Section 3 3 2 Peptide Spectrum Match for details 10lgP The peptide matching score Mass The theoretical mass of the peptide including the H2O but not the extra proton for positive charge ppm The precursor mass error calculated as 10 x observed mass theoretical mass theoretical mass m z The precursor mass to charge ratio RT Retention time e Scan Scan number Spec Number of spectra assigned to the peptide Accession The accession number of the highest scoring protein containing this peptide The other proteins containing this peptide can be examined in the Peptide Detail panel See Section 3 3 3 Protein PTM A dot indicates the peptide contains a PTM 3 3 2 Peptide Spectrum Match For each peptide the Peptide Spectrum Match shows the peptide spectrum matching details 71 PEAKS DB Scan 5341 m 2 1016 4247 2 2 RT 35 491 10lg9 125 60 ppm 89 1 All matches 1 Intensity 953 Y15 ap 1 7 s p 7 s A a a v c a v s A srx P n Ya 5n 808 38 bi3 b amp rur 4 585 27 e ees Y16 Ya b17 H20 y 90742 Maaz 1562 61 Yl 4 1649 64 bs H20 Ye bis Vig Ya 452 35 680 37 28131 1352 55 1750 67 200 400 600 anu 1000 1200 1400 1600 1800 2000 ly 1 zx 2r ErrTol 0 8 Da Y intensity threshold 3 Info Ton Match survey zb b H20 b NH3 b 24 Seq Y y H20 v
83. VOMLERAAFF GVTSNLVLYL E z 61 NSLNFNWDGQ QASRATLLFL GASYLLAPVG GWLADVYLGR FLTISLSLLL YLAASGLLLT 121 TITNDGRRSF CGEMPELPLE PACPSSSCOG SWSSPYCATT LYLVLLLLAL AASSVRSTLT acing 181 SFGADOVMDL GRDATRRFEN WFYWSINLGA ILSLLVVAFI EONISFLWGY SIIVGLVGLA 241 FFIFLFATPV FITEPPTGSO VSSMLELAFO NCCPCRRSSS RDSESAHLLP DORSNOPGPS 301 POEDMANFOV LVEKILPVMVT LYPYWMVYFO MOSTYVLOGL HLHIPNIFRT NPNISLLLRS 361 DSSNYRIPEA WLLLANVAVI LILIPVEDHL IDPLLLRCRL LPSSLORMAL GMFFGFETSII 421 VAGVLERERL OYIAANOTVP OLIGRDLYYA APLSIWWOIP OYLLIGVSEI FASIPGLEFA 117 Chapter 16 Creating a High Throughput Workflow For your convenience PEAKS provides workflows for protein identification quantification and inChorus search multi engine protein ID QWI Identification Quantification InChorus Once a specific workflow is selected a dialogue pops up to allow you to specify the analysis steps and the param eters to use in each step 1 Identification Workflow Click the workflow icon on the toolbar Ww and select Identification the identification workflow setup window will appear Click Select Data to open the Workflow Configuration dialogue where you can select the data you wish to perform identification analyze Only projects that are open in the Project View panel can be selected for analysis To select which files samples you would like to analyze either select t
84. antified proteins will appear in the top panel with homologous proteins clustered together The ratio of Sample 1 Sample 2 appears in the Ratio column and the standard deviation of Sample 1 Sample 2 appears in the SD column 99 PEAKS Q Label Free Accession ID Mass Score T Coverage Matched Description Rato D Marked E E14 Quantification Result P27924ICALE HUMAN 2d 67 569 453 5 Calnexin precursor M 1 00 106 0 00 0 01 L O O60832 DKCi HUMAN 213 S H ACA ribonudeoprote 1 00 1 10 9 01 0 00 Peake OScore 2 pss sion 1 00 0 70 e 33 47 e Multi Cluster Supports The supporting peptide is shown under the Peptides tab The retention time is shown for the specific peptide as well as the peptide ratio from Sample 1 Sample 2 Click on the beside the Outlier folder to see the peptides that were not included in the ratio To see which peptides were used to identify the protein during the PEAKS DB search select the Coverage tab The entire sequence of the protein is shown and the matching peptides are highlighted in blue In this example the total matched part accounts for 3 37 of the protein This information can be found in the Coverage column above 3 2 1 Extracted lon Chromatogram The reconstructed Extracted Ion Chromatogram chart will appear by default in the bottom panel These display the shap
85. arch Q noresults Peptide SPIDER Score RSD Mass ppm m z RT Scan FSpec Accession PTM 3 1 KMFNVGGYLQAVLDR 40 50 0 07 1709 8923 4 0 855 9500 61 621 F3 4342 12 P00489 PYGM RABIT 2 LWMNTEVVLAMPYDTPVPTFK 38 13 0 38 2451 2217 17 8 1226 6400 56 166 F1 3698 1 Q8CI94 PYGB MOUSE x 3 KWCKTQVVLAMPYDTPVPGYR 37 82 0 29 2451 2441 8 7 1226 6400 56 345 F3 3800 2 PO00489 PYGM RABIT z 4 KLLTALGDVVNHDPVVGDR 37 04 0 47 2017 0957 15 0 1009 5400 67 850 F1 4804 15 P00489 PYGM RABIT wu 5 RHMLGDVVNHDPVVGDR 36 71 0 29 1914 9482 19 4 958 5000 60 316 F3 4212 3 P00489 PYGM_RABIT E 6 RHMLGDVVNHDPVVGDR 36 71 0 29 1914 9482 19 4 958 5000 60 316 F3 4212 2 Q8CI94 PYGB MOUSE a 7 KWCKTQVVLAMPYDTPVPGYR 35 82 0 33 2451 2441 8 7 1226 6400 56 345 F3 3800 2 P11216 PYGB HUMAN 8 KEGEYGFONALLVR 35 32 0 07 1622 8416 27 0 812 4500 96 016 F3 7021 121 P49064 ALBU FELCA 9 QDYNVGGYLKAVLDR 35 22 0 20 1709 8735 7 0 855 9500 62 634 F3 4443 10 Q5MIB6 PYGB SHEEP 10 VKKKTRVSTPTLVEVSR 34 50 0 24 1927 1577 4 9 643 3900 74 775 F3 5550 58 P49064 ALBU FELCA _o 11 KLLTALGDVVNHDPVVGDR 34 04 0 47 2017 0957 15 0 1009 5400 67 850 F1 4804 15 Q9WUB3 PYGM MOUSE 12 TOSGAHWRLNVSVSEAALEASTR 33 34 0 39 2456 2043 21 9 819 7600 90 107 F1 6429 2 P00330 ADH1 YEAST E ae PY Sess 1 Da re E m 1 1 P Am am c a amm l E errr ee ag L Peptide Spectrum Match Protein P00489 PYGM_RABIT 7 Denovo KMENVESYLOAVLDR EHE E L
86. are usually dealing with non centroided survey scans mass error tolerance should be set a little wider than the parent ion error tolerance used in the PEAKS DB database search Upper Bound Precursor Charge The precursor ion of an identified peptide may have 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 identified proteins The labels used in the experiment are defined in the Label Options section To add a label click on the Add Label button To delete a label from the list select the label and press the Delete Label button Each label is defined by sample name added mass target residue and labeling efficiency If one sample has multiple labels with different mass shifts add multiple labels with the same sample name These labels will contribute to the same number in the ratio Clicking the Save As button at the top right allows the user to save parameters for ease of use when regularly performing quantification with the same parameters 89 PEAKS Q MS Level 3 Understanding the Result Once completed the protein quantifi
87. ase of use when regularly performing quantification with the same parameters All the parameters in quantification will be saved except the Parameter Table which will change from one project to another The Assign replicates button helps to assign the samples a replicate number This enables PEAKS to perform replicate analysis Refer to Section 6 Replicate Analysis in LFQ for details on how to assign replicates and perform replicate analysis 3 Understanding LFQ Result Once completed the label free quantification result will be displayed in the quantification node Q Double click on this node and the Summary view tab will appear by default Right click on the result node to find more operations supported for the a label free quantification result PEAKS supports export of the label free quantification results to Excel or HTML file by right clicking the result node and choose the corresponding function Please refer to Section 4 2 Export Label Free Quantification Re sults for details PEAKS also supports changing the normalization factor of the protein ratio Right click on the result node and select Normalization Settings the Normalization Settings dialog will pop up t Normalization Settings Linormalze Pepbde Ratios i Automatically Normalize Peptide Ratios Manual y Mormalze Peptide Ratios 1 0 0 7 If you select Unnormalize Peptide Ratios the protein ratio will be calculated from peptide ratios w
88. atabase Search id ji E Spider search E A R252 inChorus Search E A R351 fe J R352 Export DTA file Export PEL file Export MGF file Show Reports Save Copy As Close Project 105 PEAKS Q Label Free Replicate Data Comparison Select the replicate and samples on which you want to perform data comparison analysis Only two replicates can be selected for data comparison analysis Replicate Result Comparison Select the label free quantification results and samples on which you want to perform result comparison analysis You need to select one label free quantification result for each replicate and two samples you want to compare Once a sample is selected all the samples with the same index in other replicates will be selected FY Replicate Analysis Tools Y Replicate Data Comparision V Replicate 1 070828 O1 024170824 DMSO 0h LRAW 7 Replicate 2 070828 O1 03 070824 DMSO h 2 RAW 7 irt l 070828 01 02 070824 DMSO 0h 1 RAW i 070828 1 03 070824 DMSO 0h 2 RAW 4 Replicate Analysis 070829 01 02 070827 drug Oh 1 RAW 070829 O1 03 070827 drug Oh 2 RAW De Nowe PEAKS Search SPIDER Search PTM Finder Y Replicate Result Comparision 4 Replicate 1 LABEL FREE 20 ficate 2 LABEL FREE 18 Selected Sample 1 Sample 2 Sample 3 Sample 4 J Sample 1 JJ Sample 2 6 3 Understand Replicate Analysis Results Once the replicate analysis is completed a new replicate
89. ation m z 619 15 4 RT 25 33 i Intensity 2 15E8 ee The Heat Map view provides a few convenient ways to zoom and navigate the LC MS features in the data Zoom to a specific Heat Map area click the desired start m z value RT position and drag the cursor to the desired end m z value RT position release the mouse button Zoom in out smoothly place the mouse pointer at a particular m z value RT position scroll the mouse wheel button e See the whole Heat Map click the 1 1 button 4 1 Blur Unblur Heat Map The Heat Map view offers various controls to study the LC MS data features 11 CO Miro 7 E rk Feature E Show MS2 Bb show PID of none Shaw 3D View Moise Level 1 For smoother view of the Heat Map choose Blur and for sharp contrasted view choose Unblur 4 2 Highlight Feature Hide Feature Highlight Feature highlights the identified MS features by painting them with a chosen colour a a EP 7 eae a EE a z oe f Y gt ide m au i r i BE p 40 57 a _ E a r 2 F E I m L4 ME a fram EO oi E o x E m AT A E r1 pr a te ik e gr i EO a a fs Te o 4 LA HT r s ul thant 5 kt cma x LES NE rta E 5 QUA or et E 1 4 p Bao o g 3521 QUEM rre east E P uL RN r 4 i un 5 F A A q Pars For i sos d 3 i NE us re doe ces Apo EE EL x Y k p w EI zx T
90. ble proteins are displayed in the ratio columns with reporter ion mass as the header eg Ratio 117 114 This 1s the ratio calculated from the unique peptides from the protein The SD is the standard deviation of the peptide ratios in the protein The peptides of the select protein together with their ratios are displayed at the bottom half of the protein view StartPage X d QUANTITATION 14 22 Mar 11 13 03 X E Accession ID Mass Coverage Display PEAKS Score p value Coverage Querymatched Description Marked Ratio 117 115 114 112 SD 117 115 114 112 El E 5008 Search F i L P00722 8GAL _ECOLI 1 116351 600 98 8 1 99 2 4 3 0 Beta galactosidase Escherichia coli ud 4 31 0 47 E FDECOY zP COL4 CO4A HUMAN 2 192770 160 60 4 2 551 0 46 0 Complement C4 A precursor Acidic com ri xd DECOYSQSNDHS ICA1L HUMAN 3 54407 082 55 8 2 43 1 1 24 0 Islet cell autoantigen 14ike protein Amy r O QONYC9JOYHS_HUMAN 4 511935 780 i 48 4 E 0 2 0 Cliary dynein heavy chan 9 Axonemal 7 N A 0 fDECOYsP23786 CPT2 HUMAN 5 73777 055 47 5 2 43 0 Carnitine O palmitoyltransferase 2 mito T d Peptides Coverage Tool Box ID Sequence PEAKS Confidence 10Log p value Confidence M Z 2 Mr Calk Delta Mass Error ppm File RT Scan Quality Frag Mode Start End Hits 117 115 114 112 m Spectrum 1 A 144 10 PLONDIGVSEATR 100 0 119 31 0 0 1 3545 2 1600 8179 0 1234 77 0997 TRAQSam
91. cation result will be displayed in the quantification node in the project tree 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 level quantification results are summarized in one page in the Summary view In the heatmap proteins are clustered into a tree structure Move the mouse to the tree to select a cluster and left click to show the variation trend chart for that cluster 1 Heatmap View 14617 AA AH a r tu cr Cell colour represents the log ratio to the Control Sample Control Sample is marked with log2 ratio 5 0 0 0 5 0 Default group 1 QSHCD5 NCOAS HUMAN Cz84 Q5Cz84 HUMAN DT5351 VPS4B H MAN P12238 ADT3 HUMAN P18124 RL7 HUMAN P13837 ATTA3 HUMAN trlB4DNOO BADNOO HUMAN F 1817 KV204 HUMAN t 28N355 O8N355 HUMAN tr B3KNZ4 B3KNZ4 HUMAN P28232 CTNA2 H MAN t Q98BAT QS8BAT HUMAN trjOG8IUKT OSIUKT HUMAN WeB CSJWB8 HUMAN QT7LO0J3 SV2A HUMAN t B4E354 B4E354 HUMAN tr D8RBUS DERBUS HUMAN 2 Notes 3 Result Statistics Table 1 Statistics of data and unfiltered result Table 2 Result filtration parameters of MS Scans 550 Protein fold change 75 of MS MS Scans 12443 4 Other Information Table 4 Search parameters Table 5 Instrument parameters Quantification Type ICAT SILAC Fractions VPS54B1J RAW Quantification Ma
92. cid except the one in the brackets Choose where the cleave sites are by selecting after or not after and before or not before to specify the range There is also an option to Allow up to one end of a peptide to disobey the cleavage rule 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 an enzyme that was created select the appropriate enzyme and click the Delete Enzyme button Note For information on defining new enzymes on the fly for PEAKS de novo or PEAKS DB refer to sections Section 2 2 Enzyme Specificity 2 2 PTM Configuration From the Configuration window select PTM from the left hand side menu to change the PTM configuration PTM List Built In gt 4 hydroxynonenal HNE n lt Built In gt Acetylation K lt Built In gt Acetylation N term lt Built In gt Armdation lt Built In gt Applied Biosystems deavable ICAT TM heavy lt Built In gt Applied Biosystems deavable ICAT TM light lt Built In gt Applied Biosystems TRAQ TM 4plex K lt Anlt In gt Annliecd Binsystems iITRACKTM anlex Mi Delete PTM PTM Details PTM name 4 hydroxynonenal HNE Mass Monossotopic 155 11504 Neutral loss mass Monoisotopic 0 0 Residues that can be modified CHE Anywhere Middle Only N term C term Formula H 16 CS OC Rule Built in PTMs The built in PTMs within PEAKS are liste
93. ck and drag horizontally to zoom to a region e Scroll the mouse wheel button 1f your mouse has a wheel above the x axis to change peak height and below the x axis to zoom along m z e After zooming into a particular spectrum hold the scroll bar below the spectrum and slide to view the rest of the spectra Double click the spectrum to zoom back to the whole spectrum If your lab regularly requires sharing of results with a non PEAKS user you may want to try the exporting function as follows Otherwise go to Section 4 Conducting the Data Analysis for conducting data analysis 1 Click the summary view tab again Click the export button to export the results into html and csv text file viewable in Excel Select a directory in your file system where you want to export the results 1 Notes V Proteins html proteins html V Supporting peptides protein peptdes csv V DB search peptide spectrum matches db search psm csv V De novo only peptides de novo only peptides csv 2 Result Statistics De novo only Peptide Protein Summary Figure 1 The false discovery rate FDR curve X axis is the number of peptide spe REL Proteins fasta proteins fasta 5 0 i 45 Identified Peptide Spectrum Matches 211 Peptides pepxml peptides xml 4 096 ane De novo only peptides pepxml de novo only peptides xml 3 5 Save into C Users rahman PeaksExportsSILACproject_PEAKS_6 3 0
94. concnnoncononnononnononnoos 125 4 2 Export Label Free Quantification Results sees 125 2 2 1 Expott Result in Excel or HTML sess asia Modes bir p cbe Deed ita 125 4220 EX por SUMA PACS datos 126 S Export SPIDER Rest iii 126 O Export inG hors Result socios c a i rui tiles m tdi dies 127 15 Cotitigutatton and Preference Sai A ob aant A a 129 I PEAKS Enytsonment Preferentes soo bote e as ior taa oh ul ated ORA 129 11 General Preference S ense Robe ta bebe shai pa anb daa dU Ea buone Abo Vau Drame ais 129 KET IIS Dla yO PuONs ss riscos 130 DA MEC onnectl fls eode eee AA oscar tov cud dudas 130 LM Derby Database ios 130 I Ae Pertortnane c isse ye bitte bam odia Meri above abes leen iqu beue Mex a MS 131 1 2 Instrument Pre le FE S cias li disent PON 131 1 2 ABE WI NAS e i ua elis add AAA Sead ttt oae 132 12 2 Bruker yep oa Mba 132 1 273 Shimadzu Axima TUN eiae a a A A A a 132 L24 WALL CATAS SS ASS inet aun ete 132 1 3 5earch Enee PIetetetices ea E leaned N eN eats 132 123 1 Mascot SeA os einen OM a a A 132 1 372 NX anden OC DID S si li cios 133 1 93 23 OMS SA SNE S sd inusitato Altec beta ince dies 133 1 3 4 S 6QqU6St S CLAS a AS 133 4 Spectr tirxihinolation Preferentes sooo tue x at ct battute obe Leid tian lutea te oes 134 2 AP EARS COD CUR AIO Miva coute oue oleal prebuit decd smacks iaa 135 2L Enzyme COn EUr O ac icnexercunwee sh Sod od 135 2 2 BIN COSITA OA Mn tae oboe AN di buo onboar
95. ct General in the in the Preference dialog and click the Browse button below Project Folder to specify the default location 3l Chapter 5 Data Visualization 1 Overview After the project is created the spectral data can be visually examined For a typical LC MS MS fraction three views are provided MS this view shows the TIC total ion chromatogram plot and all the MS scans For each MS scan the corresponding MS MS scans are also displayed MS MS this view lists all the MS MS scans For each MS MS scan the corresponding MS scan is also dis played Heatmap this provides a bird s eye view of the whole LC MS dataset 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 E O lelp AEA QW PEAKS 3 16 May 11 lPeaksProjects Mew Pro J Intensity Tic 7 4 100 ali k 50 MS MS eL im LW i D 2 MS View The MS View contains TIC and all scans The total ion chromatogram TIC is displayed in the left of 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 Clicking on a specific position in TIC will display the corresponding survey scan The survey scans can also be navigated by using up and down arr
96. ction 4 Filtering De Novo Sequencing Result 2 Result exporting this is achieved by clicking the Export button at the top of the summary view The exporting function is discussed in a separate section Section 2 Export De Novo Result 3 Summary report several statistical charts assist the user to get an overall picture of the results and assess the result quality This function is the focus of this section The charts in the report are divided into three sections Notes A user can enter a special text note regarding the experiment Click the Notes button at the upper right corner of the summary view to edit the note 2 Result Statistics The figures and tables summarize the data and results 3 Other Information The search parameters and MS instrument information are given here In the rest of this section we discuss the charts in the summary report Denovo TLC 3 and ALC gt 30 y Apply Filters Export Notes 1 Notes De novo Summary It is the test project for user manual 2 Result Statistics Figure 1 The distribution of de novo ALC score a histogram of score b The plot of error vs score Y a 3501 50 4 4o 300 1 r z 150 2504 2004 ppm e 1504 number of PSMs 1001 30 35 40 45 50 55 60 65 70 75 80 85 30 35 40 45 50 55 60 65 70 75 80 85 90 De novo ALC scores De novo ALC scores Table 1 Statistics of data and r
97. d assigned ions Users can pick a peak on the panel and assign ions or tags to it in manual de novo e Ion 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 H20 and y NH3 ions Spectrum Alignment and Error Map Panel The Spectrum Alignment shows how the proposed ions as signed in manual de novo align with the spectrum By default the Spectrum Alignment displays b ion and y ion 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 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 To create a new peptide candidate for sequencing select the m z value in the Result Panel and right click to bring up a pop up menu 58 Peptide De Novo Sequencing Qs OrbiSample mzXML X PO gt OrbiSample mzxiM om New Candidate for Manual De Novo i 69534015 f Remove the selected Candidate E H 507 30252 f Config Error Tolerance in Manual De Nowo 3 B 547 59247 f Config PTM in Manual De Nove H 820 8849 Ri ie 798 0384 Rj Nee H 582 81006 E m E Esl 537 2491 RI 160 Add new sequence Can t Save H 733 2819 Rj Select
98. d en d 136 2 50 Database Conr urat oN eoun boast bean aat a lanes 137 24 Instrument Contursi ii e ald rema 137 2 5 Parameter Ontur is 139 vi Part Basic Operations Table of Contents Welcome TO P BARS NN T RTT m 4 TIME EWING UOTIS at dois Bd ridus 4 2 Major New Peatures 11 99 secco tedio dud E A M ue erin PIdURE 4 2 Gutdehmnes TorU sms this Mam al sore A SEU ers oett dias ta ud dus qd Oe ed dius 5 AO COPE ui E R A E E A 6 S OCV Ced SUP DOL eea ra a paid 6 2 Installation and RE a atu UR A a os 7 T Backase Contents as 7 Ze SEM REGUE DES a a iria airada 7 3 Installatom Om a Windows COMPUT sind alors f A IRC CIS ALON OPA PEU O stie true sseri A c cadet e sei MEA MdL MATRE 8 4 T Registration witht Internet CONMMSCION AA A 9 4 2 Registration without Internet Connetti oi sisisi can a si 12 2 5 Iec resisterine PEAKS an csi 14 44 Common Errors durne Resistravioni 22 3 oo ooi o eo DO Epi QUAS uaa a A Ea IETA SENARREN 14 SAdjusune PEAKS Memory Sage A A 15 OL Wihat S ANCKE aset Sur etu toca ia dat Pot ded 15 See IS Vue WOISIBEOUDD s oso ead A 16 INO cuu rrr 16 2 The Mati ser MENACE oo eoe E MIDI MPG UMEN TM MN M EIS 16 Ba o the Analysis Resulta ia dao A 17 A Conductine the Data Analysis os 21 Ja CES Cr 22 A LoadimeDoti toda PEAKS PLE serene uet oque A Aa 23 NOU c i p 23 2 Supported lata LOMAS ra pua a cali Dp Lad 23 35 Vendor Specilic Regulo MEMS
99. d in the PTM List To see additional built in PTMs from the Unimod library click the Show Unimod box Clicking on one of these built in PTMs will display the information listed about that PTMs in the PTM Details panel Note A built in PTM can not be deleted or edited and therefore the Delete PTM button and the PTM Details panel will be grayed out Create a new PTM Click on the New PTM button Now simply enter the information about your PTM in the PTM Details panel 136 Configuration and Preferences PTM Name this name will appear in the PTM list for future use after it is saved Monoisotopic mass the mass that the residue gains or losses as a result of the PTM Neutral loss mass the mass that the modified residue losses as a result of fragmentation in Daltons For exam ple 28 would signify a loss of 28 Daltons Residues that can be modified enter residues that can be modified anywhere residues that can only be mod ified if they are at the N or C terminus or in the middle only Chemical formula the chemical formula of the PTM This should correspond to the mass listed above e Rule you can enter a comment for your reference Click the Add Update button to save the changes The new PTM will now appear in the PTM List where it can be accessed later To delete a PTM that was created select the appropriate PTM and click the Delete PTM button Note For information on defining new
100. d 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 a low confidence less than 60 For more detailed positional confidence place the cursor over the sequence of interest and a Position Confidence Table will appear showing the confidence that each amino acid pair of amino acids are correctly identified FE DHPOTHYYAVAVVUK amp AMELLLINVK T 7 SHRPITHLHGHPE 1 0387 BIHSTVFDALHPPEDE Di TIT 0669 Mass Tags The low confidence residues can be displayed as mass tags by setting up the threshold score using the button uk in the title bar of the Peptide Candidates Frame If the score is set at 0 0 all of the amino acids in the peptide sequences will be displayed Increasing the threshold will display a mass in square brackets if the residues do not satisfy the threshold EE 71 show mass tag for confidence less than y O 10 20 30 40 50 60 70 80 90 Color code gt 90 80 90 60 80 lt 60 f 10 a 141 5 LAMVPSCGVE 374 Modifications Consider the following sequence SHM 15 99 TNLNGNPEDR The 15 99 in brackets refers to a position where a modification may have occurred If you forgot the PTMs you specified before running de novo check Table 3 in the summary view Search for a Peptide The peptide candidates can be searched by entering the value in the search tool located in
101. dify the license information accordingly on the server side A new license file is required to make the changes effective Select About PEAKS from the Help menu The About BSI PEAKS Studio dialogue box will appear About BSI PEAKS Studio PEAKS Studio 5 3 build 20110511 Copyright 2000 2010 Bioinformatics Solutions Inc Al rights reserved Bioinformatics Solutions Inc BSI acknowledges that Ronald Beavis is the author of the X Tandem program BSI is grateful to Dr Beavis for allowing us to share X Tandem with our users BSI distributes X 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 2010 11 19 2011 11 14 View end user license agreement a dm pe or Tae elle i Hed ore and intemational treaties Unauthorized reproduction Bee distribution of this program ds fe severe civil and criminal penalties and will be prosecuted to the 4 maximum extent possible under the law Click the License Wizard button to continue Then follow the instructions in Section 4 1 Registration with Internet Connection or Section 4 2 Registration without Internet Connection for re registering PEAKS 4 4 Common Errors during Registration 1 The registration key is case sensitive and it is recommended to copy ctrl C paste ctrl V the registration key whenever
102. dow 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 hand side menu to change the enzyme configuration Enzyme List lt Built In gt Semi Lys C lt Built In gt Semi Pepsin pH 1 3 dele lt Built In gt Semi Pepsin pH gt 2 lt Built In gt Semi Proteinase K lt Built In gt Semi Trypsin lt Built In gt Trypsin re l fil Enzyme Details Enzyme Name Trypsin Cleave Sites X all amino acids after RK and not before p or after and before or after and before Allaw up to one end of a peptide to disobey the deavage rule BudaiLpdate Built in enzymes All of the built in enzymes within PEAKS are listed in the Enzyme List Clicking on one of these built in enzymes will display the information about that enzyme in the Enzyme Details panel Note A built in enzyme cannot be deleted or edited and therefore the Delete Enzyme button will be disabled 135 Configuration and Preferences Create a new enzyme Click on the New Enzyme button to create a new enzyme 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 in this position while set brackets indicate any amino a
103. dow when setting the label free quantification parameters Project Hame replicateAnalysis 1 Project Location D test Peaks Peaks Projects Data Files EU rephcateAnalyss 1 fl R151 Sample 1 if Ly 070828 O1 02 070824 DMSO Oh LRAW B L R152 Sample 2 5 070828 O1 02 070827 drug Oh 1 RAW Add data files 2 1 R251 Sample 3 b 070828 O1 03 070824 DMSO Oh 2 RAW 5 070829 O1 03 070827 drug h 2 RAW Add data fies Delete Up Down AddSample SortbyRepkcate To assign the replicate number in the New Project window select the sample from the project view on the left hand side select the Replicate check box and click the drop down list below the check box to select a number Once assigned the name of the sample will be changed to indicate its replicate number and the sample number in the replicate The sample node icon colour also will be changed to display the replicates 103 PEAKS Q Label Free q A ia ao ay AS Juantiticatic Tools Label Free Por Basic Options io Mass Error Tolerance 0 2 Da Peptide Score Threshold 10logP 20 Label at MS MS level Se E eg iTRAQ f 20 sn Retention Time Range 3 0 min Protein Score Threshold 10logP 20 Label at MS level Upper Bound of Precursor Charge 3s do normalization eg SILAC E Parameter Table Label Free AndrenWatson C R EEE up down Assign
104. e It is assumed that PEAKS users are familiar and comfortable with using computers and their respective operating systems Given this it is beyond the scope of this manual to instruct the user on the use of Windows dialogue boxes menus file storage etc Please refer to the operating system s manual or one of the numerous computer help books for such information Similarly PEAKS users are expected to be familiar with mass spectrometry standard operating practices data acquisition and analysis 5 Service and Support In addition to reading this manual it is recommended that users take the time to view the accompanying training videos that explain the main features of PEAKS visually and in detail here http www bioinfor com products peaks support tutorials php Please send technical questions to lt support bioinfor com gt We strongly encourage users to provide BSI with any suggestions or comments as BSI is consistently improving and updating PEAKS to meet the future needs of the scientific community here http www bioinfor com corporate contactform php Chapter 2 Installation and Registration This section of the manual will guide users through the installation and registration of PEAKS 5 3 1 Package Contents The PEAKS 5 3 package contains e This manual PEAKS 5 3 Software Quick reference guide for PEAKS 5 3 Quick reference sheet for mass spectrometry 2 System Requirements PEAKS 5 3 runs and has been tes
105. e Project View Frame select the data file s or project containing the spectra that you wish to sequence by auto de novo Note that users can run de novo sequencing on a fraction or sample level by selecting the fraction node or sample node respectively Click the automatic de novo toolbar icon or select De novo from the Tools menu The auto de novo parameters dialogue window will appear FY De Novo lt i o Predefined parameters Instrument_default Error Tolerance Data Refinement Parention 0 1 Fragment ion 0 1 Replicate Analysis Enzyme X De Nova PEAKS Search SPIDER Search FTM Finder Tops Maximum allowed variable PTM per peptide 3 General Options Report up to 5 candidates per spectrum The meaning of each parameter is discussed in the following sections 2 1 Mass Error Tolerance Parent mass error tolerance The parent precursor ion mass errors that PEAKS will allow for during the analysis Fragment mass error tolerance The error tolerance for the peaks in the MS MS spectrum 48 Peptide De Novo Sequencing 2 2 Enzyme Specificity This informs PEAKS as to what type of enzyme was used to digest the sample Choose from a drop down list of enzymes Note There are several semi versions of common enzymes in the dropdown list For example semi trypsin has the same digestion rule as trypsin except that semi trypsin allows one termini to violate
106. e Databases 2 Databases to be Used in PEAKS inChorus Function Chapter 1 Welcome to PEAKS pr 1 Main Functions PEAKS is an integrated software platform for several common proteomics analyses using tandem mass spectrom etry data from all major mass spectrometry vendors It integrates the following functions in a user friendly graph ical user interface Peptide de novo sequencing De novo sequencing is PEAKS best known function It derives the peptide s sequence from the MS MS spec trum without using a protein database De novo sequencing is instrumental to the identification of novel or mu tated peptides that are not included in the database and to the study of the organisms without a protein database Peptide protein identification with database search PEAKS can also identify peptides and proteins by searching a sequence database with the MS MS spectra Variable PTMs post translational modifications are allowed for the search PEAKS uses a unique approach to achieve superior accuracy and sensitivity PTM analysis After the proteins are identified a second round search using the included PTM Finder tool can find more peptides with additional PTMs Homology analysis The included SPIDER tool can identify peptides with an inexact database Therefore when the target organism does not have a protein database the database of a closely homologous species can be used SPIDER can also help ident
107. e at the project tree of the PEAKS user interface On the computer s file system a project is saved as a directory that contains multiple files that contain the compressed spectral data and the analysis results It is possible to transfer the whole project directory to another user to open with PEAKS Studio or the free PEAKS Viewer To create a new project simply click the new project button m on the toolbar The following New Project dialog will appear where new samples and data files a k a fractions of samples can be added Users also get the chance to specify important properties of the data files such as the name replicate number and instrument type FY New Project Project Name New Praject 5 Project Location C Wsers zrahman PeaksProjects Data Files EY New Project 5 1i Sample 1 PEAKS supports different instrument vendors raw data formats A list of supported formats can be found in Section 2 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 you trying creating a project with your own data ensure that the vendor specific requirements discussed in Section 3 Vendor Specific Requirements are met Upon clicking the OK button in the New Project dialog PEAKS will make an effort to import the vendors raw MS data into the PEAKS project Once the data is loaded it becomes a part o
108. e in the protein e End the end position inclusive of the peptide in the protein Unique whether this peptide is unique to the current protein group Additionally the peptides from the protein and below the user specific score threshold are also displayed in the table but in grey color Although their correctness is questionable they are worth examining once an interesting protein is confidently identified by the other high confidence peptides 3 4 3 Coverage The coverage tab contains the protein sequence with the supporting peptide sequences represented by the under lining blue bars Placing the cursor over a blue bar shows some more details about the corresponding peptide including the sequence with PTM informtion score scan number and number of spectra reporting this peptide 5pl lP027698 ALBU BOVIN Serum albumin OS Bos taurus GN ALB PE 1 5v 4 1 MEWVIFISLL LLFSSAYSRG VERRDTHKSE IAHREKDLGE EHFKGLVLIA FSOYLOOCPFE a ae Na 61 121 181 3 4 4 Tool Box Two tools are provided to e search the current protein sequence by NCBI BLAST search the protein accession name by NCBI Entrez 3 5 De Novo Only View The de novo only view displays high confidence de novo sequences whose corresponding spectra only have low confidence database matches High confidence de novo sequence means the TLC and ALC score of the sequence passes the corresponding user specified score threshold Low confidence database match means the p
109. e of the peptide features over the retention time range where they were identified Heat Map 3D View XIC MS2 Annotation Extracted Ion Chromatograph LOEB Biene Baie _ _ _ __ _ _ ___________ 2 326 327 328 329 330 331 332 333 334 335 336 337 Retention Time mirs Sample 1 Sample 2 3 2 2 Heat Map Select Heat Map tab to view the 2D heat map When viewing the heat map in colour red represents high intensity and yellow represents low intensity The grayscale heat map displays high intensity in black while white represents low intensity If the peptide is identified in PEAKS DB there will be a star after the sample name 100 PEAKS Q Label Free Heat Map E Vira Lac mn Annotation Ieotnpe o Sample 1 Feature intensity 2 0253 Sample 2 Feature intensity 1 256 33 64 Iit 2 3349 O9 4 ZUE 4 3i 33 22 ae 33 09 SE A E 3252 L E r 3282 d i r 11345 11380 11355 1136 0 1134 5 1135 0 11355 1136 0 Vertical Axis retention ime Horizontal Axis m z identiled peptide Color Code 0 100 3 2 3 3D View Click on the 3D View tab to display a 3D View of the peptide features for sample 1 and sample 2 Intensity is displayed on the y axis m z on the x axis and retention time on the z axis Click and move the cursor to rotate around the image Notice that as you move one sample image the other sample moves to the same location HeatMap 3D View xic M52 An
110. e quantification results can be exported in Excel xls or HTML html format 4 2 1 Export Result in Excel or HTML To export the label free quantification results right click on a label free result node and choose Export HTML or Export Excel command from the pop up menu 125 Exporting Data Reports and Printing FA Project View nis D ftest Peaks Peaks ii The following dialog appears FY Export Quantification result to Excel File Select the Type of Results to Export Complete Protein List Peptide Detals Omitted i MCP complant output Export only Marked Pratein s and Corresponding Peptide s Select the Export Destination Excel File PEAKS provides two types of exporting functions complete protein list without peptide details or MCP com pliant output When you select MCP compliant output you can check the Export only Marked Protein s and Corresponding Peptides s if you are only interested in some proteins and previously marked them in the result table The output of Complete Protein List consists of two major sections one is the representations table which displays a representative protein for each cluster the other is the whole protein table which lists all the clustered proteins The MCP compliant output contains the two tables described above however it also provides more information than that in the whole protein table These additions include all of the sup
111. e title bar of the Summary view panel The following export dialog will appear 4 De novo peptides de novo peptides csv More De novo peptides pepxml de novo peptides xml All de novo candidates all de novo candidates csv Save into C Wsers wzhang PeaksExports Mew Project 7 DENOVO 9 The following exporting options are available e Result summary The Summary view page will be saved in summary html file in HTML format in the specified location De novo peptides The peptides identified by de novo sequencing will be saved in peptides csv file in Com ma separated Values CSV format in the specified folder De novo peptides pepXML In addition to CSV format the peptides will be saved in pepXML format Click the Export button to save the selected result components to the specified location 2 2 Export Images The annotated Spectrum Ion Match table Error Map and Spectrum Alignment can all 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 M Sequence LVDELTEFAK TLC 8 9 ALC 89 ppm 2 5 v Intensity 95 100 L v p s 1 7 E Fax riri image lon Config 100 zn 300 400 Sil 101 zx 2v ErTol 0 50a 4 intensity threshold This will display the Export Images dialog for selec
112. ecify the data refinement parameters first and click next 3 Wait for the analysis to complete A new result node will appear in the project tree Double click the node to open the result file 4 The result presentation is similar to the PEAKS DB result with additional information to show which peptide is identified by which engine s 2 Understanding PEAKS inChorus Result The inChorus result is displayed in a very similar format of the PEAKS DB result Section 3 Understanding PEAKS DB Result This section only highlights the differences Peptide Score The first noticeable difference is that the inChorus peptide score is not the 10l1gP 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 Peptide Prophet M Keller et al Anal Chem 2002 74 5383 92 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 with a weighted sum 3 The weighted sum scores of all peptides are converted to a probability by using the PeptideProphet M method again 83 PEAKS InChorus Protein Score The protein score is also a percentage score It s calculated as follows The
113. ecursor Mass Search Type 8 Monoisotopic Average Fragment Mass Error Tolerance 0 2 Da Enzyme Trypsin m New Enzyme SetPIM Switch type Switch type Maximum allowed variable PTM per peptide 35 General Options e Max Missed Cleavages 15 Filter Options Filter the spectra which satisfy the following conditions for use in the PTM search De novo ALC score greater than 50 b recommend 5056 Peptide score 10logP less than 15 recommend 15 3 Understand PTM Finder Result The results from a PTM finder search are presented in a similar format to those seen in a PEAKS DB search Please refer to Section 3 Understanding PEAKS DB Result for more information on interpreting the search results 112 Chapter 15 Homology Search with SPIDER After having obtained de novo sequences for peptides that are novel or from a species without a corresponding protein database it is possible to look for a homologous peptide in a database from a related species Han et al 2005 This process is called homology search and can help you to learn more about the proteins in your sample Homologous proteins can provide clues as to possible functions of your novel peptides SPIDER reconstruction can use both your de novo sequence tags and a homologous peptide to reconstruct the probable real sequence To search with SPIDER you must first have good quality de novo sequences either on their own or in conjunction with a P
114. ent ions The parent ion error tolerance can be specified in either Da or ppm and using monoisotopic or average mass Enzyme Choose the enzyme used to digest the proteins and the number of missed cleavages allowed in each peptide Refer to Section 2 2 Enzyme Specificity for details 66 PEAKS DB PTM Click the Set PTM button to choose a list of PTMs to be used during the search Refer to Section 2 3 Fixed and Variable PTMs for details Database Select the protein sequence database for the search You can select from the list of databases which have been configured and set the taxonomy if applicable To configure a new sequence database refer to Chapter 6 Adding a Sequence Database Or if you have only a few protein sequences you can choose to paste the protein sequences from a Windows clipboard Database Select database IHATRAAVEEGVVPGGGVALVRALOAIEGLEGDNEEONVGIALLRRAVESP LROIVANAGDE PSVVVDEVEOGSGNYGFNAATGVYGDMIEMGILDPAEVT RSALOAAASIGGLMITTEAMVAEIVEDEPAMGGMPDMGGMGGMGGMPM a Paste sequence De novo tag options PEAKS DB requires the de novo sequencing results to improve its search speed sensitivity and accuracy You can choose to perform a fresh new de novo sequencing or select from the existing de novo sequencing results 1f there are any Estimate FDR Selecting this option enables PEAKS DB to validate the search results with an enhanced tar get decoy method A few important statistical charts
115. eptide 10IgP score is below the user specified score threshold The table is identical to the peptide table in a de novo sequencing result node Refer to Section 3 2 De Novo Peptide View for how to use it 4 Filter PEAKS DB Result Through the summary view users can effectively filter the database search results to ensure the result quality by specifying score thresholds for peptides proteins and de novo sequences Note Whenever you changed a score threshold the Apply button changes color to remind you to apply the filter by clicking it 75 PEAKS DB StartPage X f PEAKS 5 19 May 11 10 10 x Peptides i0lg 15 FOR Proteins 10lgP 20 and 0 unique peptides De novo Only TLT 3 and ALC 2 50 and peptide 10lgP 8 Apply Filters Export Notes tein Summary Peptides The threshold here will affect both peptide and protein views and therefore has to be chosen with caution For peptide view only peptides with 10lgP score above the threshold will be kept in the table For protein view the number of supporting and unique peptides is based on the filtered peptide results If the FDR estimation option was turned on in the PEAKS DB search parameters the score threshold for peptides can be easily chosen by clicking the FDR button An FDR curve will popup Move the mouse cursor along the curve When the desired FDR is reached right click and select Copy score threshold FY Score select
116. ersa The intensity threshold check box provides an option to annotate the lower intensity peaks You can change the spectrum annotation preferences by clicking the Sa button to open the Spectrum Annotation Preferences window Refer to Section 1 4 Spectrum Annotation Preferences for more details on how to set spectrum annotation preferences o Ed E F F E 3 2 3 lon Table The Ion Match tab at the bottom panel of the de novo peptide view contains the Ion Table that shows the proposed ions with their corresponding masses If an ion is found in the corresponding spectrum it must first pass two criteria before being displayed in a specific color blue for N terminal ions and red for C terminal ions It must be found within the mass error tolerance chosen by the user and must have an intensity of greater than 2 of intensity of the ion with the highest intensity The ion types to display in the table are controlled by the same configuration as the Pean annotation Ce 3 2 2 Spectrum Aeon bH20 b NHS Seq I EEES 459 19 437 18 433 16 1278 69 1260 56 1261 66 24 538 23 533 90 T a RE ss ose emsr 7 220 0088 5 1019 43 1001 43 1002 40 Y 749 46 731 45 Ls 0 7 197 6 E 10 1189 53 1171 51 1172 50 V 515 35 487 34 Los nes 2 1355 66 1341 62 s 62 NV 345 24 En 7 EEE ha 1 as 0 1399 11
117. esult Table 2 Result filtration parameters of MS Scans 4233 De Novo ALC 30 of MS MS Scans 0236 De Novo TLC 33 Peptides after filter 9208 3 Other Information Table 3 Search parameters Table 4 Instrument parameters Parent Mass Error Tolerance 0 1 Da Fractions For ASMS Poster CID 57 16 trypsin RAW Fragment Mass Error Tolerance 0 8 Da Ion Source ESI nano spray Enzyme Semi Trypsin Fragmentation Mode CID CAD IRMPD y and b ions Fixed Modifications MS Scan Mode FT ICR Orbitrap Carbamidomethylation 57 02 MS MS Scan Mode Linear Ion Trap Max variable PTM per peptide 3 Histogram of Score ALC The histogram of ALC scores is a graphical representation showing a visual im pression of the distribution of ALC scores of the identified peptides The peptides are binned in 5 interval of scores Mass Error Distribution Mass errors in ppm of the identified peptides are plotted against their ALC scores The mass error is calculated as a ratio of observed mass error difference between observed mass and theoretical mass and the theoretical mass and is expressed in ppm 51 Peptide De Novo Sequencing 3 2 De Novo Peptide View The de novo peptide view displays the de novo sequencing results in more detail as shown in the next figure The table at the top displays all the de novo sequences and the bottom half of the view provides additional information about the peptide spectrum match The use of this view is
118. f that project so you can manipu late delete the original data files without affecting the analysis in PEAKS To close a project that you are working on select the Close Project command from the file menu or use the close 1 l project icon from the tool bar It is recommended to close the unused projects to preserve computer memory In the rest of this chapter we discuss the details of data loading and project creation 2 Supported Data Formats Following is the list of supported data formats in PEAKS PEAKS supports these formats at three different levels 23 Loading Data to a PEAKS Project Native Support PEAKS can read the following files directly without additional work e mzXML e mzData e mzML DTA file or a directory of DTA files e MGF e PKL PEAKS 5 x projects See Section 3 8 PEAKS 5 x Projects Library Level Support The instrument vendor s software library is required to be installed at the same computer PEAKS will call the software library to read the data directly e RAW file Thermo Fisher Scientific See Section 3 1 Thermo Data D directory Agilent instruments See Section 3 3 Agilent Data Convertor Level Support Third party convertors are required Users may need to install the required convertors and let PEAKS know their locations This only needs to be set up once PEAKS will call the convertor to convert the data to another supported format before loading The actual
119. figured here will also be used in 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 soft ware 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 The NCBInr and SwissProt databases are ideal for this purpose OMSSA At the time of writing OMSSA can not be used with databases that are not in NCBI or SwissProt format and have those results 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 in its path This creates problems when PEAKS creates temporary databases on your behalf To avoid this best practices suggest that all our databases are put in a folder 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 will have to be identical to the database configured in PEAKS in order for inChorus to parse Mascot results correc
120. fine on most of today s computers it is recommended to try to increase the value to determine the highest optimal value for the computer Keep in mind that trial and error may be needed as Java will not start if the value set is too large for the system Please do not hesitate to contact BSI to ask any questions about the memory usage 6 What s Next You are almost done Depending on the data formats and the type of analysis needed there may still be two additional configuration steps before data analysis can be conducted To read the instrument s raw data formats it might be required to install specific software libraries to support the instrument See Section 3 Vendor Specific Requirements for the requirement To conduct database search for protein identification a protein or EST sequence database must be configured See Chapter 6 Adding a Sequence Database Or 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 GUI and basic operations See Chapter 3 A 75 Minute Walk through 15 Chapter 3 A 15 Minute Walkthrough 1 Overview In this chapter we quickly walk through some of PEAKS basic functions by playing with a sample project included in the PEAKS installation Three concepts are covered 1 The main user interface 2 How to examine the analysis results 3 How to conduct data analysis By trying out the
121. gistration with Internet Connection Follow the following instructions when the computer has the Internet connection 1 When an unregistered PEAKS is run the following license wizard appears Select Request license file has Internet connection and click Next Request license file without Internet connection 5 I have already obtained the license file Description If you have purchased a license have the registration key or want to evaluate the software and this computer has Internet connections please use this option to request the license file 2 The following window will appear Input the required information To evaluate the software without a regis tration key select the 30 day evaluation option Click Next Important Input ONLY English characters as other character encoding will corrupt the license file Installation and Registration Your Name Email Address Email confirm Important You will receive your license file via email Registration Key Request a 30 day evaluation license No registration key required Institution 3 If the license file is requested successfully the following window will appear Otherwise an error message will occur Make sure that there is no typo in the registration key field in step 2 The registration key is case sensitive Registration request sent The software registration request has been sent to BSI successfully Please check your email to
122. h ww KPYM MOUSE De Novo eS 73 57 N gt TSDPTEAAAVGAVEASFE IEEE EE ELE EET T Database lt APT gt 08 peptides shown in blue PRPHSEAGTA FIOTOOLHAA 1 EKEMIRSGMNV ARLNFSHGIH GLIEGSGTAE VELEKEGATLK SLOVRERGAD FLVTEVENGG ASFIREAADY HEVEKVLGEK 61 121 PAFKVFLAQOK MMIGRCNRAG SGETARGDYP LEAVRMOOLI RECSGAIIVL TESGRSAHOV WAEDVDLREVN LAMDVYGEARG Color Code gt 75 gt 50 gt 0 181 3 4 Protein View The Protein View contains a table of identified proteins For each selected protein in the table three tabs are provided at the bottom half of the view MADTFLEHMC EYHAETIENY ITLDHAYMER SLGSREGVNL GRNIKIISKI EKPVICSTOMI AREAEAAITYH ARYRPRAPII FERERGDVVIV Peptides the peptides identified from this protein Coverage a graphical display of the protein coverage TSDPTEAAAVGAVEASFK RLDIDSAPIT REATESFASD CDEHILWLDY PCAAVDLPAV ENHEGVRRFD EIMIRREPRPT LOLFEELRRL AVIRNPOTAE LIGWREGSGE Tool Box useful tools such as a BLAST search on NCBI s website 73 ARNTGIICTI PILYRPVAVA EHICREVVEVG SERDIODLEKF EILEASDGIM EAREGSDVANA APITSDPTEA OAHLYRGIFP TNIMRVVPVE GPASRSVEML LOTRGPEIRT SRIYVDDGLI GVEODVDMVE VARGDLGIEI VLDGADCIMI AAVGAVEASF VLCEDAVLNA PEAKS DB Show top 7 proteins in each group accession contains searc O noresults gi 15599581 ref NP 253075 1 EAT om ETE 39185 PME b ME aps 20 20 61869 S80 gNlAS5983S8 rEFNP_251852 1L ars LI
123. he individual file sample click the All Samples or the All Fractions buttons and 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 selected files samples or all files samples respectively from the Selected Data list Click OK to proceed to the next step 118 Workflow Workflow Configuration n All Data a di Selected Data E it D test Peaks Peaks Projects TRAQoroject E i O test Peaks Peaks Prajyects spike 20 11 02 22 i ITRAQSample mzXML 1 152 sample 2 EY D ftest Peaks Peaks Projects SILACproject 1 AS 153 Sample 3 Sample 1 wie 154 Sample 4 dl SILACSample mzXML Dz test Peaks Peaks Projects spike 2011 02 22 E Me spikes 040BD6 2 RAW i SoikeO O40606_ 2 RANW A ly Spike 1_040806_2b RAW Yil p Ly Spikez 040806 2 RAW J R155 Sample 5 A Spike3 040806 2 RAW J R156 Sample 6 s Sokea 040806 2 RAW BAS Samaa Note All files loaded in a single workflow will be processed in exactly the same way using exactly the same parameters If you want to do some differently than others you must set up separate workflows Once the data is selected you can specify parameters for the downstream analysis steps one by one by clicking the other buttons in the workflow dialogue Please refer to the chapters on each individual function if you require more detail
124. ht click the mouse and select the Config Error Tolerance in Manua De Novo command from the popup menu to open a dialog where the error tolerance can be set PTM configuration To mention the types of post translational modifications PTM to include in manual de novo sequencing with the peptide candidate selected in the Result panel right click the mouse and select the Config PTM in Manual De Novo command from the popup menu to open the PTM Setup window To know more about the PTM configuration using the PTM Setup window refer to Section 2 3 Fixed and Variable PTMs 64 Chapter 9 Protein Database Search with PEAKS DB 1 Overview The PEAKS DB function identifies peptides proteins from a protein sequence database If your target proteins are in a known protein sequence database this is the recommended method for analyzing your MS data PEAKS DB belongs to the database search approach in MS analysis However it takes advantage of the PEAKS de novo sequencing results to achieve a higher sensitivity and accuracy than other software purely based on database search PEAKS DB includes a built in result validation to ensure the quality of the reported results Additionally PEAKS DB automatically generates a list of highly confident de novo sequences whose spectra do not match database peptides These are possible novel peptides in the sample The use of this function is outlined in the following section Details of each
125. i 1 a q el p n a Doh Ca ANR it b f Ute hi vd a i eee d p y bi Ade A A i i j 6 fl 4 0 a beep eee i 2875 Oy s i Os 5 VET at i ie TU 2445 ques NL Er aa n a 0d 1 i i 22 1 0 hi n El i a n h i i 40 a 1835 4 aM NH i i E i 14 71 1 T i i i 2 71 em 6 4 n 3 be d 900 1000 1100 To change the default mark colour click on the colour icon of the Mark Feature unMark Feature button to display the colour palette Select the preferred colour from the colour palette 36 Data Visualization 4 4 Show MS2 Hide MS2 Show MS2 displays the features associated with tandem Mass scan by marking them with the selected colour o g AS O o or pao E z tr 0 g ung a S a E o ee oa Be aS oy de S og ee he n g 8 E Epa A oo z o Sy um g RC a ba E 2 o a c e age o de frome o o oia a 975524 zt G Ba o 85 m ag m m Pre oo Bag Ds Dn oom e af eo Shoo sob o a a o 40 07 E o e 8 ow o amp o og 96 RO 774 M ue GiB e me T Me ege e cB 1I GE al Ob vA IS Bunt 8 8 o amp o 35 o o ol E o a tb Q 20 ab P ofa d to ae B aoo o d 060 ga la 2 o osp DE gee S T AUN QUSS i Pall Qqag SF Be So Sa Oy i 605 NA Pas a al qe a ore e o g e a a o 35 21 E F E PA a os o9 T o Y A 5 a gn o ho o a epa 2 T a oo o Ob ap 19 75 14 71 To change the defualt colour click on the colour icon of the Show MS2 Hide MS button to display the colo
126. ify single amino acid mutations and protein database errors Protein quantification PEAKS Q an optional node quantifies proteins using a large variety of methods ICAT 1TRAQ LFQ SILAC and user defined labels 2 Major New Features in 5 3 Throughout this manual new and existing features will be discussed in considerable detail Below is a short list of major new features implemented in PEAKS 5 3 Unprecedented accuracy and sensitivity for database search The revamped database search engine PEAKS DB results in substantially improved accuracy and sensitivity for peptide identification resulting in the identification of more peptides with a reduced false discovery rate FDR In particular the preview version of the PEAKS DB engine produced excellent results in the ABRF iPRG 2011 study for ETD data analysis Comprehensive result visualization Welcome to PEAKS Numerous improvements were made to support the visual examination of the results from many different an gles Being so confident about PEAKS s performance BSI is pleased to allow users to have a new powerful mechanism to examine their results so vigorously and yet so conveniently A major addition to most of the analysis results is a summary view that displays the result statistics Through the charts users can easily answer important questions such as whether the target decoy FDR calculation is reliable and whether the instrument is well calibrated Moreover the pe
127. in mass spectrometry labs The PEAKS automated de novo sequencing can process over 10 spectra per second on a moderate desktop PC Moreover users can use the manual de novo sequencing tool to assist the manual interpretation of an individual spectrum Most importantly the automated de novo sequencing results are used in several other analyses in PEAKS including PEAKS DB for database search and SPIDER for homology search For example in PEAKS DB the de novo only table lists the highly confident de novo sequences for the spectra that do not match any database peptides This provides a convenient way to identify novel or mutated peptides in your sample PEAKS assigns a local confidence score for each amino acid in the de novo sequence This local confidence ranges from 0 to 99 indicating how confident the algorithm is about the particular amino acid The whole peptide is evaluated by two measures the ALC Average of Local Confidence and TLC Total of Local Confidence scores Roughly speaking ALC reflects the average correct ratio for the amino acids in the sequence and TLC reflects the expected total number of correct amino acids in the sequence The use of automated de novo sequencing is outlined in the following Details of each step can be found in later sections of this chapter 1 Select a project a sample or a fraction on the project tree Click the automatic de novo toolbar icon i or select De novo from the Tools menu
128. in the summary view of the PEAKS DB result will depend on this Uncheck this only 1f you want to do your own result validation Perform on the fly preprocessing Check this box if you have not pre processed your data in the data refine ment step and uncheck it if you have already done so 3 Understanding PEAKS DB Result After PEAKS DB is done two result nodes will be generated One is a database searching result and the other is from the subroutine de novo sequencing when not using an existing de novo tag The result of PEAKS DB consists of e Summary outline of PEAKS DB outputs with statistics Also a place for result filtration Peptide list of peptide identification Protein list of protein protein groups inferred by identified peptides De novo only list of good de novo sequences without a good assignment from database search 3 1 The Peptide and Protein Scores Peptide score 10lgP The scoring schema of peptide identification involves matched peaks and their inten sities precursor mass error enzyme specificity de novo sequence and peptide size etc A statistical evaluation OIgP is given for each peptide spectrum match Here lg is the common logarithm with base 10 and P is the probability that a false identification of the current search has the same or better significance Protein score 10lgP The protein 10IgP score in PEAKS is the weighted sum of 10logP score of all sup porting peptides Afte
129. ine Note Some databases use one entry to represent multiple protein entries The FASTA headers are concatenated with a delimiter Since some of these databases use unprintable control codes as delimiters PEAKS will use the equivalent ASCII decimal code to represent them For example the NCBI NR database uses CTRL A as a delimiter so the user should input 1 as the equivalent decimal delimiter To be able to do PEAKS DB using a specific taxonomy corresponding files must be downloaded and then refer enced by PEAKS in the Taxonomy Options panel l To download the taxonid file click the Download button A window will appear confirming the FTP or website which has been identified as the location of the desired database To invoke the default FTP client software and download the file automatically click Yes Click No to copy the URL to the system clipboard If No 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 Be sure to save the file to a location that is accessible 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 button and selecting the file 2 Databases to be Used in PEAKS inChorus Function The database con
130. ion 2 000 3 000 4 000 5 000 6 000 number of peptide spectrum matches If the FDR estimation was turned off then an empirical threshold is needed Usually a score of 15 is a good choice At 101gP 15 the equivalent P value is 0 01 Note P value and FDR are two very different concepts In PEAKS DB search P value is defined as the prob ability 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 peaks bioinfor com doc peaks53 peaks db paper php Proteins Empirical thresholds for protein 10lgP score and the number of unique peptides are needed here A protein score of 20 or higher is recommended The unique peptides are the high confidence peptides that are unique to the group of proteins not found in other protein groups To achieve confident results at lease one unique peptide is needed for a protein group The thresholds here do not affect the peptide and de novo only views De novo Only The minimum TLC and ALC de novo sequencing scores and the maximum peptide 10lgP score for a peptide to possibly appear in the de novo only view De novo sequences with TLC and ALC scores above the threshold and whose corresponding specra only have database matches with 10lgP score below the threshold will be shown in the De novo Only view The thresholds here do not affect the peptide and protein views Again em
131. ion rule allows users to filter the results flexibly including by individual engines scores A venn diagram and the side by side FDR curves display each engine s contribution to the final result Heatmap view of quantification results The heatmap provides a bird s eye view of the protein quantity changes across different groups of samples The proteins are automatically clustered according to their quantity change patterns facilitating the quick identifi cation of possible interesting proteins and patterns Heatmap view for LC MS data The data heatmap provides a bird s eye view of the peaks in LC MS data including the peptide features high lighted New statistical peptide score A rigorous statistical score 10lgP is introduced to measure the significance of a peptide spectrum match in database search The score is 10 times the common logarithm of the P value 3 Guidelines for Using this Manual This manual is intended to assist in the use of PEAKS 5 3 It outlines functionalities provides instruction on how to customize PEAKS to a specific application provides a task based reference and offers troubleshooting recommendations Each main function of PEAKS is discussed in one designated chapter Welcome to PEAKS Note It is highly recommended that users begin by going through the Walkthrough provided in Chapter 3 A 15 Minute Walkthrough to quickly become familiar with the most commonly used features of PEAKS 4 Scop
132. ithout nor malization PEAKS will normalize the result when Automatically Normalize Peptide Ratios is selected and the normalization factor will be displayed You can also set the normalization factor manually by clicking the Manually Normalize Peptide Ratios and by inputting the ratios into the text field The format of ratios should be numbers separated by colons and the number of ratios should be the same as the number of samples in the quantification result 3 1 Summary View The label free quantification results are summarized in a one page summary as shown in the next figure 98 PEAKS Q Label Free 1 Heatmap View Ob a dues LE apunte Zi adi EL dur El ajdues p apdues OL adweg p epdiuec apes 1 i 3 ALBU MOUSE BTGZ MOUSE FSD5 MOUS PPAR MOUSE DOTS OUSE LC7TLZ MOUSE TCPH MOUSE IGBP1 MOUSE CRUMT MOUSE TIEG3 MOUSE SERA MOUSE STATE_MOUSE logZiratio 6 0 00 B Datuuit group 1 Cell colour represents the log ratio to the average intensity across different samples 2 Notes 3 Result Statistics Table 1 Statistics of data Table 2 Result filtration parameters Table 3 Sample Selection amp of MS Scans 24874 Protein fold change 2 Default group 1 of MS MS Scans 65632 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 sample 17 sample 18 4 Other Information Table 4 Search parameters Table 5 Instrument parameters Quant type Label free quantification
133. l the mouse wheel button ncrease the peak intensity place the mouse pointer in the spectrum scroll the mouse wheel button e See the whole spectrum double click in the spectrum or click the 1 1 button 3 The controls for the spectrum annotation Click the e button to decide the fragment ion types to be annotated in the spectrum e Click the ErroTol to set the mass error tolerance to annotate fragment ions Deselect the intensity threshold checkbox to turn on the low intensity peak annotation 4 The ion match table error plot and peptide spectrum alignment 42 PEAKS DB Clicking the header of an ion type column in the ion table will let the spectrum annotation and the error plot only display only that particular ion type The error plot shows the mass error and m z of each annotated peak A good peptide spectrum match should have these dots centered at error 0 line 3 3 3 Protein Besides the Peptide Spectrum Match the Protein tab shows the proteins that contain the selected peptide If the peptide is found in multiple proteins the top scoring protein is displayed by default The other proteins can be examined by selecting from the drop down list at the top of the Protein tab For each selected protein this view shows e the alignment between the de novo sequence and the database search sequence for the same spectrum and e the primary sequence of the protein Protein Peptide Sn ectrum Matc
134. le node or the data file that is to be exported and select the desired export format de Project View UU D test Peaks Peaks Projects peaksDBProject E L Sample 1 f DATA Data Refine ay DENC De novo LA DENG E 5 DEAK Peaks Database Search p SPIDE Spider search mM 8 SPIDE inChorus Search t Eg C users zrahmar 1 Hs D test Peaks Pe Export DTA file UY D test Peaks Pes p Sample 1 Export PKL file gt TRAQSAY Export MGF file DATA ls DENC Export MzXML file 4 PEAK Eporteaifto Excel el QUAN Export Result to Html Show Reports Delete fraction s Clicking Export DTA file will open a dialog prompting for the folder name and location to which DTA files will be exported For MGF and PKL the dialog will ask for a name and a location for the file Clicking Export MzXML File will open the Export mzXML File dialog FY Export mzXML File a m Start RT End RT Save as OD PeaksStudos 3 Data Orbange mra Brovise OK Cancel Enter the starting and ending retention times in the appropriate boxes Then click the Browse button to select a destination to save your file 2 Export De Novo Result PEAKS de novo sequencing result can be exported to other supported formats All export functions are available through Summary view panel 121 Exporting Data Reports and Printing 2 1 Export Summary and Peptides To export results press the Export button in th
135. les representative Instructions on how to use this tool follow System Requirements This extractor can be installed on the same machine as 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 2000 or Windows XP is recommended for use with this tool 25 Loading Data to a PEAKS Project 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 The socket used by the 4700 SERVER this is the port that the Oracle database listens to the default is 1521 e Username to access the Oracle database most likely we do not need to change this the default is tsquared Password to access the Oracle database mostly likely you do not need to change this either 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 to choose a spot set After selecting a spot set click OK to open it The job run infor
136. mation of a 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 3 5 Bruker Data D and LIFT directories from Bruker mass spectrometers can be imported provided that CompassXport software is installed on the same computer as PEAKS The spectral data will be contained in the yep baf or fid file If loading fid files you can select the top level folder to load them all at once make sure the merge option is set in the Bruker instrument preferences refer to Section 3 5 1 Instrument Preferences for Bruker Data CompassXport 3 0 is readily available on the Bruker Daltonics web site PEAKS currently cannot import raw data from CompassXport 3 You can either import line spectra instead refer to Section 1 2 2 Bruker yep baf fid or you can install CompassXport 1 3 This version is fully compatible with Peaks You may need to contact your Bruker representative to obtain CompassXport 1 3 3 5 1 Instrument Preferences for Bruker Data aos eS To set Bruker data related preferences in PEAKS click the Preferences toolbar icon Ve or select Preferences from the Window menu
137. ment and then Shimadzu AXIMA run in the menu on the left hand side will show the Shimadzu instrument preferences on the right hand side Click Browse to tell PEAKS the location of the Shimadzu run2xml exe file Shimadzu AXIMA run ABI wiff Shimadzu run2xml exe File Location E Bruker yep baf fid Came zu AXIMA rur Varian xm 3 7 Varian A conversion tool is embedded into Varian s data acquisition software which allows the conversion of Varian raw data into pkl files which can be immediately read by PEAKS The trans type data raw is converted in Varian programs by clicking File Save As and selecting the pkl file format or by clicking File right clicking Export and selecting pkl If you are viewing a chromatogram with 27 Loading Data to a PEAKS Project the Varian software all the spectral data in the viewed chromatogram is converted to the pkl 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 click the Pref erences toolbar icon o or select Preferences from the Window menu to open the Preferences window Clicking on Instrument and then Varian xms in the
138. menu on the left hand side will display the Varian in strument preferences on the right hand side Click Browse to tell PEAKS the location of the xmlrai exe file Varian XM5 Default xmirai exe Location ABI wiff Bruker yen baf fid E Shimadzu AXIMA nur Varian xms H Search Engine Ton Config 3 8 PEAKS 5 x Projects Projects created in any PEAKS 5 series software can be opened in PEAKS 5 3 To convert the project toa PEAKS 5 3 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 popup gus i A This project was created by an older version of PEAKS Do you want to convert it now 3 Ho thanks FI Project Convertor Converted Projects Identification workfow_versianS_3 Project Location d Peaksstuda5 3 serverDe 0 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 1s not altered 4 Creating a New Project 1 i3 To create a new project select New Project from the file menu or using the new project icon on the toolbar The New Project dialog appear 28 Loading Data to a PEAKS Project LY New Project Project Name New Project 5 Project Location C Users zrahman PeaksProjects Ei New Projec
139. n enhanced target decoy method that is more conser vative in keeping results performed together with the PEAKS DB search The Estimate FDR checkbox must be checked in the search parameters to enable this function Note The decoy hits are removed from the counting of the number of PSMs in the FDR curve Similarly unless otherwise specified all the counts in the summary view have excluded the decoy hits By default the false hits are also excluded from the peptide and protein views as well as the exported results PSM Score Distribution Figure 2 a and b help you assess the quality of the results and the effectiveness of the enhanced target decoy method decoy fusion It is strongly recommended to turn on the Estimate FDR 68 PEAKS DB checkbox in the search parameters so that both the target and decoy PSMs are shown in the same figure with different colors Figure 2 a shows the number of PSMs at each score interval If the target decoy method worked as promised then you should observe a similar number of the target blue and decoy matches brown in the low score region If the search result is of high confidence then you should observe very few decoy matches brown in the high score region The vertical dashed line indicates the user specified score threshold 1000 500 600 400 number of Pops 0 10 20 30 40 50 60 70 80 530 100 110 PEAKS peptide score 10lgP A Decoy B Target Figure 2 b plots the precur
140. n instruments Select the manufacturer of the instrument from the Manufacturer drop down list The names of the instruments will then appear in their vendor specific formats Select an instrument to view the detail instrument information in the Instrument details panel below Select General in the manufacturer list and the instruments will be listed in a general format Note The details of a built in instrument cannot be deleted or edited and therefore the Delete PTM button and the Instrument Details panel will be grayed out Create a new instrument 1 Click on the New Instrument button 2 In the Instrument Details panel provide a name for the instrument 3 Next fill in the details in the Basic Options panel In the Manufacturer drop down list select a specific vendor or General 4 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 to decide the charge of the ions 138 Configuration and Preferences 5 Use the MS Precursor Scan drop down list to select the type of MS scan that was performed This selection will tell the PEAKS Data Refine tool whether the survey scan is of sufficient resolution to determine the charge and the monoisotopic peak from the examination of the survey scan 6 Use the Fragmentation Type drop down list to select the method of fragmentation used Thi
141. n the project tree Click the PEAKS Quantification tool bar icon Q Note Refer to Section 2 Quantification Workflow for how to conduct PEAKS DB and quantification in a single workflow 2 Select the quantification protocol Label at MS level and specify the PEAKS quantification parameters in the dialog and click OK Wait for the analysis to finish A new quantification result node 9 will appear in the project tree Double click the node to open the result file The quantification result display is similar to the PEAKS DB result A ratio is added to each quantifiable peptide and protein 2 Setting Parameters The following parameter dialogue pops up when clicking the quantification tool bar icon Q Select Label at MS level eg SILAC from the left hand side 88 PEAKS Q MS Level Quantification Basic Options Mass Error Tolerance 0 1 Da T Upper Bound of Precursor Charge Label at MS MS level i ppe g Pme Retention Time Range 10 mi Peptide Score Threshold 10logP a a ne Label Options Added Mass Residues Labelling Efficiency 9 0 0R 0 0K 4 03K 6 02R B8 01K 10 01R C Label Free Add Label Delete Label The following parameters are available in the Basic Options section of the quantification window Mass Error Tolerance This parameter is used to locate the precursor ion peak group of an identified peptide in the survey scans Since in a SILAC or ICAT experiment we
142. nconcnncncnncnnoncnncnnononnonconannononos 42 Bate Dota ASS ls 43 TData Reinement C UU E 45 MES ui TREE 45 2 Data Refinement Paratelets A obice Pala qe o vac Plum o adesto aue Qu edges 45 o Peptide De NOVO SEQUE soree elec soto thites ies tau deal amie note hioc quoda a tcu Devon uet itdur 47 l OCT VIC TT u H 47 2 De Novo Sequencino Parameters dl dana 48 2 As Mass Eror Tolerance SA A AN Pau melde A Ms wad 48 2 2 Eoy meS pece I so oett a Moet Ra a 49 2 3 ICA and V able PENIS ari 49 Dia OEE Paranoicos 50 2 5 Savin the Parameters Tor Future Use ous ace ti boob aw edet antea ete 50 3 Understanding PEAKS De Novo Sequencing Result essere 50 SUMMA WCW Dr ias 51 32 TIO NOVO PODES NICW islas idas 32 3 24 Peptide Table cea ac elio ed aee Mn puce sis Denies ide aum 52 3 2 2 5 DEGLU DD ANNOLAUIOMN tii ida 53 De LOM TaBe ia 55 9o dc 1 10 10 0 e E ON S6 LS PEU AAA A A A S6 Di Os Patel SCAM EA UI II II iau rhe o bicoefut S6 4 Hiltermo De Novo Sequence Result rss a iii 56 I export e NOVO RESUS ir lt 57 6 Run Auto De Novo Sequencing on a Single Spectrum eese 3I de Manual De NOVO SCQUCNCING 2 es 57 7 1 Manual De Novo Graphical User Interface ccc cece sce eecec ences eneeneeseeeaeneeas 57 4 2 Manual De Novo Operations esscr aE E E T 59 OPS Paty DB EAS oe bo ait A A E A 65 ES Cua A 65 2 Sel TEARS DB Pal aime ters ooi nies Tuo ode mete
143. ng export dialog will appear 123 Exporting Data Reports and Printing Y Proteins html proteins him W Supporting peptides protein peptdes csv DB search peptide spectrum matches DB search psm csv De novo only peptides de novo only peptides cev More Proteins fasta proteins fasta Peptides pepxml peptides xml De novo only peptides pepxml de novo only peptides xml Save into C Users zrahman PeaksExports peaksDBProject PEAKS 3 Browse The following exporting options are available Result summary The Summary view page will be saved in summary html file in HTML format in the specified location Proteins html A list of protein identifications will be saved in proteins html file in HTML format in the specified location Supporting peptides A list of supporting peptides for each protein identification will be saved in pro tein peptides csv file in Comma Separated Values CSV format in the specified folder DB search peptide spectrum matches A list of peptide spectrum matches with scores greater than the thresh old will be saved in DB search psm csv file in CSV format in the specified location De novo only peptides A list of good de novo sequences that do not have good or no database matches will be saved in de novo only peptides csv file in CSV format in the specified location Proteins fasta A list of protein identifications will be saved in pr
144. nly I L isoform are counted as one Since the same peptides may be identified by multiple spectra due to data redundancy and different charge states this number is usually smaller than the number of Peptide Spectrum Matches Protein Groups Table 3 PEAKS DB groups the proteins identified by the same set of peptides or a subset into the same group as there is not enough information to determine which of them contribute to the identified peptides in the sample This number in the table shows the number of protein groups in the filtered result Proteins Unique Peptides Table 3 These show the number of identified proteins with the specific number of unique peptides A unique peptide is a peptide that passes the user s peptide filtration score threshold and appears in only one protein group e PTM Profile Table 4 Beside each PTM is the number of the identified peptide sequences not PSMs containing the PTM Experiment Control Figures 3 a and 3 b plots the precursor m z error of the identified PSMs These plots can help determine whether the MS instrument functioned properly Figure 3 a is the histogram of the mass errors If the instrument worked properly then the histogram should be concentrated around 0 ppm Figure 3 b plots each PSM using its m z x axis and mass error y axis For a well calibrated instrument the data points should be distributed within a narrow horizontal band centered at the 0 ppm horizontal line Table
145. node at the beginning of the row indicates that the group has multiple proteins To expand the group click the button at the left The table s columns are Accession The accession number of the protein entry in the database e 10lgP Protein confidence score e Coverage The number of amino acids spanned by the assigned peptides divided by the protein length x 100 The blue blocks indicate assigned peptides at particular positions in the protein Darker blocks indicate high confi dence passing the user s filtration score threshold peptides Peptides The number of high confidence peptides assigned to the protein Unique The number of high confidence peptides that are unique to the group of proteins not found in other protein groups Mass The calculated mass of this protein Description The part of the protein s header information as parsed from the database Note For the counting of Peptides and Unique two peptides with the same starting and ending positions in the protein are counted as one regardless of their PTM forms This seemingly counter intuitive counting rule is to follow the MCP Molecular amp Cellular Proteomics guideline 3 4 2 Peptides The Peptides tab displays the supporting peptides assigned to the protein The table is almost the same as the peptide table in the Peptide View except that three additional columns are added 74 PEAKS DB e Start the start position of the peptid
146. notation Isotope Sample 1 Feature intensity 2 0268 Sample 2 Feature intensity 1 2588 3 2 4 MS2 Annotation Select the MS2 Annotation tab to view a graphical representation of the spectrum annotation This is similar to the de novo results and PEAKS database search results spectrum annotation Please refer to Section 3 2 2 Spectrum Annotation for more details 101 PEAKS Q Label Free Heat Map 30 View xic M52 Annotation Isotope Intensity 94 LOC 1077 92 20 111 52 ba H20 v b8 NH3 ju miz 00 1000 1500 000 ER ee be ee LFOSample1 mzxML ms 2 mze1134 91 2 2 Ba al 1 1 ax zr Errol 0 5De V intensty threshold arias se 9700702102 40 3 2 5 Isotope Select Isotope tab to view the isotope distribution detected in the samples Heat Mag 32 view xic M52 Armataton Tsatope Sample 1 Feature ntensity 2 0268 Sample 2 Feature intensity 1 2558 Inkerediy 951 nitenaty 575 100 100 RT 32 65 AT 532 91 1135 41 1124 1135 1136 4 Filter LFQ Result PEAKS quantification results can be filtered to show all peptides with a certain fold change You can set the appropriate value for the filter by changing the filtration parameter value from the drop down list in the title bar of the Summary view panel Click the Apply button to refresh the results The results will be updated in all views accordingly O LABEL FREE 15 22 Mar
147. o Section 3 4 1 QSTAR or QTRAP for details on ABI wiff preferences 1 2 2 Bruker yep baf fid Clicking on Instrument and then Bruker yep baf fid in the menu on the left hand side will display the Bruker instrument preferences Note Refer to Section 3 5 Bruker Data for details on Bruker instrument preferences 1 2 3 Shimadzu Axima run Clicking on Instrument and then Shimadzu AXIMA run in the menu on the left hand side will show the Shimadzu instrument preferences Note Refer to Section 3 6 Shimadzu Data for details on Shimadzu instrument preferences 1 2 4 Varian xms Clicking on Instrument and then Varian xms in the menu on the left hand side will display the Varian instrument preferences Note Refer to Section 3 7 Varian for details on Varian instrument preferences 1 3 Search Engine Preferences This section allows users to configure preferences for the following search engines Mascot X Tandem OMSSA and Sequest 1 3 1 Mascot Settings Clicking on Search Engine and then Mascot Settings on the left hand side will display the Mascot preferences Mascot Settings Hostname or IP address Port 80 Virtual Directory Version Mascot Server 2 2 X User name Password Email Test Connection Save Password 132 Configuration and Preferences The settings parameters specify how PEAKS accesses the Mascot server if a
148. o add data to an existing project choose the project from the Project View panel and select Add Data command from the file menu or use the add data icon E from the toolbar The original project window will open Project Name O ftest Peaks Peaks Projecte peaksDEProject Project Location D test Peaks Peaks Projects Data Files Sample Details EY DO test Peaks Peaks Prajects peaksDBProject 5 A Replicate OrbiSample mzXML 1 eg DATA REFINE 1 01 Apr 11 10 16 20 dy DENOVO 6 08 Apr 11 10 12 Instruments i DENOVO 2 01 Apr 11 10 16 a PEAKS 3 01 Apr 11 10 16 JE SPIDER 7 08 Apr 11 10 12 E SPIDER 9 08 Apr 11 10 40 Add data files Sort by Replicate 3 You can add more files to an existing sample using Add data files button or create additional samples using Add Sample button 4 You will need to select the instrument vendor type For more information on adding files samples or setting up the instrument configuration refer to Section 4 Creating a New Project 6 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 1 P Click o from the toolbar The following Preference dialog pops up 30 Loading Data to a PEAKS Project WM Preferences General Default Input File Directory Search Engine Spectrum Annotation 2 Sele
149. oject To do such a comparison select those PEAKS DB nodes and right click Click on Compare Results and the comparison will be done automatically D temp LFQ_Heatmap_Blank C LABEL FREE 13 27 Apr 11 21 25 Jl Sample 1 EL PanTumorSCX 1 RAW 4 DATA REFINE 2 27 Apr 11 15 50 22 jy DENOVO 6 27 Apr 11 17 05 al PEAKS 10 27 Apr 11 19 14 gt 4l Sample 2 Compare Results E PanTumorSCX2 RAW DATA REFINE 1 27 Apr 11 Delete Result fig DENOVO 5 27 Apr 11 16 30 e PEAKS 9 27 Apr 11 19 00 6 1 Comparison Result After comparison is finished a comparison node will be added to the project as shown in the following picture d PEAKS DB AE D ftemp LFQ Heatmap Blank ff g Compare run 9 10 12 20 May 11 16 45 C LABEL FREE 13 27 Apr 11 21 25 gt A Sample 1 o ES PanTumorSCX1 RAW DATA REFINE 2 27 Apr 11 15 y DENOVO 6 27 Apr 11 17 05 8 PEAKS 10 27 Apr 11 19 14 J Sample 2 E PanTumorSCX2 RAW DATA REFINE 1 27 Apr 11 15 iiy DENOVO 5 27 Apr 11 16 30 m PEAKS 9 27 Apr 11 19 00 J Sample 3 E PanTumorSCX3 RAW DATA REFINE 4 27 Apr 11 15 The result panel will be opened automatically after completing the comparison Since the comparison run is done on the fly it won t be saved it 1s suggested to export the results before closing the result panel The details of exporting will be given in the next subsection The result consists of th
150. olecta bend 85 1V PEAKS Studio User Manual v5 3 Il PEAKS Protein Quantification id M LAM aii 86 TE KS FE MS la rabos die pi 88 1 OVERVIEW SA A ATI ADA AA AA AAA A Al 88 2 Se UNOS Orammie EP Sr sil 88 3 Understandine the Result api aco bars Py die ba eth eb E 90 Dele E tees eut 90 SA iM VIEW TER 90 OO seit VIENA ins 91 I2 PEAKS Q MS MS Level A A a it 92 ANC ti edo 92 2 SCUING Pai aie et S oos A A sta Maced wan ha ecu sen Meher 92 5 WnGerstancine the Rest 93 3 Tu SUMMA VI ss 94 2 2 S O tete e d Enc ei 94 9 9 PeDEdE VIEW eo oett ten A uu ron adesione e tnt olawentelys 94 I5 PEARS O Ladbel Pri 96 PES Ua T A 96 PAR EIS HCAS gl ROTE UE Lo E NE O e ET TO UT 96 3 Understandine LEO RESU 235 2 A AE acu elatus dede A embeds 98 SS SUMMIT AY VIS WEA A A E 98 Lo LOS VIEW TE T s 99 22 T Extracted Ton Chromalosral dd iii 100 3 2 2 IS MIP A AAA AS AA RSS 100 Ded O E 101 3 2A NES AMO LALO rai id dais 101 O OE 102 a ETE LFO RES UM A A A A AE AAA AAA AA AA 102 5 Export Quantification Result to Other Formats sess emere 102 G Replicate Ania ysis 1 EO s rss rai 103 6 1 Assign Replicate Number to a Sample ccccceccec cece ences ence eeeeeeeeeeeeeeneeees 103 6 2 RUM Replicate AM Aly S18 a2 d ette mete A A A aues ean d sects 105 6 3 Understand Replicate Analysis Results ooooooococococconoccononconcnnoncnnonnoncnnnnnnnoos 10
151. olerance Enter the range of fragment mass error in Daltons that PEAKS will allow for when determining the peptide sequences The two additional checkboxes let SPIDER match the two pairs of amino acids without any penalty as they have identical or very similar mass values PTM Clicking the Set PTM button will bring up a separate window for PTM configuration The PTM con figuration is the same as it is in de novo sequencing Section 2 3 Fixed and Variable PTMs 114 Homology Search with SPIDER Filter The filter option asks for the minimum de novo tag score ALC and maximum database search score 1OIgP for a spectrum to be used If the ALC is too small then the spectrum is unlikely to provide a significant hit If the database search score is very high then the spectrum was already assigned confidently in the PEAKS DB search step 1 2 Run SPIDER Independently The configuration panel of an independent SPIDER Search will be invoked by selecting either a de novo result or a data file and clicking the SPIDER icon on the toolbar X or choosing SPIDER Search from the Tools menu Running SPIDER independently allows you to find protein matches from a homologous database even when there were no results in a conventional database search or when a database search wasn t run The configuration panel in this case is appears as follows FY SPIDER Search SPIDER Search Predefined parameters default Save as General Options
152. olour Red blue and purple respectively 1 Set SPIDER Parameters 1 1 Run SPIDER on PEAKS DB Result The configuration panel of a restricted SPIDER Search will be invoked by selecting a PEAKS DB result and click ing the SPIDER icon on the toolbar X or choosing SPIDER Search from the Tools menu Running SPIDER on a PEAKS DB result searches only proteins that have been identified by PEAKS DB This allows you to efficiently expand coverage of your proteins and explain additional spectra that may not have matched to the database using database search The configuration panel in this case appears as follows 113 Homology Search with SPIDER FY SPIDER Search SPIDER Search Predefined parameters default Save as General Options Data Refinement Query Type Tag Match ia Homology Match Report Top Replicate Analysis Mass Error Tolerance De Novo Fragmention tolerance 0 2 Da 4 Leucine Isoleucine W Lysine Glutamine PEAKS Search PIM SPIDER Search PTM Finder Maximum allowed variable PTM per peptide 35 Filter Use the spectra which satisfy the following conditions for use in the SPIDER search De novo ALC score greater than 50 recommend 5055 Peptide score 10logP less than 15 recommend 15 Note that when a SPIDER search is launched in this matter a protein database or a de novo run does not need to be configured The SPIDER search will search based on the de novo run used for a par
153. on time between and Quality value greater than suggest 0 65 Data Preprocess peak centroiding charge deconvolution and deisotope yes no Retention Time Window It defines the maximum difference of retention time between two spectra to be merged Precursor m z Error Tolerance The tolerance of the difference in m z between two spectra to be merged Min Charge It defines the minimum charge of a precursor ion Max Charge It defines the maximum charge of a precursor ion Precursor Mass Range It defines the precursor mass region to select scans for further analysis Retention Time Range It defines the retention time region to select scans for further analysis Quality Threshold It defines the spectrum quality threshold to select scans for further analysis The recom mended value is 0 65 Data Preprocess If the data is already pre processed select no already done Otherwise select yes to pre process the data for all further analysis Or select no to do on the fly pre processing for further analysis Once all parameters are set press the OK button to initiate data refinement process 46 Chapter 8 Peptide De Novo Sequencing 1 Overview De novo sequencing is not only a preferred method for identifying peptide sequences yet to be included in databas es but also a proven method to measure alongside database findings PEAKS is the most adopted tool for de novo sequencing
154. on window should automatically appear after the CD ROM 1s inserted If it does not find the CD ROM 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 5 3 installation dialogue appears click the Next button Sm PEAKS Studio 5 3 ER Introduction Introduction Install amp nywhere will quide you through the installation af PEAKS Important Information iS License Agreement tis strongly recommended that vau quit all programs before Choose Install Folder continuing with this installation Ghoose Shortcut Folder STERNEN Click the Next button ta proceed to the next screen If you want to Fre Installation Surnrnary change something on a previous screen click the Previous button Installing Install Complete You may cancel this installation at any time by clicking the Cancel F button InstalAnywhere by Macrovision Cancel Previous 5 Follow the on screen instructions to finish the installation 6 Adjust the amount of memory utilized by PEAKS as outlined in Section 5 Adjusting PEAKS Memory Usage 4 Registration All users are required to register in order to use PEAKS A license wizard will appear to guide the registration process the first time PEAKS is run Normally the registration process is straightforward and involves
155. or a sample node Click the PEAKS inChorus button on the tool bar WN PEAKS Studio m TR File Tools Window Help i la E ld a 4 ARS O W 2 Check each engine that you would like to use at the left column of the parameter setting panel Specify the search parameters for each engine in the right side of each panel Each engine s parameter setting interface in PEAKS is kept very similar to their native interface Please refer to third party software s user manuals for how to use them For PEAKS DB refer to Section 2 Set PEAKS DB Parameters If you already have a search engine s result in a separate file or opened in current PEAKS project check the Import Result at the bottom of the engine list Important The other search engine s result should be based on the same refined data node in order to do inChorus 82 PEAKS InChorus inChorus Search Database Search Predefred parameters ranGamole SPIDER Error Tolerance Parent ian 20 0 Enzyme OMSSA Trypsin A XiTandem E Masest Manamum missed deavages per pepbde l PTM Kf Oxidation M T Phosphor lation 5TY F Carbamidomethylation Sequest Import Result Maximum alowed variable PTM per peptide 3 Database ay Select database Database Semple DA Paste sequence Taxa all species De Hove Tag Options Available de nove tage de nowo with current parameter m Note If your data is not refined by PEAKS yet you will be asked to sp
156. or project on the project tree and all the fraction s under the selected node will be refined The use of this function is outlined in the following 1 Select a project sample or a fraction node Click the Data Refinement button EN on the tool bar TECDE 2 Specify the Data Refinement parameters in the popup dialog and click OK Most of the parameters are self explanatory and the default parameters provide a good starting point for the analysis 3 Wait for the analysis is done A new Data Refinement node will appear at the project tree Later analysis on this fraction will be based on the refined data FN Project View QU C Users bshan PeaksProjects New Project 3 3 L Sample 1 data0109172008_TP10_25fm_ BSA_D1 RAW de DENOVO 2 30 Mar 11 11 14 DO bara REFINE 1 30 Mar 11 11 13 26 PEAKS 3 30 Mar 11 11 17 2 Data Refinement Parameters After selecting a data node in the project tree click the data refinement toolbar icon R The Data Refinement Parameters dialogue window will appear 45 Data Refinement FY Data Refinement ou o MS MS Data Refinement Merge Scans Y Data Refinement Retention time window for raw files only Replicate Analysis Precursor m z error tolerance De Novo Correct Precursor Charge States PEAKS Search E Min charge 1k Max charge 3 3 SPIDER Search Filter Scans PTM Finder Only keep scans satisfying Precursor mass between Retenti
157. oteins fasta file in FASTA format in the specified location Peptides pepxml A list of peptide spectrum matches will be saved in peptides xml file in pepXML format in the specified location De novo only peptides pepxml A list of good de novo sequences that do not have good or no database matches will be saved in de novo only peptides xml file in pepXML format in the specified location Click the Export button to save the selected result components to the specified location 3 2 Export Images From the Peptide view and the De novo only view the Annotated Spectrum Ion 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 2 2 Export Images for details 4 Export Quantification Results PEAKS Qdabeled and label free quantification results can be exported to other supported formats All export functions are available through Summary view panel 124 Exporting Data Reports and Printing 4 1 Export Labeled Quantification Results To export the result press the Export button in the title bar of the Summary view panel The following export dialog will appear Select control sample 51 i J Result summary summary html v Proteins protems csv J Supporting peptides protein peptdes csv
158. ow of the keyboard The tandem scans associated with the current survey scan are shown in the bottom right panel 32 Data Visualization Internity 591 10 a o Survey Scan 50 miz 200 400 600 600 LOI 1200 1400 1600 calles Poo le E AIR Spiked 040806 2Z RAW ms 1RT 0 0094 scan 1 a all isi 2x Fr ErrTol intensity threshold ne pare 593 09 1 247 07 1 430 984 0 Intensity 95 100 T 5 09 Tandem Scan 583 47 574 93 50 551 9 af p s r z y H mz 100 200 00 400 500 600 eT 0 0094 69 Seed min A IN 1 1 2 2 ErrTol intensity threshold Pe id aaa The survey scans and tandem scans provide a few convenient way to zoom and navigate in 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 the peak intensity place the mouse pointer in the spectrum scrool the mouse wheel button e See the whole spectrum double click in the 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 and its survey scans are shown on the right Zoom options are the same as described in Section 2 33 Data Visualization
159. 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 61 Peptide De Novo Sequencing D2 90807 2 Manual De Novo nteroicy C LOD 344 25 1241 55 1375 71 71 1150 64 5p 1042 6 ums inm 1090 47 226 12 246 18 iii inii 1604 81 1359 65 1480 76 bi H30 bi i J T T T T T ET r rz 100 200 300 400 500 600 700 en 200 1000 1100 1200 1300 1400 1500 1600 1700 da dh 11 2x 2 Errtol 0 502 F intensity threshold OrtiSample mzXML ms 2 mz 802 90607 7 2RT 0 0729T1C 1 79E7 1260 5649 62 7 a 0 5 1 y n s 0 5 i 0 5 500 1000 1500 mz 344 25 1241 55 d 251 IRE E1241 8 3 1344 25 Hia Intenaite 5 307 1000 1520 Note that the manual de novo candidate information is updated in the Result panel Ion Table panel and Spec trum Alignment and Error Map panel The selected ions are also annotated and colour coded in the Spectrum Annotation panel After setting two ions PEAKS will estimate the residue found between them if a residue corresponds closely to the mass difference The peptide sequence candidate name will change to show
160. pirical thresholds are needed A peptide 10lgP score of 8 is recommended Recall that roughly TLC is the estimated number of correct amino acids and ALC is the estimated percentage of correct amino acids in the de novo sequence Check Section 1 Overview for more explanation about TLC and ALC 76 PEAKS DB 5 Export PEAKS DB Results for Publication The Export button at the top of the summary view allows exporting of the filtered results into a list of html text file readable in a web browser and csv text file readable in Excel files This provides the opportunity to supplement the results in a publication or put up the results on your website Note A better way to share results is to share the whole PEAKS project directory It can be opened in our free PEAKS Viewer http www bioinfor com peaks viewer index php that has the same GUI as PEAKS Studio Note Labs with in house software can easily make use of the csv files in their own analysis workflow To export the filtered results 1 Click the Export button at the top of the summary view A file chooser appears 2 Choose the location and directory name where you want to put the exported files Click OK This will create a collection of files in the target directory Refer to Section 3 Export PEAKS DB Result for details 6 Comparison of PEAKS DB Results In PEAKS 5 3 we support comparisons on at most three PEAKS DB results including filtered results in one pr
161. ple mzXML 0 047 2 0 79 CID CAD 787 800 4 59 a I Spectrum 5 D 144 10 WENPGVTQUNR 100 0 104 26 0 0 786 8787 2 1571 7814 0 0386 24 5423 TRAQSemple mzXML 0 42 8 0 79 CID CAD 15 26 3 94 L Spectrum 7M 4144 10 SGIFR 99 7 30 55 0 0 427 7355 2 853 4602 0 0037 4 2909 TRAQSample mzXML 0 59 11 0 777 CID CAD 205 210 LI Spectrum 8 V 144 10 DEDOPFPAVPK 144 10 100 0 60 06 0 0 15 4257 2 1628 8654 0 0284 17 4618 TRAQSample mzXM 0 763 13 0 791 CID CAD 506 517 3 3 Peptide View The peptide view displays all the identifiable peptides and their quantification ratios The interface is similar to the peptide table in a PEAKS DB result The quantification ratios of those quantifiable peptides are displayed in 94 PEAKS Q MS MS Level the ratio columns with reporter ion mass as the header eg Ratio 117 114 Right click on a peptide and use show original spectrum popup menu to jump to the data view to check the reporter ions in the original spectrum Note 4 1 10000f 1398 scan search Q O no results Peptide 10lgP Mass ppm m z RT Scan Spec Accession 115 11 114 11 116 11 114 11 117 11 114 11 PTM i T 144 10 AGIQIVADDLTVTNPK 144 10 97 06 2043 1455 2 9 1022 58 48 812 F1 2894 70 P00924 ENO1 YEAST 2 21 0 51 1 48 H 2 D 144 10 AIPENLPPLTAD
162. porting peptides and their coverage within the protein False discovery rate FDR estimation is also displayed if PEAKS DB was run with a decoy database The results also include the Single Peptide Based Protein table which contains all the proteins with only one supporting peptide detected 4 2 2 Export Summary Page The Summary view page can be exported in HTML format To export the summary page click the Export button in the Summary view This opens a Save dialog box for specifying the filename and location of the result 5 Export SPIDER Result PEAKS SPIDER search result can be exported to other supported formats To do so select the SPIDER result node right click and select the mapu Result command from the pop up menu a PEAKS ce 11 10 16 Delete Result The following export dialog will appear with the available exporting options listed below 126 Exporting Data Reports and Printing SPIDER supporting peptides SPIDER protein peptides csv SPIDER peptide spectrum matches SPIDER psm csv More Proteins fasta proteins fasta SPIDER peptides pepxml SPIDER peptides xm Save into D test Peaks Export Files peaksDEProject_SPIDER_7 Browse e Proteins html A list of protein identifications using SPIDER search will be saved in proteins html file in HTML format in the specified location SPIDER supporting peptides A list of supporting peptides fo
163. possible 2 The user information can only contain English characters letters digits and symbols Characters from a non English encoding may cause a registration to fail 14 Installation and Registration 3 If the computer is behind a firewall or has other internet connection problems the registration may fail Please follow the on screen instructions or refer to Section 4 2 Registration without Internet Connection 5 Adjusting PEAKS Memory Usage By default PEAKS will utilize up to 1GB 1024M B memory on the computer If the system is equipped with more memory it is recommended that this limit should be adjusted as a larger memory will allow PEAKS to take more advantage of the computing power provided by the modern multi core processor After installation the PEAKS directory e g C PeaksStudio5 3 contains a file called Memory Utility exe Click to open this file it also can be accessed from the Windows Start Menu and the following window will open W Memory Utility Set memory heap size for Java VM in MB Optimally the heap size should be greater than 1GB 1024MB and higher values will yield better performance However the Java VM will not start with values that are too large for your system As an example PCs running 32 bit Windows XP should have access to between 1200 to 1500 MB of memory 0245 48 Selected file C PeaksStudio5 3 StartPeaksStudio lax While this default value 1 024 MB works
164. pplicable Enter the hostname or an IP address port virtual directory Mascot server version as well as your username password and email address To make sure that everything is entered correctly and that the server is working click the Test Connection button To save the password and avoid entering it every time check the Save Password box 1 3 2 X Tandem Settings Clicking on Search Engine and then XTandem Settings on the left hand will display the X Tandem prefer ences X Tandem Settings Launch Server o Local Search X Tandem Server Settings Hostname for IP address Port Test Connection x Tandem Local Settings d PeaksS tudo5 31 tandem Browse Start by selecting whether PEAKS should access a server or local version of X Tandem For the server version enter the hostname or IP address as well as the port To make sure that everything is entered correctly and that the server is working click the Test Connection button 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 1 3 3 OMSSA Settings Clicking on Search Engine and then Omssa Settings on the left hand will display the OMSSA preferences Omssa Settings Default Omssa Path Browse D PeaksS tudio5 3 omssa
165. ptide and protein tables are rewritten to provide convenient sorting and searching functions allowing easy location and examination of certain par ticularly interesting peptides or proteins e Built in result validation PEAKS DB provides a seamlessly built in result validation with an enhanced target decoy method The score threshold can be conveniently selected from the FDR curve avoiding the guess work familiar in competitive products Enhanced result reporting The new summary view provides a central place for specifying the score thresholds to filter the results Results can be filtered quickly and efficiently with the summary view showing the changes at a glance The filtered result can be easily exported to several csv and html files for publication or result sharing with non PEAKS users Alternatively the results can be saved as a PEAKS project that a collaborator can view with a free PEAKS Viewer De novo sequence tag generation By specifying a confidence threshold with a user friendly sliding bar users can promptly convert the acclaimed PEAKS de novo sequencing results to high confidence de novo tags Users can even export these tags for the integration into their own in house analysis workflow mproved inChorus search The inchorus function combines the search results of several search engines It now supports Mascot 2 3 An intuitive engine icon displays the different engines that identified the same peptide The new filterat
166. quantification result node will appear at the project tree 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 except that a ratio is added to each quantifiable peptide and protein 2 Setting Parameters This is for quantification based on the relative intensities of fragment peaks at fixed m z values within an MS MS spectrum Select Label at MS MS level eg 1TRAQ from the left hand side under Tools heading in the quantification window theEnter the relevant MS MS level labeling quantification parameters on the right hand side of the window 02 PEAKS Q MS MS Level Quantification ITRAQ Basic Options Mass Error Tolerance Peptide Score Threshold 10lpgP a Label at MS MS level CUL age eg ITRAQ TMT Label Options Label at M5 level eg SILAC Sample Name Reporter Ion Da Labelling Efficiency 92 114 11 2 115 11 Label Free 53 116 11 117 11 AddLabel Delete Label The following parameters are available in the Basic Options section of the quantification window Mass Error Tolerance This parameter is used to locate the reporter ion peaks in the MS MS spectrum If the MS MS spectrum is centroided use the fragment ion tolerance set in the PEAKS DB database search Otherwise the mass error tolerance should be set a little wider than the fragment ion error tolerance in the PEAKS D
167. r WinRar The desired result is a FASTA format text file a fas or a fasta file Move the database file into a directory that PEAKS can access Click Browse to inform PEAKS about 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 blank Based on the selected format from the FASTA Format Database list in Step 2 the accession number infor mation and parsing rules for the database headers are automatically entered in the textboxes in the Advanced Options Fasta Title Format panel below If Other was selected in Step 2 enter the parsing parameters into the corresponding textboxes Alternatively if the database format is similar to one of the public databases such as NCBI nr the parsing rules can be filled up by selecting the similar database from the drop down list and edited to set the desired parsing rules If the configuration dialog was invoked from the toolbar click the Add Update button and then OK If the configuration was invoked when specifying DB search parameters simply click OK 41 Adding a Sequence Database Note Apart from starting with a greater than 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 l
168. r each protein identification will be saved in SPIDER protein peptides csv file in Comma Separated Values CSV format in the specified folder SPIDER peptide spectrum matches A list of peptide spectrum matches will be saved in SPIDER psm csv file in CSV format in the specified location e Proteins fasta A list of protein identifications will be saved in proteins fasta file in FASTA format in the specified location e SPIDER Peptides pepxml A list of peptide spectrum matches will be saved in peptides xml file in pepXML format in the specified location Click the Export button to save the selected result components to the specified location From the Peptide view the Annotated Spectrum Ion 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 2 2 Export Images for details 6 Export inChorus Result The inChorus result can be exported to other supported formats To do so click the Export button in the title bar of the Summary view panel The following export dialog will appear J Proteins html proteins htr 4 Supporting peptides protein peptides csw Inchorus peptide spectrum matches inchorus psm csv More Proteins fasta proteins fasta Peptides pepxml peptides xml
169. r removing redundancies PEAKS DB sort those peptides from the same protein according to their IOIgP scores In the weighted sum the k th ranked peptide gets a weight 1 k 3 2 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 a separate section Section 4 Filter PEAKS DB Result 2 Result exporting this is achieved by clicking the Export button at the top of the summary view The exporting function is discussed in a separate section Section 5 Export De Novo Results 67 PEAKS DB 3 Summary report several statistical charts assist the user to get an overall picture of the results assess the result quality and examine the reliability of the mass spectrometer This function is the focus of this section The charts in the report are divided into four sections 1 Notes A user can enter a special text note regarding the experiment Click the Notes button at the upper right corner of the summary view to edit the note 2 Result Statistics The first three figures provide important information for validating the database search result Given the large volume of MS data we cannot over emphasize the importance of statistical result validation Without it the analysis result is simply not trustworthy Then four tables summarize the data and results such
170. rameter Configuration From the Configuration window select Parameters from the left hand side menu to change the parameter configurations Please note that parameters can only be viewed and deleted from within this parameter window From the Parameter Type drop down list at the top of the window select de novo PEAKS parameters SPIDER parameters or other parameter categories The parameters that have been saved within the selected category will be displayed in the list below Select the desired parameter set from the list to view the parameter details 139 Configuration and Preferences Parameter List Parameter Type De nove Instrument default Parameter Details De Hovo Predefined parameters Instrument default Save as Error Tolerance Parent ion 0 1 Danir Fragmention 0 2 Trypan E Maximum allowed variable PTH per peptide 3 General Options Report up to Hal candidates per spectrum Creating a new parameter set Create and save new parameters during or before setting up auto de novo sequencing see Section 2 5 Saving the Parameters for Future Use PEAKS DB see Section 2 Set PEAKS DB Parameters or SPIDER see Section 1 2 Run SPIDER Independently These references will provide explanations of all of the parameters Deleting a previously saved parameter set To delete a parameter set select the parameter set and click
171. ree parts peptide comparison protein comparison and statistical charts Below is an outline of each 6 2 Peptide Comparison All the peptides identified in up to three PEAKS DB searches are displayed in the table We show m z retention time peptide score charge and whether there are multiple hits for each peptide The cover map is a quick graph ical illustration of the presence of the given peptide in one or both PEAKS DB results A solid icon indicates a successful detection of the peptide You can also select to show only the common peptides of those PEAKS DB results or the unique peptides of each PEAKS DB result by changing the display settings at the bottom of the panel We also provide filters on the peptide comparison results After inputting the PEAKS score threshold on each PEAKS DB result and clicking the Apply Threshold button those peptides below the threshold will be filtered out The following screenshot is a typical peptide comparison result 78 PEAKS DB NI PEAS 9 27 Aor 11 19 00 i m PEAKS 12 27 Apr 11 19 54 us wz m mw z Mam itlog tee O wr wr o mee e aer USE x mimi 5 291 Ag 26504 Es 37 EY amp 11 82666 35 0541 fase 34 a a ee ae Ra l m mi p FAS as 3 1353 x 2 bu LII TER TC o asa rae a NN am 3s le i e 3138 27723 60 77 i fic tr B 152 39 7 437 56839 148758 AR
172. rtainty or mutation signs represent more likely mutations Square brackets indicate an equal mass substitution common non critical de novo errors While Angle brackets indicate an equal mass substitution and a mutation When simply identifying exact peptides from the database using PEAKS DB SPIDER tag match or inChorus there is no need to reconstruct the real sequence 2 2 SPIDER Protein View Clicking on Protein View will yield a similar display as was seen for PEAKS DB When running SPIDER on its own all peptides in the Coverage column will appear red However when SPIDER is searched based on a PEAKS DB result the column will hold additional information A red region indicates areas of homology and potential mutation identified by SPIDER Blue regions indicate areas of homology from the database search and purple indicates that the spectrum was identified by both SPIDER and database search 116 Homology Search with SPIDER Accession 10lgP Coverage FSPIDER Fe FSPIDE DBP FDBU Mass Description B OOWUBSIP 63 95 29m 30 2 2 97285 een pertot Lex Eo o 3 a 3 IT cai famiy v s Rs S E E Click on the Coverage tab The highlighted colors are the same as those shown in the Display column above Coverage Tool Box 5plO8BPX98 515A3 MOUSE Solute carrier family 15 member 3 05 gt Mus musculus GN Slcl5a3 PE 2 SV 1 1 MSAPRAEEOP SRSGEROPLV ARGPRGPREW RRTAAAAVLL
173. s and any protein identification using that database will fail If an update is 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 ight gray A light grey color could also mean that the database does not have header information Configure databases for use with other search engines in PEAKS inChorus function The database config ured here will also be used in PEAKS inChorus function with other search engines Refer to Section 2 Databases to be Used in PEAKS inChorus Function for details on configuring databases for use with other search engines 2 4 Instrument Configuration From the Configuration window select Instrument from the left hand side menu to change the instrument configuration 137 Configuration and Preferences Instrument List Manufacturer General New Instrument Heete Instrument FT trap ecd cid FT trap etd FT trap pad FTMS FTMS ecd FTMS ecd cid Instrument Details Instrument Name FI trap Basic Options Manufacturer General Ion Source ESI nano spray MS Precursor Scan FT ICR Orbitrap I Fragmentation Type CID CAD IRMPD y and b ions MSn Product Scan Linear Ion Trap Advanced Options Precursor mass search type a Monoiostopic Average Parent mass error tolerance 0 01 Da Fragment mass error tolerance p 5 B J Add update Built i
174. s on setting up the parameters Note PEAKS DB search PTM Finder and SPIDER are the optional functions in Identification Workflow 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 except after an optional PEAKS DB you can define quantification parameters to perform labeled or label free quantification 119 Workflow e PEAKS Search 3 inChorus Workflow The inChorus workflow is similar to the identification workflow except that after Select Data and Refine Data steps you can specify inChorus parameters to start an inChorus search 120 Chapter 17 Exporting Data Reports and Printing PEAKS 5 3 offers a rich collection of exporting functions to allow users to create reports to share the analysis results with collaborators colleagues and clients The supported formats include HTML Comma Separated Values CSV pepXML 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 Export Data Data can be exported in a number of file formats including mzxml mgf DTA MGF and PKL To do so right click on the samp
175. s selection will tell PEAKS the type of ion series to expect for PEAKS auto de novo sequencing and PEAKS DB search Select CID ECD if alternating fragmentation was used to allow the algorithm to determine the type of fragmentation from each scan header 7 Use the MSn Product Scan drop down list to select the type of MSn 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 8 Use the Advanced Options to specify additional parameters 9 Select Monoisotopic or Average as Precursor Mass Search Type For ion trap instruments it is usually beneficial to allow the PEAKS DB search to use an average mass 10 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 options screens when the instrument is selected 11 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 an instrument that was created select the appropriate instrument from the Instrument List and click the Delete Instrument button 2 5 Pa
176. scores of the peptides from a protein are added up by a weighted sum Then the PeptideProphet M method is applied to the weighted sum scores of all proteins to convert to a probability score Engines Icon For each peptide in the peptide view an engines icon is displayed to show the engine s that identified the peptide Each engine is represented by a letter code and the block background color StartPage X DATA REFINE 5 x 2 INCHORUS 27 27 May 11 13 35 X 1 10000f1784 v scan search Q0 noresults E 5 Peptide Scan m z RT Mass ppm FSpec Score fhigines P El se E Ma H XT o D El Pm El SHC 57 02 IAEVEN 98 DEMPADLPSLA cpm S 2 2 HLSNVSSTGSIDMVDSPQLATLADEVSASLAK F7 1 1081 8 29 3242 5 3 11 99 96 BENE 53 63 3 84 82 44 1 2E 4 2 48E 10 amp 3 FFESFGDLSTPDAVMGNPK F3 835 1030 0 23 2057 9 9 10 99 97 a CRE 61 27 102 35 1 2E 5 2 818 9 76 42 w 4 AQPVQVAEGSEPDGFWEALGGK F1 868 1136 5 23 2271 0 8 99 97 O CRE 68 33 87 31 1 9E 7 5 16E 9 63 64 a EQLQDMGLVDLFSPEK F1 976 924 9 26 1847 8 1 5 99 97 O CS 63 14 112 54 4 5E 5 1 43E 6 60 27 6 KVPQVSTPTLVEVSR F9 513 820 5 13 1638 9 10 18 99 97 a CES 54 25 70 58 6 2E 5 2 79E 5 50 35 7 SC 57 02 HLPWASGLETLDSLGGVLEASGY F9 1 1188 2 44 3560 7 28 40 9
177. sor mass error verses score for all the PSMs This figure is the most useful for the high mass resolution instruments Generally you should see that the high scoring points are centered around the mass error 0 And only below a certain score threshold the data points start to scatter to have bigger mass error The vertical dashed line indicates the user specified score threshold 0 10 20 30 40 50 60 70 80 5390 100 110 PEAKS peptide score 10lgP B Decoy Target Statistics of Data and Results Tables 1 4 shows the statistical numbers of the data and results Table 1 Statistics of data Table 2 Result filtration parameters ofMS scans 7737 Peptide 10lgP 220 9 De novo TLC 23 of MS MS scans 20319 Protein 10lgP 220 De novo ALC 250 Proteins unique peptides 0 Table 3 Statistics of filtered result Table 4 PTM Profile Peptide Spectrum Matches 7036 Q 0 98 17 03 3 Peptide Sequences 1820 Q 17 03 71 Protems Groups 632 Q 0 98 51 Protems 638 M 15 99 82 Proteins unique peptides 220 72 117 722 287 Eb N 0 98 94 FDR Peptide Spectrum Matches 1 0 Cor57 02 660 FDR Peptide Sequences 2 9 denovo only 2022 69 PEAKS DB Most entries in these tables are self explanatory A few worth mentioning are Peptide Sequences Table 3 This is the number of distinct peptides in the filtered result Peptides with the same primary sequence but dif ferent PTMs are counted separately But several peptides differentiated with o
178. sort the results by clicking on any of the titles of the columns For example to sort the peptide sequence candidates by the scan number click on the title bar of the Scan column The following list describes the contents of the columns in the Peptide Candidates Frame The first column is a unique index for the peptides in the list e Scan Scan number Peptide The amino acid sequence of the peptide as determined by de novo sequencing 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 TLC Total local confidence It is calculated by adding the local confidence for each amino acid in the peptide sequence ALC Average local confidence TLC divided by the peptide length m z The measured mass charge value in Daltons for the spectrum e z The calculated charge value for the peptide RT Retention time elution time for the spectrum as recorded in the data ppm The precursor mass error calculated as 106 x observed mass theoretical mass theoretical mass 52 Peptide De Novo Sequencing Confidence Scores Next to the proposed sequence candidates the auto de novo Total Local Confidence TLC and Average Local Confidence ALC confidence scores are shown The local confidence scores for each amino acid that is confidence that the correct residue in each position has been identified are represented by color coding Re
179. ss Tolerance 0 1Da Ton Source ESI nano spray Quantification KT Range 1 0min Fragmentation Mode CID CAD IRMPD y and b ions Upper Bound Charge 4 MS Scan Mode FT ICR Orbitrap Peptide Score Threshold 15 0 MS MS Scan Mode Linear Ion Trap No Label K 100 0 R 100 0 Light K 4 03 100 0 R 6 02 100 0 Heavy K 8 01 100 0 R 10 01 100 0 3 2 Protein View The Protein view shows a list of proteins that are identified in the database search together with their identified peptides in the window below The quantification ratios for quantifiable proteins are displayed in the ratio columns with sample names incorporated into the header e g Ratio Heavy No Label The ratio is calculated from the unique peptides of the protein Proteins with no unique peptides will not be assigned a ratio The SD is the standard deviation of the peptide ratios in the protein The peptides of the selected protein together with their ratios are displayed at the bottom half of the protein view 90 PEAKS Q MS Level top v proteins in each group accession contains search OQ 9 no results Unique Mass Description Ratio Light No Label Ratio Heavy NoLabel SD Light No Label SD Heavy No Label Mark R E E S2 mu Uma m E sm 14 m 32 m o Ovpracemacjo 92 rem yl s 4 wozo nomaron 9 9m 9m iz META CN E EA CI ETT IC RR E E
180. step can be found in later sections of this chapter 1 Select a project node or a sample node Click the PEAKS DB button on the tool bar ITUPEAKS studio NR File Tools Window Help as ig Note Refer to Chapter 4 Loading Data to a PEAKS Project for how to create a project 2 Specify the PEAKS DB 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 Note If your data is not refined yet you also need to specify the data refinement parameter first and click next Refer to Chapter 7 Data Refinement for data refinement 3 Wait for the analysis to finish A new result node will appear in the project tree Double click the node to open the result file FY Project View M PEAKS 6 15 Apr 11 16 44 X Eig C Users binma Dropbox tmp paper 0415 cid E Jl Sample 1 El 20090115 SMPA14 2 RAW 4 amp DATA REFINE 1 15 Apr 11 15 23 jy DENOVO 2 15 Apr 11 16 09 pf PEAKS 4 15 Apr 11 16 21 NT BPEAKS 6 15 Apr 11 16 44 Show top proteins in each group Summary Accession 10lgP F Coverage El Proteins gi 15599581 ol gi 15599462 MNT EET gi 15598358 gi 15599955 gi 15595984 gi 15599466 gi 15600749 gi 15596793 20 gi i5599461 4 The result contains four different views Summary Peptides Proteins and De novo only These provide dif ferent angles for examining the analysis
181. t 5 3 ERE Add data files Add Sample Sort by Replicate ox canos Use the Project Name field to name your job Click Browse to set the location to save the project in the Project Location text box Note Refer to Section 6 Changing the Default Project Location for changing the default save location for projects Use the Add data files button to browse to the location of the files you wish to load Select the files you wish to load and click Open Once the data file appears select the Instrument Vendor and Instrument Type that was used to generate the experimental data from the drop down lists Selecting the All Instruments option from the Instrument Vendor drop down list will display some general instrument types in the Instrument Type drop down list If no fragmentation mode is specified in the instrument type name in brackets the default setting is CID If you would like to apply the same instrument configuration to all of the files in the sample or to all of the samples in the project click on the whole sample button or whole project button respectively To add another sample click on the Add Sample button To add a data file to Sample 2 click on the Add data files button Select the instrument vendor and type from the drop down menus unless you had previously applied the instrument config
182. t d E Decoy B Target 1 n LI Q Did You Know awe of MS scans 2076 Peptide 101gP 215 wel calbrated A well calibrated instrument should have very narrow distributions fhe mass error axis in both figures The instrument control section of the summary view in the PEAKS D6 result 5 provides two figures that can help determine a whether the mass spectrometer is Protems unique peptides gt 0 3 Examining the Analysis Results Next let us open a sample project included in the PEAKS installation 1 Click the open button at the toolbar File Tool Window Help Hd ii 200 220 240 260 280 300 320 340 360 380 400 420 440 Figure 2 The distribution of PEAKS peptide score 10tgP a histogram of score b The plot of precursor mass error vs score O Table 1 Statistics of data Table 2 Result filtration parameters of MS MS scans 900 Protem 10igP 220 De novo ALC 250 b 15 20 25 30 35 40 45 50 55 PEAKS peptide score 1019P a Decoy 9 Target De novo TLC gt 3 2 Select PEAKS_HOME derbyServer PEAKS DB Tutorial in the file chooser Here you need to replace PEAKS HOME with the directory where you installed PEAKS If you have not changed the default directory during installation it should be the C PeaksStudio5 3 directory 3 Click OK in the file chooser The project is now opened in the project tree as shown below F
183. ted on Windows XP Vista and 7 PEAKS runs with the following hardware requirements Minimum A dual core processor 2GB RAM and 100GB free hard drive space Recommended A quad core processor 4AGB RAM 500GB free hard drive space and 64 bit OS Note When configured correctly one CPU license of PEAKS 5 3 can take the most advantage of the computing power provided by a quad core processor Refer to Section 5 Adjusting PEAKS Memory Usage for adjusting the memory usage of PEAKS 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 directory 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 3 Vendor Specific Require ments for the vendor specific requirements for raw data loading 1 Close all programs that are currently running 2 Insert the PEAKS 5 3 disc into the CD ROM drive Or double click on the downloaded PEAKS installation file and move ahead to step 4 Installation and Registration 3 The installati
184. ticular PEAKS DB run and will search against the proteins identified in that result Since SPIDER can be computationally intensive filters are provided to limit the SPIDER search to high quality results based on user specified thresholds The rest of this section will describe the different options that you have when setting up the parameters for your SPIDER search After setting up the desired parameters we can save them for future use Click the Save Param eters button and define name for these preferences for future use reference Any parameters that you save will be available in the drop down list at the top of the window To retrieve parameters select a saved parameters file and the corresponding settings will be shown Query Type There are two query types for SPIDER e Tag Match This is not a true mutation search instead it will search the database with the de novo tags peptides while taking into account possible de novo sequencing errors This search mode allows for the use of fixed modifications Homology Match This is a more rigorous and a more computationally intensive search mode taking into account all types of mutations and the positional confidence scores This search mode also creates reconstructed peptides that use information from both the de novo sequences and the database sequence in order to characterize the real sequence This search mode allows for the use of both fixed and variable modifications Mass Error T
185. tification with Labels at MS MS Level e g TRAQ and TMT 1 Overview Quantification with isotope labels at MS MS level is one of the three quantification modes that are supported by the optional PEAKS Q module of PEAKS Studio 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 different samples will have the same precursor m z and is fragmented together However in the MS MS labels from the different samples will produce different reporter 10ns which can then be used to calculate the quantification ratio between samples User defined labels are also supported in PEAKS Q as well as commercial labels such as 1TRAQ and TMT The quantification analysis is based on a PEAKS DB database search result See Chapter 9 Protein Database Search with PEAKS DB Ensure that you specified the isotope labels as PTMs when you performed the database search After database search is done follow these steps L Select a PEAKS DB result node in the project tree Click the PEAKS Quantification tool bar icon Q Note Refer to Section 2 Quantification Workflow for how to conduct PEAKS DB and quantification in a single workflow 2 Select the quantification protocol as Label at MS MS level and specify the PEAKS quantification parameters in the dialog box on the right and click OK Wait for the analysis to finish A new
186. ting the result items to export 122 Exporting Data Reports and Printing m Export Images Image Types 3 Error Map j Ion Table and Error Map ig Current 5pectrum View 5 Spectrum Alignment View Annotated Spectrum with Alignment Image Options Basic Options Advanced Options Print Smallest images suitable for viewing online Select the desired result elements from the Image Types list The Basic Options panel offers two choices for image size Web small images that are suitable for online viewing and Print oversampled images that are suitable for printing Image Options Basic Options Advanced Option Scale lon Oversample 1 X Fle Format png Width 956 H Height 268 5 Save As D grahman peaks peaks53 build dasses images 582 8 1006_ png Browse The Advanced Options panel offers choices for scaling factor file format resolution oversample factor and location to save the images 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 3 Export PEAKS DB Result PEAKS DB results and PTM Finder results can be exported to other supported formats All export functions are available through Summary view panel 3 1 Export Summary Proteins and Peptides To export the result press the Export button in the title bar of the Summary view panel The followi
187. tly 42 Part Il Basic Data Analyses Table of Contents Te Data Retenes idad did 45 BOV NISW PR omens neeeeays 45 2 Data Re tibementPartametetes eai dale Genie aua Hed TAS ina o edad cias 45 5 Pepulde De NoVOxSeEQuetic IP AE 47 ime cui rcm 47 2 De NOVO Seguen ae Para meters ad lod 48 2T MASS Enor LOAN a eo qM 48 2 2 IBZ YING Spe IC soe t ut atuetexee a 49 2 9 brxed and Variable FTM Souri aaa 49 ZOC EPA merid ede inr Ried Ee naman bone a ureter puna adams et nose ac tie Dus cel Gus E dod 50 2 3 SAVN the Parameters Tor Putre Use iia 50 3 Understanding PEAKS De Novo Sequencing Result ooocooococoocccconcnconcnncncononnononncnnnnonos 50 Selle SUMMA VIEN en Peer 51 DO NOVO EPS MI e ENTO 52 o2 oPeptide Table esee an ea E E O EE R A 52 3 252 PEC ADO IO AA od uu RU MON 53 2 25 9 LOW PAD x recat a cies Sato vetet tatk note ei setup o cetus dete d Cote sister de ose ite toss 55 o2 be BIEODONIOD veta edu A AT do eut E 56 322 3 OPECUN NITE DETICHE ci ibi dum at Piece ps 56 Die 08 Patent S Cab EA A II 56 4 Liltetiie De Novo sequencing Result stas oui ditt rore die iut Praes At iaa 56 Sd EXPO DN Es ME 57 6 Run Auto De Novo Sequencing on a Single Spectrum sess eene 27 Te Mantal De Noro Seduce T soc bes A A A 57 Ri Manual De Novo Graphical User Interface nit starters De ete ree ret etae te Fo ibas 57 72 Manual De Novo Operations 2 502 425 anno 39 O 65 ike
188. to open the Preferences window Clicking on Instrument and then Bruker yep baf fid on the menu on the left hand side will show Bruker instrument preferences on the right hand side 26 Loading Data to a PEAKS Project Bruker yep baf fid files E ABI vaff Default Compass File Location Bruker yenjbar 2 Shimadzu AXIMAT rur Varian xm Raw File Converter Options Compassxport wil export g Raw data Line spectra Bruker fid file may contain several files do you want to merge them into one data set 7 yes a no Default Compass File Location Click Browse to tell PEAKS the location of the CompassXport file con verter Raw File Converter Options CompassXport by default will export raw data If you attempt to load raw data and no spectra are displayed choose to export line spectra Bruker fid files may contain several samples By default these samples are not merged into one data set Select yes if you would like PEAKS to merge all the samples into one data set 3 6 Shimadzu Data 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 click the 2 Preferences toolbar icon o or select Preferences from the Window menu to open the Preferences win dow Clicking on Instru
189. ur palette Select the preferred colour from the colour palette 4 5 Show PID Show PID displays the peptide identifications from a PEAKS DB search Select the PEAKS DB search result from the dowp down list E Show PID of The peptide identifications are marked with the selected colour Placing the cursor on a marked peptide displays more information on the identifed peptide in a pop up window 37 Data Visualization 10 97 35 21 29 75 24 65 14 71 821 4 To change the default colour click on the colour icon of the Show PID button to display the colour palette Select the preferred colour from the color palette ee E Oe ae A Week fee Go to peptide detail panel 2465 8 4 XE a Show 3D View 3 x r i Highlight Feature ps u 12 21 poh Mark Features p Unblur Aa a aes a 001 00 600 500 To view the peptide details of a peptide place the cursor on a marked peptide right click to display a pop up menu and select the command Go to peptide detail panel This will show the peptide details in the MS MS View panel see Section 3 MS MS View 4 6 Show 3D View The selected Heat Map area will be displayed in a 3D view 38 Data Visualization Intensity Mt ulii E kp b EI LE at E 1 l re FH P 7 PUDE anc l im E mm i eD0000 15 o 0 0 1 OOO ES l 4 7 Noise Level Select the appropriate threshold
190. uration to the whole project in step 3 These separate samples can be used to get batch results for multiple files in the samples They can also be used to batch export dta mgf or pkl files containing all the data in the sample Separating into samples is also necessary for label free quantification refer to Chapter 13 Label Free Ouantification LFO To declare a sample as a replicate click on the sample node and select the replicate check box and set a replicate number using the replicate drop down menu You can set up to 3 samples to be replicates of the same experiment Setting replicates allows you to use the Replicate Analysis tools refer to Section 6 Replicate Analysis in LFOQ To delete a sample or data file select the appropriate node sample or data file and click the Delete button You can also change the order of the samples within a project or data files within a sample using the Up and Down buttons Click the OK button once all data files and samples are added to the project 29 Loading Data to a PEAKS Project 8 The project will appear in the Project View panel The outlined G symbol indicates that the file is still loading The solid e symbol indicates that the file has finished loading 5 Adding Data to an Existing Project m To open a saved project select Open Project or Open Recent Project command from the file menu or from the toolbar 2 T
191. ut directory where PEAKS HOME DIRECTORY is the location where PEAKS is installed Select the Browse button to change this location Default Configuration File Directory Configuration files for PEAKS can be found at USER HOME peaks by default These files locations cannot be changed Default Log File Location Log files for PEAKS can be found at USER HOME peaks by default These files locations cannot be changed 1 1 1 Display Options Clicking on Display Options on the menu on the left hand side will display interface preferences on the right hand side Display Options Show Decoy Hits Show Percentage Score Check Show Decoy Hits to display protein and peptide hits from decoy database in PEAKS DB results PEAKS uses 10lgP to display its results by default To view the percentage score along with 10lgP in peptide and protein view as well as the exporting files for PEAKS DB result check Show Percentage Score 1 1 2 RMI Connections Clicking on RMI Connections on the menu on the left hand side will show the RMI connections preferences on the right hand side RMI Connections Server Host localhost Server Port 33003 Chent Port 31003 Worker Port 35003 The default port numbers for the Server Client and Worker will appear The port numbers can be changed if conflicts arise Contact technical support at BSI for more information 1 1 3 Derby Database Clicking on Derby Database
192. x 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 You can also replace the file PeaksStudio5 3 wolf exe with the program compatible with MassLynx 4 0 For links to different versions of Wolf visit the link below 24 Loading Data to a PEAKS Project http www bioinfor com products peaks support watersmicromass php 3 3 Agilent Data Agilent QTOF data can be loaded provided that MassHunter software is installed on the same computer Agilent Ion Trap data can be loaded provided that CompassXport is loaded on the same computer as PEAKS The instrument produces a d folder The spectral data within this folder is in the yep file Select this file to load the raw data 3 4 Applied Biosystems Sciex Data 3 4 1 QSTAR or QTRAP WIFF data from Applied Biosystems Sciex QSTAR or QTRAP mass spectrometers can be loaded Analyst QS for QSTAR or Analyst 1 4 for QTRAP and the MSX plug in must be installed on the same computer as PEAKS The MSX tool is produced and sold by Infochromics Ltd and is available at cost from Bioinformatics Solutions Inc Please contact a BSI sales representative to obtain an evaluation or full license 3 4 2 Instrument Preferences for WIFF E i To set WIFF related preferences in PEAKS click the Preferences toolbar icon or select Preferences from the Window
193. xport Quantification Result to Other Formats iri e erp IR UII isa 102 6 Replicate Analy sis In DEC A ESA ESA CR E MEUM M MD DUE 103 6 1 Assien Replicate Number 16 4 Sample sata ads 103 0 2 RUM Repucale MASIA Sa 105 6 3 Understand Replicate Analysis Results 22522544299 ains aiii 106 60 4 Export Replicate Analysis Res cade nre ren ev v NN EUOKende Suen eda doceat ov eka ueboeds 108 87 Chapter 11 Quantification with Labels at MS Level e g SILAC and ICAT 1 Overview Quantification with isotope labels at the MS level is one of the three quantification modes that are supported by the optional PEAKS Q module of PEAKS Studio In this mode the isotope labels with different mass values are introduced to 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 error tolerance set by the user The ratio is calculated from the intensities of those peaks PEAKS Q supports user defined labels and commercial quantification labels The quantification analysis is based on a PEAKS DB database search result See Chapter 9 Protein Database Search with PEAKS DB Ensure that you specified the isotope labels as PTMs when you performed the database search After database search is done follow these steps 1 Select a PEAKS DB result node i

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