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PEAKS 6 User Manual

1. 802 90607 2 E Manual De Novo A Ease suo Pea oma 802 910 Note The pop up menu will not be accessible 1f you have highlighted any of the results in the Result Panel The figure below shows the main panels related to manual de novo sequencing The five main panels are indicated in the figure below StartPage X amp Orbisample mzxM x gt OrbiSample mzxML gt 802 90507 2 Tag P A Searched T ag Fane n Manual De Novo Result Panel gt 4M OMSSA 8 08 Mar 11 11 00 gt 695 34015 RT XJ XITANDEM 9 08 Mar 11 11 00 gt 507 30252 RT i amp gy DENOVO 16 09 Mar 11 13 52 547 59247 RT 5 bg DENOVO 7 08 Mar 11 11 00 Jy DENOVO 17 09 Mar 11 13 53 gt 820 00409 AT PEAKS 10 08 Mar 11 11 00 798 0384 RT i PEAKS 3 07 Mar 11 14 24 gt 582 81006 RT ied INCHORUS 11 08 Mar 11 11 00 gt 537 2491 RT t 2 PEAKS 5 07 Mar 11 17 00 733 2819 RT Edi DENOVO 2 07 Mar 11 14 11 Selected Tags 8 8 cl Heat Map MS MS Ms e e i ff Y A c D E YY YD YL YM YA YYA YYY E E E Intensity 95 100 692 29 1893 51 Spectrum Annotation Panel 1375 71 1260 56 1150 64 50 1049 6 Y1 1090 47 1189 53 226 12 246 18 zx ii 1021 49 1604 81 1359 66 1489 76 b1 H20 miz 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Se y 22 2X 2r Esrtol 0 5 Da
2. P PEAKS DB PEAKS PTM and SPIDER M Mascot S Sequest X X Tandem O OMSSA 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 The FDR curves for all selected search engines are placed together in a single figure in the sum mary view The black marks on the search engines FDR curves denote the thresholds of the corresponding engines The red mark denotes the inChorus FDR threshold 0 500 1000 1500 2000 2500 3000 3500 4000 number of peptide spectrum matches PEAKS Mascot OMSSA Sequest Inchorus X Engine Threshold Note Since overlap exists between different engines the FDR threshold for the inChorus result is higher than the respective FDR threshold for each individual engine Venn Diagram A Venn diagram shows the combination result for the three engines which identified most PSMs under the current FDR threshold setting 95 PEAKS InChorus PEAKS p Mascot 595 0 lt 0 1 1612 18 E 1 1 10910 target decoy FDR o 1291 0 267 0 lt 0 1 lt 0 1 gt 122 8 6 6 OMSSA 3 Filtering PEAKS inChorus Result The filtration differs from the PEAKS DB filtration mostly by the peptide filters The peptides can be filtered by the target inChorus FDR or each individual engine s score If the target inChorus FDR is used PEAKS will calculate t
3. Peptide Score Threshold 10lgP Only peptides with a score above this threshold are used to quantify the identified proteins Choose the appropriate quantification method used in experiment from the Select Method drop down list that contains predefined quantification methods The details of the selected quantification method will appear in the Quantification Method Detail panel To create a new quantification method click the New button to display the New Edit Quantification Method dialog Refer to Section 2 3 Labeled Q Method Configuration for how to create or edit a quantification method 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 z Once completed the protein quantification 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 99 PEAKS Q MS Level 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 Proteins are clustered if they exhibit a similar expression trend across samples Move the mouse to the tree to select a cluster and left click to show the variation
4. 100 200 300 400 S00 Ma 111 1 2x 2 ErrTol 0 8 Da V intensity threshold Moving the cursor 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 be found on the right hand side of the bottom bar of the spectrum annotation panel The annotation provides a few convenient ways to zoom and navigate within 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 cursor on a particular m z value right below the x axis line scroll the mouse wheel button ncrease Decrease peak intensity Place the cursor in the spectrum and scrolling the mouse wheel e See the whole spectrum Double click in the spectrum or click the 1 1 button 57 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 ulu buttons to switch the Spectrum View from profile mode to peak mode and vice versa The intensity threshold check box provides an option to annotate lower intensity peaks To change the Spectrum Annotation Preferences click the Sa button to open the Spectrum Annotation Pref erences window Refer to Section 1 4 Spectrum Annotation Preferenc
5. Measure the m z difference between two peaks Select a peak blue arrow with the Freeze Bar and move the mouse to the left or right Hold the Position Bar green triangle above another peak A pop up window displays the difference between the two peaks in the example below the difference is 109 92297 Fe 1 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 ErTol 0 5 Da 7 intensity threshold Cerpi 29 TIC a1 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 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 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 63 Peptide De Novo Sequencing intensity 55 100 1585 79 1375 71 Set y ion Set b ion Remove ion Set other io
6. 14 71 To change the default colour click on the colour icon of the Show MS2 Hide MS button to display the colour palette Select the preferred colour from the colour palette 4 5 Show PID Show PID displays the positions of peptide identifications from a PEAKS DB search Select the PEAKS DB search result from the drop down list I3 Show PID of The peptide identifications are marked with the selected colour Placing the cursor on a marked peptide displays more information on the identified peptide in a pop up window 40 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 aS Go to peptide detail panel 2465 8 4 XE a Show 3D View 3 x r i Highlight Feature ps u 12 21 A a Mark Features p Unblur Aa 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 Noise Level Select the appropriate threshold for noise filtering Once selected the Heat Map view will reflect the changes 41 Data Visualization d 1e8
7. 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 View False Discovery Rate FDR Curve Figure 1 in the Summary View 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 FDR r3 0 0 500 6000 6500 7000 7500 8000 m 0 500 1000 1500 2000 2500 3000 3500 4000 4500 45000 number af peptide spectrum matches L Normally a lt 1 FDR is recommended for score filtering If you notice a rapid growth of FDR around 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 an enhanced target decoy method that is more con servative in keeping results performed together with the PEAKS database search tools The Estimate FDR with decoy fusion checkbox must be checked in the search parameters to enable this fu
8. 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 work flow This will create a collection of files in the target directory which are also indexed by an html file Refer to Section 4 Export Database Search Result for details 6 Running PEAKS PTM and SPIDER Separately PEAKS PTM and SPIDER can also be run independently based on PEAKS DB results In both cases the search is invoked by selecting a PEAKS DB result and clicking the appropriate icon on the toolbar Note SPIDER can also be run based on PEAKS PTM result Under this case select a PEAKS PTM result node then click the SPIDER button on the toolbar 6 1 Run PEAKS PTM on PEAKS DB Result Invoke PEAKS PTM by selecting a PEAKS DB result and clicking the PEAKS PTM icon on the toolbar A or choosing PEAKS PTM from the Tools menu Running PEAKS PTM on a PEAKS DB result is functionally equivalent to running the two together in an integrated search The parameters used in a PEAKS PTM search are very similar to a PEAKS DB search The only difference is that no protein database needs to be selected and some parameters previously in the Advanced Setting need to be set 86 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PT
9. ES General General Display Options Default Input File Directory RMI Connections SS C Users peaks Browse Derby Database Raw file convertor Default Project Folder ABICwiff C Users peaks PeaksProjects Re Bruker yepjbaf fid Temporary File Directory Shimadzu AXIMA run _ temp Browse Varian xms Default Log File Location Waters raw Search Engine Mascot Settings SERVER LOG log CLIENT LOG log COMPUTENODE_LOG log XTandem Settings Omssa Settings Spectrum Annotation ok Cancel Heb 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 Default Project Folder PEAKS uses USER HOME PeaksProjects as the default folder for project files where USER HOME is the user home directory in your system Select the Browse button to change this location Please make sure this directory is readable writable by PEAKS 130 Configuration and Preferences Temporary File Directory PEAKS uses PEAKS HOME DIRECTORY temp as the default temporary file output directory where PEAKS HOME DIRECTORY is the location where PEAKS is installed Select the Browse button to change this location Please make sure this directory is readable writable by PEAKS Default Log File Location Log files for PEAKS can be found at PEAKS HOME DIRECTORY by default These files locations cannot
10. IPI 25 6 MB Downloading 32 Cancel Selected Instrument Software AB SCIEX MS Data Converter Downloads The Downloads table shows all the downloadable requests their status and available actions on them The status of an ongoing download is displayed in the Progress column To cancel an ongoing download click the Cancel button in the Action column Once completely downloaded the Install button appears in the Action column The corresponding software can be installed or the corresponding database can be configured by clicking the Install button The Next button remains disabled until all the downloads and the installations are completed An already installed item can be reinstalled by clicking the Reinstall button that appears in the Action column once the corresponding item 1s installed Selected Instrument Software The configuration wizard cannot download some vendor specific software as the corresponding vendor needs to be contacted to get that software If any of those software packages were selected in the instrument selection panel then their information appears in the Selected Instrument Software table Clicking the Display Information button shows information on how to get the software from the vendor in a popup dialog Click Back to go back to instrument selection or the database selection panel to change the selection Click Cancel to cancel the wizard anytime A
11. and Peptide views display the absolute intensity or relative intensity of the quantifiable proteins and peptides To change the reference sample select the appropriate sample eg ratio to 114 from the dropdown list beside Show in the Summary view PEAKS also supports changing the normalization factor of the protein ratio Select auto manual or no normalization factor from the dropdown list For manual normalization provide the normalization factors in the textbox to the right Show ratio to 114 Mormalization factor auto 1 0 0 9826379 C i E E a Ln E m 105 PEAKS Q MS MS Level Note Whenever you changed a filtration parameter the Apply Filters button changes color to remind you to apply the filter by clicking it 4 Export Quantification Results PEAKS Q Summary view and results can be exported to various supported formats Refer to Section 5 1 Export Labeled Quantification Results for details 106 Chapter 13 Label Free Quantification LFQ 1 Overview Label free quantification is one of the three quantification modes supported by the PEAKS Q module This quan tification type is based on the relative intensities of extracted ion chromatograms XICs for precursor 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 corr
12. eg SILAC TRAQ 4plex Name iTRAQ 4plex Method Type Reporter Ion Quantification Madification Target C Label Free New N Terminal Modification C Terminal Modification Side Chain Modification at Modification Mass 144 10207 Label Options Name Reporter Ion Mass Da 114 114 1107 115 115 1077 116 116 1111 117 117 1144 The 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 A little wider tolerance than the fragment ion error tolerance set in PEAKS DB is recommended Peptide Score Threshold 10lgP Peptides with a score above this threshold are used to quantify the identified proteins Peptide level quantification results are still shown for peptides with a score below this threshold Choose the appropriate quantification method used in the experiment from the Select Method drop down list that contains predefined quantification methods The details of the selected quantification method will appear in Quantification Method Detail panel To create a new quantification method click the New button to display the New Edit Quantification Method dialog Refer to Section 2 3 Labeled Q Method Configuration for how to create or edit a quantification method Clicking the Save As button at the top right allows the user to save parameters for ea
13. 4 Export Quantification Results PEAKS Q results can be exported to other supported formats All export functions are available through the Summary view panel To export the quantification results press the Export button in the title bar of the Summary view panel Refer to Section 5 1 Export Labeled Quantification Results for details 101 Chapter 12 Reporter lon Quantification e g iTRAQ and TMT 1 Overview Reporter ion quantification with isotope labels at MS MS level is one of the three quantification modes that are supported by the optional PEAKS Q module of PEAKS Studio This is based on the relative intensities of frag ment peaks at fixed m z values within an MS MS spectrum In this mode isotope labels with the same mass are introduced to several samples The samples are then analyzed together in an LC MS MS experiment The same peptides from different samples will have the same precursor m z and are fragmented together In the MS MS scans labels from the different samples will produce different reporter ions which can then be used to calculate the quantification ratio between samples User defined labels are supported in PEAKS Q as well as commercial labels such as 1TRAQ and TMT The quantification analysis is based on a PEAKS DB result See Chapter 9 Peptide PTM and Mutation Identifi cation PEAKS DB PEAKS PTM SPIDER Ensure that you have specified the isotope labels as PTMs in the database searc
14. 4 Other Information Table 4 Search parameters Quantification Type ITRAQ Quantification Mass Tolerance 0 2Da Quantification RT Range 0 0min Upper Bound Charge 0 Peptide Score Threshold 20 0 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 protein coverage of their identified peptides in the window below The quantification ratios of the quantifiable proteins are displayed in the ratio columns with label name as the header eg 114 A protein is considered quantifiable if it was identified by a unique peptide above the peptide score threshold set in the parameters The ratio is calculated from the unique peptides of the protein Proteins with no unique peptides are not be assigned a ratio The denominator sample can be changed from the Show drop down menu in the Summary view The normalization mode can also be selected in the Summary view see Section 3 4 Filtering Quantification Result 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 104 Table 2 Result filtration parameters Protein fold change gt 2 Table 5 Instrument parameters Fractions injl RAW inj2 RAW inj3 RAW inj4 RAW in
15. 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 ratios of the peptide of label free quantification result A and the y axis 1s the ratio of the same peptide relative intensity ratios in corresponding samples of label free quantification result B Ratio LABEL FREE 18 sample 2fsarnple 1 E cn m c led hd ditm 00 m 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 the 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 sample both in ascending order of size and scaled from O to 100 In the ideal case both replicates should result in the same protein ratios and thus the expected result is represented by the diagonal line in red 117 PEAKS Q Label Free 80 au FO 50 d 30 Protein ratio of LABEL FREE 86 sample sample
16. Clicking on Mascot Settings on the left hand side will display the Mascot preferences Mascot Settings Hostname or IP address myMascot mySite com Port 60 Virtual Directory mascot Version Mascot Server A ttt B User name myMascotAccount Password essees Email test amp bioinfor com Test Connection These parameters specify how PEAKS accesses the Mascot server if applicable 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 The port and virtual directory match the above settings for most servers 1 3 2 XITandem Settings Clicking on X Tandem Settings on the left hand will display the X Tandem preferences XTandem Settings 2 Launch Server Local Search X Tandem Server Settings Hostname or IP address Port Test Connection X Tandem Local Settings Actandem Browse 133 Configuration and Preferences 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 Brow
17. Export Summary Proteins and Peptides for details Text Formats The text format export options are the similar to those for PEAKS DB For PEAKS Q only quantifiable proteins and peptides are used in result exports Unlike PEAKS DB exports the PEAKS Q export includes a Reagent intensity option and excludes de novo only peptides options The reagent intensities will be saved to reagent intensity csv See Section 4 1 Export Summary Proteins and Peptides for details of other options Select the output location and click the Export button to save the selected result components to the specified location Export Images 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 3 2 Export Images for details 5 2 Export Label Free Quantification Results 5 2 1 Export Result in Excel or HTML To export the label free quantification results in Excel xls or HTML html format right click on a label free result node and choose Export HTML or Export Excel command from the pop up menu 127 Exporting Data Reports and Printing V Project View hp D test Peaks Peaks ii ci a i i QE AB lorsalzalipn Setting H Sample 2 2 Export Excel The following dia
18. Lis Mascot Semino S ade 133 1 572 XT Ange se WIN OS iaa E T tea EAE AN 133 1337 OMS SA SeN a SI EAA RNN aN 134 4 Spectrum Annotation PreferencES uds Ud 134 2 PEAKS CONS ur O tad caia 135 2 T EZ Me SONO Ura ON a add o clesd 135 2 2 PTM Conii SUIT ON TA A TAS au n upon tu und 136 2 99 I abeled O MEMO Conn uralom Ai 139 2 4 Database Con ura ON dns veces ionem pavet e a inca api odd ipe aiti dead tuas diio deo Ege 140 2 eInstrument COn Eura OT sd 142 vi Chapter 1 Overview Welcome to PEAKS PEAKS 6 1 How to Use This Manual This chapter of the manual provides an overview of PEAKS distinctive features and describes a typical data anal ysis Workflow in PEAKS Users are strongly recommended to read this chapter to get a big picture of what PEAKS provides and how PEAKS is used Other parts of this manual are intended for reference and do not need to be read from cover to cover Many contents of this manual can be read from the software s inline help An electronic and most up to date version of this manual can be found at http bioinfor com doc peaks6 htmlmanual index html The installation of the software is covered in a separate chapter Chapter 2 Installation and Activation 2 What Is PEAKS PEAKS is a complete software package for proteomics mass spectrometry data analysis Starting from the raw mass spectrometry data PEAKS takes care of every step of data conversion peptide and protein identification PTM and
19. The top control pane has two additional buttons Export and Notes The result can be exported by clicking the Export button The Notes button allows you to type in a text note about the project which will be displayed in the result summary report After applying filters the statistics report page at the bottom of the summary view will be updated accordingly We only explain two statistical charts here see screenshot below Figure 2 a shows the PSM score distribution If the search result and the peptide 10lgP score threshold is of high confidence then you should observe very few decoy matches brown in the high score region Additionally if the FDR estimation method decoy fusion worked properly then you should observe a similar or larger number of decoy brown matches than target blue matches in the low score region Figure 2 b plots the precursor mass error v s score for all the PSMs above the 10lgP score threshold This figure is the most useful for high 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 Figure 2 The distribution of PEAKS peptide score 10lgF a histogram of score b The plot of precursor mass error vs score a ppm number af PSMs 10 20 30 40 50 60 FO a0 10 20 30 40 50 60 FO ag PEAKS peptide score 10lgP PEAKS pep
20. 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 Jl 1 1 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 3 398 23 399 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 m lt 44 25 31M 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 To 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 panel and the Spectrum Alignment and Error Map panel One or more tags can be inserted by highlighting the desired tags clicking Select to move them into the Selected Tags list and then clicking the Apply button Press the Cancel button at any time to exit the search and discard an
21. ends of the searched peptides can violate the enzyme s cleavage rules Note None enzyme search is implemented as a enzyme that can cut at every position allows non specific cleavage at both ends and by default allows resulting peptides with lengths up to 65 amino acids 70 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER PTM Click the Set PTM button to select from a list of PTMs to be used during the search Refer to Sec tion 2 3 Fixed and Variable PTMs for details The PEAKS DB tool only supports a limited number of variable PTMs This number should not exceed 7 Database Select the protein sequence database for the search Select one from the list of databases that have been configured in PEAKS and set the taxonomy if applicable To configure a new sequence database refer to Chapter 6 Adding a Sequence Database if you have only a few protein sequences you can choose to paste the protein sequences from a Windows clipboard Database Select database gt P62258 14335E HUMAN 14 3 3 protein MDDREDLVYOAKLAEOAERYDEMVESMEKEVAGMDVELTVEE Q Paste sequence Note The pasted protein sequences should follow FASTA format Each sequence follows a description line which starts with gt PEAKS omits invalid characters white space and numbers in a sequence A maxi mum of 1000 protein sequences can be pasted De novo tag options PEAKS DB requires the de novo sequencing results to improve its
22. while manually de novo sequencing a spectrum start by selecting the peptide candidate in the Result panel Right click the mouse and select the Config PTM in Manual De Novo command from the pop up menu to open the PTM Setup window To know more about the PTM configuration using the PTM Setup window refer to Section 2 3 Fixed and Variable PTMs 67 Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM SPIDER 1 Overview The PEAKS software package provides a complete set of database search tools to do in depth protein analysis With the help of powerful tools such as PEAKS DB PEAKS PTM and SPIDER users can identify all the proteins which are present in the sample with high sensitivity while also finding all the possible PTMs and mutations located on the protein of interest With the embedded support for multiple enzyme digestion users can achieve almost full coverage for single protein study An automatic validation mechanism is also included in each PEAKS database search tool to ensure only valid results are reported PEAKS DB is a database search tool uniquely assisted by PEAKS de novo sequencing technology to achieve high sensitivity and accuracy Note For more details check paper PEAKS DB De Novo sequencing assisted database search for sensitive and accurate peptide identification Mol Cell Proteomics 2011 Dec 20 PEAKS PTM is a dedicated tool for searching unspecified PTMs and mutat
23. 1 0 20 1 1 9 T 1 T 1 0 10 20 20 40 50 60 ri BO 90 100 Protein ratio oF LABEL FREE 85 sample 4sample 1 95 6 4 Export Replicate Analysis Result The replicate analysis plots and diagrams can be exported as image files 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 3 2 Export Images for details 118 Chapter 14 Creating a High Throughput Workflow For your convenience PEAKS software provides workflows for protein identification quantification and inChorus search multi engine protein ID QU Identification Quantification InChorus Once a specific workflow 1s selected a dialog pops up to allow you to specify the analysis steps and the parameters to use in each step 1 Identification Workflow Click the workflow icon Ww on the toolbar and select Identification The identification workflow setup window will appear FY Identification Work Flow Click Select Data to open the Workflow Configuration dialogue where you can select the data you wish to perform identification analysis 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 the individual file sample click the All Samples or the Al
24. 7 E As x A F p f E a 7 ir Sm F _ E EB a 3521 2o mae za vd z Ey gt 1 EU E s a2 x F amp TN Lr MED e r Tari E E a gt a 5 xi i RS is 1 aa rz m n i i i er B a gt b k a ans HN E a T ur E E T3 i 4 gan F T Kk E A E E STA gt 29 75 a 1 i Bam A n e sri E md 3 n L TE TE uh d EIE OE ra hs E j k if SOLOS e Ba Be B PA A 4 E ae E a Ld ps 4 T RC F P i ee Wo e PLE me ee oo EA no zx at gu 1 u Ta s pL a x 2485 A TREAT VE is dier lp AE a d n Pa 543 4 42 48 V m AA ai E Lia uar eit wg sE a zr st A A Lp i nl i Lx 1 72 P H a Ww du Pie EAE C dye aia C sa x FR Ee i MOD c o ETE TEE i r 5 4 P TIE LI t m s in A m 7 Qu sn tis kh Es A E i E gt e mp rp mi E po E 4 E z z a T amp T es 7 E 211 E iF Fa d I T ES J Ea EL hi x is R E t5 5 ae de 2 8 E VE ati aL EA Pa 5 er T E no a a pa i i A wy Ta E i j t k a wu m ta a X te gt i m ZZ E A E Ak h i T zi E be m E Pa O een A eee cpu ME LEUTE JE La JE S00 500 70 300 900 1000 1100 38 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 i LI Sample Text Sample Text 4 3 Mark Feature Unmark Feature Mark Feature marks the identified features by circling around
25. AB SCIEX wiff preferences 1 2 2 Bruker yep baf fid Clicking on the Bruker yep baf fid option under the Raw file converter section in the menu on the left hand side will display the Bruker instrument raw file converter preferences Note Refer to Section 3 5 Bruker Data for details on Bruker instrument preferences 1 2 3 Shimadzu AXIMA run Clicking on the Shimadzu AXIMA run option under the Raw file converter section in the menu on the left hand side will show the Shimadzu instrument raw file converter preferences Note Refer to Section 3 6 Shimadzu Data for details on Shimadzu instrument preferences 1 2 4 Varian xms Clicking on the Varian xms option under the Raw file converter section in the menu on the left hand side will display the Varian instrument preferences 132 Configuration and Preferences Note Refer to Section 3 7 Varian for details on Varian instrument preferences 1 2 5 Waters raw Clicking on the Waters raw option under the Raw file converter section in the menu on the left hand side will display the Waters instrument preferences Note Refer to Section 3 2 Waters Micromass MassLynx Data for details on Waters instrument preferences 1 3 Search Engine Preferences This section allows users to configure preferences for the following search engines Mascot X Tandem OMSSA and Sequest 1 3 1 Mascot Settings
26. ALBU_BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 vx F outline il tl E 1 169 16334 E 173 185 19132 196 198 200 214 2 223 ae ee BERS d d 161 YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE 1 spectrum KVLT sub A SSARQ Scan F1 565 m 2 495 2886 2 2 R 212 79 10lgP 17 33 ppm 3 9 by PEAKS PTM gd 96 K v r v s s ar o 4 ho Eu A EM gt amA O M a EE EE ale n 31 4 T b2 NH3b2 b3 H20 Ya Y3 4 100 200 300 400 500 600 700 800 900 wis 2X 2Y ErrTol 0 5 Da preprocess Y low intens label 21 ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK n AAA AAA zo Pp c 4 5 Creating a PEAKS Project SI S Bud ISI 80 AAs per line Y de novo only tags sharing confident PTM min ion intens 9 coverage O V 10AA gap 6 AAs 5 296 AM 157 02 PTM Carbamidomethylation 889 462 0 98 43 01 Carbamylation 57 02 Carbamidomethylation D Deamidation NQ Dehydration 40 I co 48 00 Dethiomethyl m N 15 99 Oxidation M 162 05 Hexose T X N term 17 03 Ammonia loss N 17 03 Pyro glu from Q 42 01 Acetylation N term 21 98 Sodium adduct 31 99 Dihydroxy 37 95 Replacement
27. Browse button and selecting the file Note Please do not rename the taxonomy files otherwise PEAKS cannot recognize the files Delete a previously configured database To delete a database file select the database to be deleted from the Database List and click the Delete button at the bottom Moving Updating a database To move a database to another directory the location must be updated in PEAKS Select the database and 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 databases 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 gray color could also mean that the database does not have header information Configure databases for use with other search engines in PEAKS inChorus The databases configured here will also be used in PEAKS inChorus to call the X Tandem and OMSSA search engines However Mascot search depends on Mascot s databases only When using these third party software tools note the following with care e X Tandem At the time of writing X Tandem has difficulty in searching through large databases and may crash It is therefore suggeste
28. 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 confidence passing the user s filtration score threshold peptides If SPIDER has been run SPIDER peptides will be represented as blocks coloured in various shades of red 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 e PTM All the PTMs that occurred on the protein displayed in color coded icons Avg Mass The average mass of this protein Description The part of the protein s header information as parsed from the database Mark Allows the selection of specific proteins This allows the selection of proteins for multiple sequence alignment as well as selecting which proteins are exported from the export feature in the summary view 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 3 2 Coverage Tab The coverage tab characterizes the protein sequence at the amino acid level It has three major components Protein sequence
29. Distribution Mass errors in ppm of the identified peptides are plotted against their ALC scores The mass error 1s calculated as a ratio of observed mass error difference between observed mass and theoretical mass and the theoretical mass and is expressed in ppm 3 2 De Novo Peptide View The de novo view displays the de novo sequencing results in greater detail as shown in the next figure The table at the top section displays all the de novo sequences and the bottom section provides additional information about the peptide spectrum match 54 Peptide De Novo Sequencing B 1 90f9 gt abos Y scan search QO no results Scan ft Peptide TLC ALC miz z RT ppm o i A 9 2 4 MGNADLINWMPLK 8 4 70 695 34 2 0 320 4 8 2 3 5 ANELLLNVK 7 6 84 507 30 2 0 369 2 3 4 7 SHRMTNLNGNPEOR 8 8 63 547 59 3 1 344 3 3 5 8 HSTVFDNLNPPEDR 11 6 83 820 88 2 1 422 3 0 6 10 WAWFFQLYLAMVPSCGVCDR 13 1 65 798 04 3 1 551 3 5 z 12 LVDELTEFAK 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 4 4 Sequence DNPQTHYYAVAVVK TLC 11 4 ALC 82 ppm 1 8 v Intensity 100 V1 y12 p nfe o r a v v a v a v v k 1375 71 Y11 H20 b11 Y10 1260 56 V9 1150 64 50 1049 6 Ye b10 912 52 1189 53 y Y7 y 12 ya y9 H20 246 18 3H20 Y4 749 46 1031 49 bi Y13 D umo V6 b7 1359 66 1489 76 327 17 16 29 l b 356 36 v y3 ys 585 44
30. Images for details 5 Export Quantification Results PEAKS Q labeled and label free quantification exporting function is also similar to that of de novo or PEAKS DB All export functions are available through the Summary view panel 5 1 Export Labeled Quantification Results PEAKS Q results can be exported to other supported formats To export the quantification results press the Ex port button in the title bar of the Summary view panel The following export dialog will appear Export summary view Proteins proteins csv Export protein coverage Supporting peptides protein peptides csv 4 DB search peptide spectrum matches DB search psm csv Proteins fasta proteins fasta Ej 4 Export supporting peptides Export best unique PSM Peptides mzidentml version 1 0 0 peptides 1 0 O mzid Put all protein details in a single html Peptides mzidentml version 1 1 0 peptides 1 1 0 mzid Peptides pepxml peptides xml Reagent intensity reagent intensity csv Save into D test Peaks60 PeaksExports Sample Project Labelling Browse Save into D test Peaks60 PeaksExports sample Project Labelling Browse Export Cancel Export Cancel HTML Report The options are the same as those for PEAKS DB For PEAKS Q only quantifiable proteins are used in result exports See Section 4 1
31. Name Digest 2 E Replicate 1 silicio RAW Enzyme Oz x a Gen 5 Ab Instrument Orbitrap Orbi Orbi View i BS Tops RAW Fragmentation Copy to whole p 4 6 Conduct an Identification Analysis To conduct an identification analysis 1 select a project sample or result node from the project tree 2 Click the desired analysis tool button Here we show the PEAKS complete identification analysis workflow PY PEAKS Studio os A File Tools Window Help la lu HEA 0 SH QW sr DENOVO 3 rez 18 53 PEAKS 4 07 Jun 12 18 53 PEAKS PTM 5 07 Jun 12 18 53 A SPIDER 6 07 Jun 12 18 53 El J GluC EM F1 BSA GluC 1 mzxml DATA REFINE 1 07 Jun 12 18 53 B J Trypsin Eh F2 BSA Trypsin 1 mzxml DATA REFINE 2 07 Jun 12 18 53 Overview A search parameter pane will pop up Most search options are standard and straightforward More details are provided in the following see screenshot below l If the proteolysis enzyme was specified for each sample at the project creation step one can choose to use the enzyme specified in each sample This makes it possible to use multiple enzymes in a single project and a single search Specify the fixed PTMs and a few common variable PTMs expected in the sample Select a protein sequence database or copy and paste the protein sequences for the database search Conduct de novo sequencing using the same parameters
32. Pyro glu from Q 17 03 6 N term 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 only 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 e 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 For each type of PTM delta mass number of PSMs containing this PTM and PTM locations presented in the sample are listed Experiment Control Figures 3 a and 3 b plot the precursor m z error of the identified PSMs These plots can help determine whether the MS instrument functioned pro
33. Y 1392 66 219 V6 2238 97 691 40 5 y3 604 37 389 24 miz 500 1000 1500 2000 2500 3000 de d li 1 1 2X a ErrTol 0 02 Da V preprocess low intens label 3 Ton ath X bh b H20 b NH3 b 242 Seg y y H20 yHHS v 24 143 03 144 01 rmm ns se uae EEEE Hi ssuo 525 03 330 06 17405 D 2580 268028 288127 1348 55 25 0 02 5 61 23 600 22 601 20 309 62 R 2468 25 2450 23 2451 22 1234 62 21 6 689 27 671 26 672 24 345 13 A 2312 14 2294 13 2295 12 1156 57 20 n 7 804 29 786 29 787 27 40265 D 2241 11 2223 10 2224 08 1121 05 19 gi oar TEE Sh dE hus iu o fungi o rg a a ee o 1 0 I aL id d Eod d pa e E Error da e 1000 E The default display is divided into four areas 1 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 Clicking the Protein button shows a drop down list of all the proteins which contain this peptide Left click one protein it jumps to the protein in the protein table 2 Thespectrum annotation The annotation provides a few convenient ways to zoom and navigate in the spectrum Zoom to
34. 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 cursor 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 Cursor over an amino acid to see the fragment ion peaks for this amino acid 3 The controls for the spectrum annotation 83 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER Click the Se 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 Check the preprocess checkbox to switch between pre processed spectrum and original raw spectrum e Deselect the low intens checkbox to turn on the low intensity peak annotation 4 The ion match table error plot and peptide spectrum alignment 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 5 De Novo Only View The de novo only view displays high confidence de novo sequences
35. algorithm used by PEAKS has been demonstrated by publications and or third party evaluations By combining four complementary algorithms together the sensitivity is further improved Multiple enzyme Project To maximize a protein s sequence coverage it is advantageous to use different proteolysis enzymes to digest the protein sample PEAKS supports the use of different enzymes in different samples of a single project The software will automatically use the enzyme specified in each sample and combine all samples results together Multiple Instrument Vendor Support PEAKS supports most major instruments raw file formats which saves the trouble for file format conversion by the users More importantly the algorithms parameters are optimized for each different instrument type respectively to ensure the accuracy and sensitivity Result Exporting The analysis result can be exported to a variety of text files The website format makes it easy to share the results on a website as html pages whereas other text formats such as csv make it easy to post analyze the results with Excel or users in house software Additionally PEAKS can export to standard result formats such as pepxml and mzIdentML inChorus If your lab already acquired other database search engines PEAKS can import the other engines results and combine all the results together The inChorus function supports the result filtering of all engines results with a unique FDR
36. 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 TLC reflects the expected total number of correct amino acids in the sequence z The precursor charge Confidence Scores m z The precursor mass to charge ratio Mass The calculated mass for the peptide ALC Average local confidence TLC divided by the peptide length RT Retention time elution time for the spectrum as recorded in the data ppm The precursor mass error calculated as 10 x observed mass theoretical mass theoretical mass PTM Indicates the types and numbers of PTMs present in the peptide with color coded icons 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 55 Peptide De Novo Sequencing amino acid that is confidence that the correct residue in each position has been identified are represented by color coding Red represents a very high confidence greater than 90 purple represents a high confidence 80 to 90 blue represents a medium confidence 60 to 80 and black represents low confidence less than 60 For a more detailed positional confidence place the cursor over the sequence of interest and
37. b6 H20 Y l Y1 A 675 28 1278 69 b13 H20 147 11 345 24 515 35 1440 2 rj 100 200 300 400 500 600 70i 800 300 1000 1100 1200 1300 1400 1500 1600 1700 Aa y 1 1 2X 2r ErrTol 0 5Da J intensity threshold 1nfo Ion Match Survey Immonium b 30 5 0 5 88 04 116 03 133 06 gt 87 06 230 08 247 10 1471 77 1472 74 E 0 70 07 327 17 344 16 1357 71 1358 71 472 22 1260 56 05 qq 10107 455 19 437 573 26 710 32 1132 62 1031 49 1261 66 1133 60 1032 55 873 39 894 51 895 49 1036 45 1 1107 49 1206 55 1277 59 731 45 568 29 497 34 398 28 732 43 569 32 458 33 399 26 1376 66 328 22 1 1475 73 Ai lt lt lt p lt lt TAO vizio 10111 3 2 1 Peptide Table 229 15 130 09 131 09 1000 KU n oe ae atis K pani F wf saar rT T 1500 miz 500 1000 PEAKS displays the peptide sequence candidates at the top of the screen The results can be sorted by clicking any of the column s titles 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 1s a unique index for the peptides in the list Scan Scan number Peptide The amino acid sequence of the peptide as determined by de novo sequencing If there is
38. 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 Show inChorus Score Show PTM with frequency greater than 3 in PTM profile table Show Decoy Hits Check this to display protein and peptide hits from the decoy database in PEAKS DB results Show Percentage Score PEAKS uses 10lgP to display its results by default Check this to view the percentage score along with 10lgP in peptide and protein view as well as the exporting files of PEAKS DB results Show inChorus Score Check this to display the percentage score in peptide and protein view of inChorus results Set the PTM display threshold by selecting the minimum PTM frequency in the PTM profile table If there are fewer instances of a PTM identification in a protein identification that the minimum it will not be displayed in the PTM profile table 1 1 2 RMI Connections Clicking on RMI Connections on the menu in the left hand side will show the RMI Java Remote Method invocation connections preferences on the right hand side RMI Connections Server Host localhost Server Port 33003 Client 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 lt support bioinfor
39. choices for scaling factor file format resolution and oversample factor PEAKS supports BMP GIF JPEG PNG and SVG image formats After setting all parameters click the OK button to export the selected result item to an image 4 Export Database Search Result The exporting mechanism for PEAKS DB PEAKS PTM and SPIDER results are the same as that of a de novo result with the exception of the number and type of available export options All exporting functions are available through the Summary view panel 4 1 Export Summary Proteins and Peptides To export the result press the Export button in the title bar of the Summary view panel The following export dialog will appear 125 Exporting Data Reports and Printing i HTML Report Text Formats HTML Report Text Formats 7 Export summary view Proteus wen E IM za pe e 4 Supporting peptides protein peptides csv IF Export supporting peptides 4 DB search peptide spectrum matches DB search psm csv m rtbest unique PSM 4 De novo only peptides de novo only peptides csv Proteins fasta proteins fasta _ Put all protein details in a single html Peptides mzidentml version 1 0 0 peptides 1 0 0 mzid Peptides pepxml peptides xml De novo only peptides pepxml de novo only peptides xml Save into D test Peaks60 PeaksExports Sample Project Identif
40. com gt for more information 1 1 3 Derby Database Clicking on Derby Database in the menu on the left hand side will show the derby database preferences on the right hand side 131 Configuration and Preferences Derby Database Derby Host localhost Port 15270 Derby Server Start Memory 512 Derby Jar Location Derby Host The name of the Derby Host as well as the Port number will come up by default The port number can be changed Derby Server Start Memory The amount of memory used to start the derby server will also come up by default but can be changed if more memory is available however it is not recommended to change this from the default setting To increase performance use the performance configuration utility see Section 5 PEAKS Performance Configuration Derby Jar Location The Derby Jar Location panel will list the location of the derby jar file by default This is displayed to find its location This location cannot be changed 1 2 Raw File Converter Preferences This section allows users to change preferences for the raw file converters of the following instruments AB SCIEX Bruker Shimadzu and Varian 1 2 1 ABI wiff Clicking on the ABI SCIEX wiff option under the Raw file converter section in the menu on the left hand side will show the preferences for the AB SCIEX instrument raw file converter Note Refer to Section 3 4 1 QSTAR or QTRAP for details on
41. data 7 aa Instrument FTMS FT Trap Fragmentation Data File D Data OrbiSample RAW Copy to whole project 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 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 of that project so that the original data files can be manipulated or deleted without affecting the analysis in PEAKS 25 Loading Data to a PEAKS Project To close an open project select the project and choose Close Project command from the file menu or use the close project icon from the tool bar It is recommended to close the unused projects to preserve computer memory The rest of this chapter discusses the details of data loading and project creation 2 Supported Data Formats The following is a list of supported data formats in PEAKS PEAKS supports these formats at three different levels Native Support PEAKS can read the following files directly without any additional tools e mzXML e mzDa
42. display area This area displays protein header information and protein sequence If one region of the protein sequence is covered it will be displayed in bold font and grey background All the confident PTMs and mutations are displayed above the protein sequence on their occurred positions PTMs are displayed as small color coded icons with the first character of the PTM a star is displayed if the PTM is a combination of the other two PTMs Mutations are displayed as white framed icons with the amino acid the position mutated Mouse over these icons to show the names of the PTMs and mutations The number above the PTM or mutation is the index of the position in the protein sequence TI Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER gt sp P02769 ALBU_BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 68 76 d c 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQOCPF DEHVKLVNEL TEFAKTCVAD 96 101 a 116 144 154 d s o Al d 21 ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK E 9g El T z o YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA 284 310 d 241 RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA 321 IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL 469 421 428 449 g 47172 474 d c 211 KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VST
43. figure is the most useful for 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 SO 100 110 PEAKS peptide score 10lgP s Decoy Target Statistics of Data and Results Tables 1 4 shows the statistical numbers of the data and results 74 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER Table 1 Statistics of data Table 4 PTM Profile of MS Scans 3113 Name AMass PSM Position of MS MS Scans 3592 Carbamidomethyl 57 02 894 E Carbamidomethyl 5702 319 DEHK N term Table 2 Result filtration parameters Deamidation 08 48 NQ Peptide 10lgP z15 De Novo TLC 23 Dehydration 18 01 43 DSTY Proteins Unique Peptides gt 2 E CIL Carbamylation 43 01 27 KN term Dethiomethyl 48 00 20 M Table 3 Statistics of filtered result Acetylation 4201 17 N term Peptide Spectrum Matches 1793 Hydroxylation 1599 11 DKPRY Peptide Sequence 833 RR E Hexose 16205 11 TN term Frotein Groups 16 Oxidation 15 99 8 M Protems 18 Proteins Unique Peptides 11 2 7 2 0 1 quem FDR Peptide Spectrum Matches 0 5 Acetylation 42 01 8 K FDR Peptide Sequences 1 1 Ammonia loss 17 03 7 N FDR Protein 0 0 Label 15N 1 1 00 6 EIL nM nis
44. is achieved by specifying the filtration rules in the area at the top of the Summary View The filtration function is discussed in Section 4 Filter PEAKS 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 Section 5 Export PEAKS Results for Publication 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 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 Four tables summarize the data and results such 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 A table concerning enzyme digestion efficiency for each sample is also displayed in this section
45. mutation characterization as well as result validation visualization and reporting data analysis result raw data gt database search visualization gt reporting validation and ee filtration homology search quantification PEAKS helps in every step of the proteomics data analysis Overview The following is a partial list of the distinctive features of PEAKS software Complete Analysis PEAKS combines four complementary algorithms for de novo sequencing database search characterizing un specific PTMs and detection of peptide mutations The combined use of the four algorithms maximizes the number of identifications Built in Result Validation A decoy fusion method is used to validate the peptide identifications automatically The decoy fusion method is an enhanced result validation method that avoids several pitfalls existing in the commonly used target decoy method With an informative result summary view the results can be easily filtered by false discovery rate FDR Protein Coverage View All the identified peptides are mapped to the identified proteins and displayed in a consolidated protein cover age view All the PTMs and mutations on the protein sequence are highlighted The interactive graphical user interface GUI allows the in depth and effortless examination of every amino acid in the identification results and every peak in the data Accuracy and Sensitivity The superiority of each identification
46. of 2 protons Ubiquitin 58 01 114 04 Carboxymethyl KW X amp N 1 00 SILAS 15N 1 129 04 Monoglutamyl 27 99 Formylation 0 98 Amidation 58 01 Carboxymethyl 9 03 13C 9 Silac label 14 02 Methyl ester 43 99 Carboxylation DKW 46 01 ISD a series 128 11 4 trimethyllammoniumbut 38 02 Acrolein addition 38 15 99 Hydroxylation 125 90 Iodination dE Een d LESE ES REO ERES NE oe dE dE por Jo E 01 54 07 15 01 ISD z 2 series 54 01 Methylglyoxal derived hyd HE 1 N l vcina nvidatinn ta aminn To create a new PEAKS project from raw data files follow the following steps see screenshot below 1 Click the new project button at the tool bar Cad and Land Land Land Land Land Land Land Lad A LIOS e ajaj ajuia iJi 2 Click the Add sample and Add data file buttons to add samples to the project and data files to each sample Overview 3 For each sample specify the sample details In particular each sample can use a different proteolysis enzyme Using multiple enzymes to analyze the same proteins can produce overlapping peptides and therefore increase the protein coverage File Tools Window Help Bi HUg X 6Qw Project Name BSA 2 enzyme Project Location CAusersibinmalPeaksProjects s Sample
47. provided in PEAKS to share results with colleagues and collaborators Activate PEAKS manually Important Keep your license key safe After a computer hardware upgrade it might be required to re activate the software 4 1 Activate PEAKS with a trial or purchased license key The software activation process is very simple If your computer connects to the internet you can activate PEAKS by clicking on the first option in the wizard In the Enter the License Key dialog paste or type in the license key and click Activate button If the activation is successful PEAKS will then start In the situations where the activation failed with the message An error occurred while communicating with BSI licensing server refer to Section 4 4 Activate PEAKS manually Enter the License Key The license key is a 20 character long case insensitive alphanumeric string You may find the key in the software package or from your BSI sales representative YOUR LICEMSE KEY 4 2 Register to get a free 30 day trial license key This option allows new PEAKS users to evaluate the software before purchase If the computer 1s connected to the internet clicking on this option will bring up a web form in the default web browser Please provide your full name institution email address and phone number in the form After the form is submitted by clicking the 14 Installation and Activation Register button an email from support bio
48. replicate analysis result node will appear in the 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 label free quantification The x axis is the log intensity of the feature detected in the first data file and the y axis 1s 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 116 PEAKS Q Label Free 28 3 5 Da _ 3 97 5 2h m um a i S 0 25 3 amp 22 5 ma 7 pa S 204 5 a 52 gc xS l l l 16 18 20 22 24 2b 78 30 Log intensity of fraction A Pearson correlation coefficient 0 884 Fraction A 070828 01 02070824 DMSO h 1 Frachon B 070623_01_03 070924_DM50_0h_2
49. representative to obtain CompassXtract 3 1 Instrument Preferences for Bruker Data This type of Agilent data uses a Bruker converter To set Bruker data CE related preferences in PEAKS click the Preferences toolbar icon or select Preferences from the Window menu to open the Preferences window Click on Instrument and then Bruker yep baf fid in the menu on the left hand side This will show the Bruker instrument preferences on the right hand side Bruker yep baf fid Raw File Convertor Options Bruker file reader will export Raw data Line spectra Bruker fid file may contain several files do you want to merge them 9 yes no CompassXtract by default will export raw data If the attempt to load raw data results in no spectra then choose Line spectra A Bruker fid file may contain several samples By default these samples are not merged into one data set Select Yes to merge all the samples into one data set 3 4 Applied Biosystems Sciex Data 3 4 1 QSTAR or QTRAP ABI data can be loaded into PEAKS provided the following converters are installed Analyst QS is required for QSTAR data Analyst 1 4 is required for QTRAP data AB SCIEX MS Data Converter is required for ABI 5600 data PEAKS Config Wizard can download and install mzWiff automatically A link is also provided in the PEAKS Config Wizard to the AB SCIEX MS Data Converter download site see Section 2 Instrument Selection
50. spectrum will be saved to all de novo candidates csv file in CSV format 3 2 Export Images The annotated Spectrum Ion Match table Error Map and Spectrum Alignment all can be exported to image files To do so position the cursor on any of those items in the result panel and click the right mouse button to view the pop up menu and select the Export Image command from the menu newco S ETATE Is 2 vl o Export image Print image Copy b Set As Anchor Peak 50 Remove nchor Peak Annotation Settings 100 00 300 Ma y li 1 1 2X FY ErrTol 0 5 Da v preprocess low intens label This will bring up the Export Images dialog for selecting the result items to export 124 Exporting Data Reports and Printing ad Export Images Image Types 3 Error Map 3 Ion Table and Error Map Current Spectrum View 3 Spectrum Alignment View j Annotated Spectrum with Alignment Image Options Basic Options Web Smallest images suitable for viewing online Print Oversampled images suitable for printing Save As D test peaks60 PeaksExportslimages 563 2941 2 png Select the desired result elements from the Image Types list The Basic Options panel offers choices for the location to save the images and the image size Web Smallest images suitable for viewing online or Print Oversampled images suitable for printing The Advanced Options panel offers
51. successfully message please check your email 16 Installation and Activation BSI Product Licensing Center Q O www bioinfor com Ics20 Index jsp BSI Products Online Registration Request file Choose File license request Visual verification Input the characters shown below case sensitive Sup RD 8 request another image 4 If the license request is sent successfully an automated BSI service will generate the license file License lcs and send an email from lt support bioinfor com gt to the email address provided to the License Wizard Either save the attached license file or copy the content between gt and lt in the email to the Windows clipboard 5 Transfer the license file to the computer running PEAKS and import the license file into the license wizard Manual Activation Gather Paste the license Paste from Clipboard Information Save License Request File Upload License Request File Import the license file the email attachment Import License Activation Complete 4 5 Re registering PEAKS Re registering PEAKS may be necessary when an additional software module was purchased or SPS was renewed BSI will modify the license information accordingly on the server side A new license file is required to make the changes effective Select About PEAKS from the Help menu The About BSI PEAKS Studio dialogue box will appear 17 Installation and Act
52. 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 i 1 a q el p n a dd 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 HE y OALE N a 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 39 Data Visualization 4 4 Show MS2 Hide MS2 Show MS highlights scans with associated tandem scan by marking them with the selected colour a 7 a j E 2 ae aa Saga d e og 2 S 00 Auc s E a oo ait me e o a a9 8 5 fe we oo P be We PO stots of a apa o 7 gt on 5g a foo 6 F og cm E S oo Eno 2s aa e om oc 0 PI SE a o o o o y b e TITAN EL EA AAA OS og SP 0 92 B ao F B o o db a Oo a Ga gt a 0 ao a i ME uk 8 o0 on r oe o la i o Y np tn oe on Pg o o e o Padua Fo a a a B o a ci too 3 dh a Oo ga e ES 8 dubie ed Q9 Dan cH oO UB o One cog e X S qP Se Bo HUP EN aor a ci 19 75
53. trend chart for that cluster 1 Heatmap View 14617 AA AH a r tu o m A M QSHCDSINCOAS_HUMAN tjQ5Cz84 Q5Cz84 HUMAN DT5351 VPS4B H MAN P12238 ADT3 HUMAN P18124 RL7 HUMAN P13837 ATTA3 HUMAN tB4DNOO B4DNOO HUMAN F01817 KV204 HUMAN t 28M355 O8N355 HUMAN tr B3KNZ4 B3KNZ4 HUMAN P286232 CTNA2 HUMAN t Q95BAT QS6BAT HUMAN trjQG8IUKT OSIUK T HUMAN trjcS jWwee cS JWweB amp HUMAN QT7LO0J3 SV2A HUMAN t B4E354 B4E354 HUMAN tr D8RBU5 DSRBUS HUMAN log2 ratio 5 0 0 0 5 0 Default group 1 Cell colour represents the log ratio to the Control Sample Control Sample is marked with 2 Notes 3 Result Statistics Table 1 Statistics of data and unfiltered result Table 2 Result filtration parameters of MS Scans 5591 Protein fold change 5 of MS MS Scans 12443 4 Other Information Table 4 Search parameters Table 5 Instrument parameters Quantification Type ICAT SILAC Fractions VP54B1J RAW Quantification Mass Tolerance 0 1Da Ion Source ESI nano spray Quantification RT 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 the
54. 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 Use the Advanced Options to specify additional parameters Select Monoisotopic or Average as Precursor Mass Search Type For ion trap instruments it is usually beneficial to allow PEAKS DB search to use an average mass Specify the values for Parent mass error tolerance and Fragment mass error tolerance in Daltons or ppm These will appear on the PEAKS de novo and PEAKS DB options screens when the instrument is selected Click the Add Update button to save the changes The new instrument will appear in the Instrument List where it can be accessed when creating a new project file To delete an instrument that was created select the appropriate instrument from the Instrument List and click the Delete button 143
55. 0 y Protein im FDR Peptide E 7 De novo only e e ES 0 200 400 600 800 1000 1200 1400 1600 1800 2000 EI number of peptide spectrum matches Figure 2 The distribution of PEAKS peptide score 101gP a histogram of score b The plot of precursor mass error vs score Q a 200 Tasks Running Info Properties 1501 ppm e 5 O PeaksProjects BS Project Name O PeaksProjed 501 Total Samples 3 Fractions BSA GluC 1 RA T T T T T T T T 7 j r T ae ESKnano spra i 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 Fragmentation Mode CID CAD IRM PEAKS peptide score 10IgP PEAKS peptide score 10lgP MS Scan Mode FT ICR Orbitra ll Decoy li Target MS MS Scan Mode FT ICR Orbitra _ 100 1 number of PSMs a Decoy Target 4 m 4 Li 4 3 Result Summary and Filtration After opening a result node by double clicking it 1 e the SPIDER node in the sample project the default view of the opened result node is the summary view The summary view provides mainly three functions 1 Specify score thresholds to filter the results 2 Examine the result statistics 3 Export results The top region of the summary view is a control pane and the bottom region is a statistics report page The result filtration 1s controlled at the top control pane see screenshot below e The peptide identific
56. 1 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 bo 200 400 600 1000 Ma 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 gt 653 36176 RT 25 0268 gt 523 23615 RT225 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
57. 25 B Sample 1 o ES PanTumorSCX 1 RAW A DATA REFINE 2 27 Apr 11 15 jy DENOVO 6 27 Apr 11 17 05 M PEAKS 10 27 Apr 11 19 14 mil Sample 2 Ely PanTumorSCX2 RAW DATA REFINE 1 27 Apr 11 15 5 i DENOVO 5 27 Apr 11 16 30 A PEAKS 9 27 Apr 11 19 00 B gn Sample 3 o ES PanTumorSCX3 RAW i DATA REFINE 4 27 Anr 11 15 The result panel will be opened automatically after completing the comparison Since the comparison run is done on the fly it will not be saved and therefore it is 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 three parts peptide comparison protein comparison and statistical charts Below is an outline of each 7 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 coverage map 1s a quick graphical 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 PEAKS provides filters on the peptide comparison results After inputting the PEAKS score threshold o
58. 39 99 5E 4 amp 3 SEVAHRFK 414 487 2649 22 28 972 5142 1 1 2 99 92 BI sili 45 42 1 33 42 05 4 LVAETEDRK 465 354 1932 23 07 1059 5559 1 7 3 99 92 als B 28 04 2 27 45 01 1 01E 2 amp 5 QGVTEAAEK 167 466 7378 18 14 931 4611 0 1 3 99 92 Ek E 2783 111 24080 52E3 amp 6 IGYGSN 4 98 KK 220 434 2328 18 96 866 4498 1 4 2 99 91 H E 47 37232 L 7 QC17 03 AKHEEIDTK 568 394 5337 24 46 1180 5725 5 7 1 99 66 B m 4005 2458 P o 8 EAHEIVSK 17 456 7428 15 30 9114713 0 2 1 99 54 B M 24001 30 76 8 9 PGGGQVEVK 561 435 7363 24 38 869 4607 3 0 1 99 38 ASME 29 06 0 90 21 06 7 1E 2 g 10 NPTAFKK 142 403 2322 17 77 804 4494 0 5 1 99 25 AGM 20 29 0 91 35 37 11 AALEAARDSK 405 516 2780 22 17 1030 5408 0 6 2 99 16 20 95 0 83 32 96 12 VISQDTQPHQQK 557 704 8610 24 33 1407 7107 2 3 1 97 93 P 34 71 The blue background means the engine identified the peptide with high confidence above the engine s own filtration score threshold See Section 3 Filtering 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
59. 49 Apply Export Edt De nova 20 25 30 35 4D 45 5D 55 60 55 70 75 80 B5 De novo AL C scores 5 You can optionally export the results to other formats by using the Summary view 2 De Novo Sequencing Parameters In the Project Tree select the data file s or project containing the spectra that you wish to have auto de novo sequenced 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 9 A a Parention 50 0 ppm Enzyme Specified by each sample PTM IF Carbamidomethylation QT Oxidation M T Deamidation NQ Maximum allowed variable PTM per peptide B Report up to 5 candidates per spectrum The meaning of each parameter is discussed in the following sections 2 1 Error Tolerance The acceptable levels of mass variance for the parent precursor and fragment ions in the respective fields The parent ion error tolerance can be specified in either Daltons or ppm 50 Peptide De Novo Sequencing 2 2 Enzyme Specificity This informs PEAKS as to what type of enzyme was used to digest the sample Utilize the drop down list to select an enzyme Note It is also possible to use the selection Use Sample Enzyme which a
60. 52E5 2 01466 2 049E5 1 322E6 0 00 0 00 0 00 0 00 V wiimaal 111 NA a i m m amm A n 231rn i n n n a nan nan nan nan ral m Coverage Peptides sp P02769 ALBU BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 va amp O outine 0 coverage 44 58 6536 75 APPS 6 AAs fl c ma 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHREKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD V confident PTM location 80 7 AAs per line 7 10AA gap 92 99 00 106 117 131 138 145 147 151 m ci i a a al i ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK AM PTM fil 144 10 iTRAQ 4plex K N 10 IB 457 02 Carbamidomethyla B 415 99 Oxidation M 58 48 m HSS ISS S fil 144 10 TRAQ 4plex Y id 40 98 Deamidation NQ 161 169 179 1838485 189 19132 Y a LLE d ag 161 YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA _ _ 4 Li i M i ii e au 221 RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA F 3 3 Peptide View The Peptide view displays all the identifiable peptides and their intensities The interface is similar to the peptide table in a PEAKS DB result The intensities of the quantifiable peptides are displayed in the intensity columns with label name as the hea
61. 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 37 Data Visualization 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 Or Nr ork Feature 0 Show MS2 J show PID of none Shaw 3D View Moise Level 1 For a smoother view of the Heat Map choose Blur and for a 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 es E Y E z E _ 4 a P a laps x i A E T ns _ 2 IO RUE i arc gy 1 i i Biche ca s X X LI 40 97 a E E a aF e z i u SN MS y E a 3 a iit oL ot E E I 1 z ik je E i EO la a A E La i 5 i amp a 58 Peck 7 m ts
62. ACIBL 474606 469 65544 938 9 EAATK 6 02 35 4 9 76 524 2901 14 1 525 2900 0 956 78 1 B6YR21 ACP AZOPC 116141 000 1673340 375 B 10 EEEMSR 35 4 9 68 779 3120 39 4 780 3500 0 194 19 1 Q85535 Y4A0 ENCCU 60051 859 54727 203 11 VADCIAR 10 02 IK 34 2 7 94 997 5744 81 8 998 5000 0 044 4 1 Q7WKR4 SYFB BORBR 940 608 2470 384 12 DVVSATVAR 34 2 7 83 916 4978 8 4 459 2600 0 123 12 1 Q8WZ42 TITIN HUMAN 347738 094 54600 363 3 4 Filtering Quantification Result The Quantification result can be filtered based on the number of fold changes between samples You can set the appropriate values of the filter by changing the filtration parameter values from the drop down lists in the title bar of the Summary view panel and clicking on the Apply Filters button The result will be updated in the Summary view the Protein view and the Peptide view accordingly The intensity columns of the Protein and Peptide views display the absolute intensity or relative intensity of the quantifiable proteins and peptides To change the reference sample select the appropriate sample eg ratio to light from the dropdown list beside Show in the Summary view To change the normalization factor of the protein ratio select auto manual or no normalization factor from the dropdown list For manual normalization provide the normalization factors in the textbox to the right
63. Database Search id ji E Spider search E A R252 inChorus Search E A R351 fe R352 Export DTA file Export PEL file Export MGF file Show Reports Save A Copy As Clase Project 115 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 per form replicate 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 T Replicate Analysis Tools Y Replicate Data Comparision F Replicate 1 070828 O1 02 070824 DMSO Dh LRAW Y Replicate 2 070828_01_03 070624_DMSO_0h_2RAW 7 A 070828 01 02 070824 DMSO 0h RAW 070828 01 03 070824 DMSO 0h 2 RAW 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 the Replicate Analysis Results Once the replicate analysis is completed a new
64. E NEN 138 91 12 5 5 17 94 112 38 153 41 20 5 5 7 94 1 155 03 22 7 7 11 11 114 34 Q280R4 H4 XENTR NEN 50 05 5 1 1 4 1L65 10449 10 3 3 12 31 07 7791 4 2 2 19 42 77819 ROCK 1 RABIT oo 73 n 1 1 47 10 81 Q25533 1433 NEOCA Oos 4 13 3 2 p 05 6 77 QSNDFBIPAPOS HUMAN BOO 16 93 2 1 1 1 52 3 5 QSVAFA TEB9 GOSHI CIC 55 2 1 1 p 2 7 3 15 P22492 HIT HUMAN HEN 55857 4 1 1 11 92 15 31 173 17 8B 1 1 1 92 5 31 5 17 4 1 1 192 5 31 P12303 TTHY SHEEP CICINI n 1 6 67 10 88 Q92597 NDRG 1 HUMAN uu ps 1 1 1 3 7 3 55 67 78 2 2 2 7 41 7 61 4 4 n 1 1 3 7 3 55 Q00799 REP2_PLAVB goo 1 1 1 0 23 0 59 Q64 94 PSME 1 PIG See 124 43 B lg lg 110 20 48 119 77 9 3 3 7 5 15 26 137 5 16 5 5 12 5 23 68 P 1349 1 LDHA_RASIT UN 39 91 2 1 1 2 63 2 71 3 13 2 2 2 5 26 5 42 P63112 HBA PAPCY WEN 595 2 1 1 6 25 645 6237 2 1 1 6 25 8 45 595 7 2 2 125 1479 Q3U952 CO5A2 MOUSE CICNI 9 02 3 2 2 L6 2 74 P00739 HPTR HUMAN wam 59 47 3 1 1 2 86 3 45 7206 5 2 2 5 71 7 18 3 49 6 2 2 5 71 16 32 P0SS14 PTMS_BOVIN NEN 51 56 3 1 1 5 88 10 728 6696 7 1 1 5 88 10 73 5135 3 1 1 5 88 110 78 Q5R887 MYL3 PONAS CICNN 57 09 3 1 1 ls 6 15 Q4QQU6ISPFE30_RAT goo 36 46 2 1 1 2 86 4 2 043103 SID2 USTMA goo b 1 1 1 0 26 0 32 ABYUIIJATPA_LACHS WIL 39 42 1 1 1 11 92 2 39 Q7V382 RPOBC_HELHP CINCO 0 n 1 1 0 26 0 31 2 7 4 Statistical Charts PEAKS provides a number of statistical charts which are easi
65. E El ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKEY l 97 bos 171 173 1 g EN RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVE B 15 IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH acystvet lt m 421 E 12 474 KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSI 48 NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKI 571 27 KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA 76 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 3 3 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 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 drop down list above the protein table controls which proteins are shown for each protein group all shows all the protein in the group top shows only the top proteins which have the most significant peptides in this group first shows only one protein for each group this protein is one of the top proteins for this group The table s columns are Accession The accession number of the protein entry in the database e 10lgP Protein confidence score
66. Error Database Missed Cleavages A PEAKS is not selected inchorus combination will not be performed Note If the data is not refined by PEAKS a data refinement with default parameter will be performed first 92 PEAKS InChorus Important To get the inChorus FDR the same target decoy database needs to be searched by all the engines For PEAKS X Tandem and OMSSA this target decoy database is generated automatically For SE QUEST and Mascot a target decoy database needs to be exported from PEAKS and added to their database list Use the tool from Configuration gt Database to export the target decoy database Enzyme Database List PTM Labeled Q Method Database Database Details aa a ExportDecoy DB OcX S FASTA format database UniProtKB Swiss Prot Basic Options Database name SampleDB Validated Path D PeaksStudio5 3 Data SampleDB fasta or EST database Advanced Options Fasta Title Format Rule to parse accession id from FASTA title spy Rule to parse description from FASTA title Hn Accession id URL http Ferna uniprot org uniprot lt Accession ID gt Delimiter s Taxonomy Options xno Then check the Search decoy database from PEAKS checkbox for each third party engine in their parameter setting or importing dialog for PEAKS check the Estimate FDR with decoy fusion check box 93 PEAKS InChorus Mascot Predefined paramet
67. 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 OK Click Cancel to copy the URL to the system clipboard If Cancel was selected click OK on the dialog detailing the copy to the clipboard Next open a browser and paste the URL into the address bar When the file download window opens click Save Use the same way to download the taxdmp file 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 Note Please don t rename the taxonomy files otherwise PEAKS cannot recognize the files 2 Databases to be Used in PEAKS inChorus Function The database configured here will also be used in the PEAKS inChorus function to call the X Tandem and OMSSA search engines However Mascot search depends on Mascot s databases only When using these third party soft ware tools note the following with care 44 Adding a Sequence Database e 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
68. For each identified peptide PEAKS also displays all the engines that identified the peptide as well as each engine s identification score 3 What Is New in PEAKS 6 Here 1s a list of the noteworthy new features in PEAKS 6 Highlights Overview nteractive protein coverage view In this beautiful graphical display all peptides identified for a protein are mapped to the protein sequence The PTMs and mutations are highlighted Individual peptide spectrum matches can be examined with simple mouse clicks Easy workflow for complete analysis With a single mouse click the PEAKS PTM blind search for PTMs or the SPIDER mutation detection algorithm can be included in the analysis workflow Multiple enzyme support One project can include multiple samples each with a different proteolysis enzyme The overlapping peptides from different enzyme digests maximize the protein coverage e Blind search of PTMs Users can turn on all of the more than 600 variable PTMs in the Unimod database and let the software find what PTMs are present Algorithm Improvements New nonspecific enzyme digestion support Now one can allow nonspecific enzyme digestion at 0 1 or both ends of a peptide Having more nonspecific digestion ends will increase the search sensitivity at a reduced search speed Neutral loss of phosphorylation and sulfation PTMs are considered in the scoring functions of PEAKS DB and PEAKS PTM New support for prote
69. GJ Run PEAKS PIM on PEAKS DB Resultados ias 86 6 2 Run SPIDER on PEAKS DB or PEAKS PTM Result esse 87 T Compatison Or PEAKS Results iS A A A A au 88 J ly xcomparison INCSUI x oodd erba aon aa veutand oo miei e 88 7 2 Pepude COMpars OM aai ecce oen Eois baii 89 159 Proci C OMpPAnSOn ia sir ciao 90 1V PEAKS 6 User Manual prassi EIE NTC 90 17 2 bxpottine Comparison Re sul o v2 onte t rude is isa 9 IO PEAKS ICO ASA waned ANDES A bead au A 92 I BEARKS E Or OVerVIe W sta 92 2 Understanding PE ARS inGhor s Result sit dis 94 3 Eterno PEAKS in nOrus Result a ii dc os 96 4 Exporinognchorus ReSult 2232055 ESA A AA AT ES 97 WTP I TS roca 98 Ws OVERVICW er PH 98 ES ETIN E OO ET T DO TD 98 3 Understandine the ResUl eni SE SN ANA ea alls NE NA 99 AS SA A O II DELI ui scende 100 LOL VIEW A A ii 100 o O al est n db Renee ee Tee rem m male elato d oce aee eunte ee ere of 101 3 4 Palt rine Quantticatlon Result uu oett A t dtt Palin qul be A Ur NA 101 4 Bxpott Quantitthicattonm Results 25 tuisse dete beutie t Ut otdt aleae ud tebsatliee m etetulca be seu tud 101 I2 PEAKS OS MS MS We vel iced iii iia 102 POVE ON EE 102 AS Paramete ea A a Maas NO 102 3 Understandi o The Rebost a a dima e aite abate 103 SS VIEW A O ice bau a entia etudes io bees 103 S APUOtCTH MV C IO O tmd ite aoe ur cupere col ns hte S Ed es calc 104 DSP CPUS AL MNT HP EN 105 54 Filter
70. IN BIBIBIB FEAKSDS GLVLIAFSQYLOQC 57 02 PFDE 57 7485 2548 2783 L4 850 4322 45 27 F2 2471 1 POZ7GSIALBU BOVIN B PEARSPTM VEKEC 57 02 C 57 02HGD 457 0 7435 2668 1792 L2 890 3993 23 78 ri 1212 2 POZ7GSIALEU BOVIN BEEE PEAXSPTM GLVLIAFSQYLOQC 57 02 PFDEHVK 7428 24912568 1 0 _ 1246 6345 45 49 F1 2624 51 POZ SSIABU ROVIN B PEAKSDB L ax In Tai 7 ES ru lama d aona nana ap naa nann mm as 73 373 move a TS ITE Emm 1 ee ifa The table provides the following controls Sorting by 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 Jumping to the spectrum in the data view Right click on one row to show the pop up menu Select Show original spectrum to jump to the spectrum in the data view to check other results for this spectrum 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 a
71. M SPIDER Predefined parameters Parent Mass Error Tolerance 20 ppm Precursor Mass Search Type Fragment Mass Error Tolerance 0 3 Da Monoisotopic Average Enzyme Specified by each sample Allow non specific deavage at one end of the peptide Maximum missed deavages per peptide 3 PIM Search with all 679 built in modifications 3 Search with preferred modifications VI Dioxidation M VI Deamidation NG g Oxidation M Remove VI Carbamidomethylation Maximum allowed variable PTM per peptide 3 Filter the spectra which satisfy the following conditions for use in the PTM search De novo ALC 95 score greater than 30 recommend 30 6 2 Run SPIDER on PEAKS DB or PEAKS PTM Result Invoke SPIDER by selecting a PEAKS DB or a PEAKS PTM result and clicking the SPIDER icon on the toolbar X or choosing SPIDER Search from the Tools menu Running SPIDER on a PEAKS DB ora PEAKS PTM result can be functionally equivalent to running the two together in an integrated search However the configuration panel in this case appears as follows and allows for a few additional options 87 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER SPIDER Search Predefined parameters IF Carbamidomethylation Use the spectra which satisfy the following conditions for use in the SPIDER search De novo ALC 95 score greater than 30 h recommend 30 Note For users
72. PEAKS 6 User Manual PEAKS Team PEAKS 6 User Manual PEAKS Team Publication date 2012 Table of Contents MADE E mem l How 10 Use This Ma a a E E l Zi Wiha lS FEARS uuena A UU l 5 What e New 1n PEARES Orune At De MEE 2 A Quick W AOUS E seoa E E sqm LIRE iat ta aiu A 4 2T Open at Bxisunes Project mas a a dolia 4 AD PEAKS Mano Ulsa e E A ERTE 5 4 5 Result Summary and FIAN seso T r But T E ETA 6 AA Result EI A a edi a A AA 7 Foren a PEAKS LO so ERR aaa a aa a a 8 46 Conduct am ddeti ticatioH ZXBalyste ridad Vd tudque e AIL ER 9 2 Installation and Zeb AG OM A salen vile Ca adu MILD iS DV M DUI ER LM LE 12 Package Contes esta E aida AUR QE dies 12 SS A A S posse po tuac RS O D Su adu cL cEU edu SU ines 12 3 Installattobi omn as Windows COMPUTE sti ida ed Ma S SHEER EN dua du aes Ci da Neto ED dois 12 A INCA MT A A A ea but ing Sateenanes 13 4 1 Activate PEAKS with a trial or purchased license Key eese 14 42 Re cister to 9et aires 0 day tral license Key as ai 14 45 Use URE AIS Asay VIS WE dica ds danita LAS taria 15 44 Activate PEAKS Manual AS ALEA 15 2 5 Ree rebisterins PEAKS ista data its as colas dfn COLUIT Ras 17 2 5 Common Errors queno Registration nd daras 18 5 PEAKS Pertorma nce C ODHS Uf atioTm ta tii epe aiti teet ides do 18 O Whats NO usos oe qui tastes qum AAA 19 3 Configuration Wizard Configure Instruments and Public Databases
73. PTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL 548 506 C h d 221 NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP 7273 802 o sid d 561 KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA Control area This area controls what to display in the protein sequence display area These are the following controls 78 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER E outline A coverage de novo only tags sharing 6 confident PTM min ion intens 3 3l F E 80 AAs per line 4 10AA gap AM PTM 57 02 Carbamidomethyla 1671 B 157 02 Carbamidomethyia 722 Z H 0 98 Deamidation NQ 86 v 18 01 v 343 01 45 00 KA 162 05 Hexose T XBN t 24 V B 17 03 Ammoniedoss N 19 V B 15 99 Oxdaion M 17 WJ roo suas Ni 15 V B r 03 PmoguftomQ 12 VB 342 01 Acetylation N term 8 B 4201 Aet aon 5 v I 458 01 Carboxymethyl K 5 Y mi 125 04 Monoglutamyl 5 Yi 27 99 14 02 Methylester 4 E 490 17 EDTodoaceyi PE 4 443 99 Carboxylation DKW 4 IB 37 95 Replacementof2 4 vB 114 04 KA 162 05 Hexose Ml 46 01 ISD a series VB 0 98 amidation 2 WJ 58 01 Carboxymethyi 2 V B 599 Hydroxato 2 Mode control The protein sequence display has two modes Outline mode The outlin
74. Please see the specified software package to find out the requirements for it 3 4 2 Convertors for WIFF Three Wiff convertors are supported by PEAKS They are 1 AB SCIEX s MS Data Converter 2 mzWiff and 3 MSX Note Before defining the converters in PEAKS please make sure these software packages are installed cor rectly and convert successfully from command line on your computer QN To set WIFF related preferences in PEAKS click the Preferences toolbar icon or select Preferences from the Window menu to open the Preferences window Select the Raw file convertor section in the menu on the left hand side then ABI wiff This will show the preferences for ABI instruments 28 Loading Data to a PEAKS Project ABI wiff a AB SCIEX MS Data Converter Location D VAB SCIEXMMS Data Converter AB SCIEX MS Converter exe Browse Options 7 Centroid amp Profile mzWIFF mzWiff Location Browse Options 5 Survey Spectrum Centroiding F Product Spectrum Centroiding Product Spectrum Deisotope Precursor Charge Determine t MSX converter Location D MSX msx exe Brawse Options ABI raw filed may contain serveral samples do you want to merge all the samples into one data set i Yes ia No 4 Survey Spectrum Centroid 4 Product Spectrum Centroid Select one of the convertors as the default convertor for WIFF file loading AB SCIEX MS Data Converter Clic
75. R A T 30 01 bo 310 Y7 H20 971 56 B Y7 NH3 QDTISSKLKE CCDKPLLEKS HCIAEVEKDA 788 40 yo NH3 RLAKEYEATL EECCAKDDPH ACYSTVFDKL 1000 55 m B 47172 474 pi bs 1085 64 d 843 50 RSLGKVGTRC CTKPESERMP CTEDYLSLIL 548 y2 y5 303 18 b4 H20 b2 H20 424 29 nen h 210 16 TFHADICTLP DTEKQIKKQT ALVELLKHKP miz 200 400 600 3800 1000 1200 uli 1 1 2x 2v ErrTol 0 02Da V preprocess Y low intens label Coverage mode Under the coverage mode all the supporting peptides and matched de novo tags will be shown 68 vi d c 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD I AA EA A A AAA AAA e C a a De novo only tags sharing X AAs This controls whether to show the grey bars for the de novo only tags Only when the de novo only tag has at least X consecutive amino acids matching the protein sequence it will be shown as a grey bar Confident PTM A PTM location is confident only when at least one pair of ions which fragment on both sides of the PTM location exists The peak intensity of this pair of ions must be greater than the threshold set in this control AA per line and 10AA gap This controls how many amino acids to show per line and whether to show a gap for each group of ten amino acids e PTM table The PTM table shows all the PTMs that occurred on this protein For each PTM the delta mass and the number o
76. To select a PTM as Fixed or Variable click the PTM from the list and click the arrow beside the Selected Fixed PTM box or the Selected Variable PTM box respectively To remove a selected PTM click the PTM from the Selected Fixed PTM or Selected Variable PTM lists and press the Remove button The Switch Type button can adjust 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 New PTM window will open allowing you the ability to enter the information pertaining to your particular PTM The newly edited PTM will be displayed in the Customized list FY New PTM Residues that can be modifed peptide protein Maximum Number of Variable PTMs per Peptide This parameter limits the quantity of variable PTMs in a peptide sequence In the de novo sequencing result peptides with more variable PTMs are removed 52 Peptide De Novo Sequencing 2 4 Other Parameters Report up to peptides Set how many peptide sequences PEAKS will report per spectrum in the de novo sequencing analysis 2 5 Saving the Parameters for Future Use After setting up the desired parameters you can save them for future use Click the drop down list at the top right of the window select Save as and provide a name for the current set of parameters Saved parameters are available within this drop
77. When using NCBInr or SwissProt databases with X Tandem it is best to use a sub taxonomy OMSSA At the time of writing OMSSA cannot be used with databases that are not in NCBI or SwissProt format in a way that is available to inChorus Also a bug in OMSSA prevents easy use of databases with OMSSA when they are stored in a folder that contains a space 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 has to be identical to the database configured in PEAKS in order for inChorus to parse Mascot results correctly 45 Chapter 7 Data Refinement 1 Overview Raw LC MS MS data often contains noise redundancy as well as errors due to sample preparation and instrument approximation The PEAKS Data Refinement tool can be used to improve the overall quality of the data All or some of the following functions can be applied to the data in a project according to the user s requirements Correct data refinement especially the precursor m z correction can often result in
78. Y intensity threshold OrbiSample mzXML ms 2 m2 802 90607 2 2RT 0 0729 TIC 1 79E7 Info lon Match Survey Seq y y H20 y NH3 1 693 30 675 28 676 27 692 29 2 893 51 912 52 894 51 895 50 1 lon Table l soo 1500 692 2913 51 L a lt r 893 51 652 29 yMax m i M I i DD n LJ E a fom The panels are briefly described below 61 Peptide De Novo Sequencing Result Panel The Result Panel shows all 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 and assigned ions Pick a peak on the panel with the cursor and assign ions or tags to it in manual de novo e on 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 ions and y ions The b ions are shown right to left in blue while the y ions are shown left to right in red The Error Map displays the confidence assigned to each ion Tag Panel Th
79. a Position Confidence Table will appear showing the confidence that each amino acid pair of amino acids are correctly identified EEDHPOTHYYAVAVVK 1 F SHRMTNLNGIPES 0387 p E ag Mass Tags The low confidence residues can be displayed as mass tags by adjusting the scoring threshold using the button 2 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 Peptide LJ 128 1 L L 128 1 o 10 20 30 40 50 60 70 80 90 Color code gt 90 80 3075 60 80 lt 60 F 8 HSTVFDNLNP 497 7 10 1141 MPA 1 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 Peptide candidates can be searched by entering the value in the search bar located in the top right corner of the title bar of the Peptide Candidates Frame Peptide candidates can be searched by scan number subsequence m z value retention time RT value and PTMs by mass difference The reported peptide candidates can be iterated by clicking the circled up and down arrow buttons in the s
80. al analysis report consists of four pages e Summary Outline of PEAKS database search results with statistics This is the place to examine the overall performance of the experiment and adjust filters e Protein Protein sequence characterization at amino acid levels e Peptide List identified peptides e De novo only list of quality 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 length etc A statistical evaluation 10lgP 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 All the PEAKS database search tools use this 10lgP score They are comparable through different search tools Protein score 10lgP The protein 10lgP score in PEAKS is the weighted sum of 1OIgP score of all supporting peptides After removing redundancies those peptides from the same protein are sorted according to their 10lgP 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 Ta Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 1 Result filtration This
81. all 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 disc into the CD ROM drive Or double click on the downloaded PEAKS installation file and move ahead to step 4 3 The installation window should automatically appear after the CD ROM is 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 installation dialogue appears click the Next button 12 Installation and Activation Introduction S Introduction InstallAnywhere will quide vau through the installation af PEAKS important Information Studio B License Agreement occ eee Itis strongly recomme
82. an be put directly on a website 1 Export Data Spectrum data can be exported in a number of file formats including mzxml mgf DTA MGF and PKL To do so right click on the sample node or the data file that is to be exported and select the desired export format PA Project View E hyd D test Peaks60 PeaksProjects test1 Gt JA Sample 1 E STITT i DATA RE Data Refine M e De novo a PEAKS 10 un Peaks Database Search Spider Search in Chorus Search Export DTA file Export PKL file Export MGF file Export MzAML file It to Excel Expo Export Result to Html 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 start RT End RT 120 Save as D test Celegans FT G6ITDDOT O1 mzxml Ies Enter the starting and ending retention times in the appropriate boxes Then click the Browse button to select a destination to save your file 122 Exporting Data Reports and Printing 2 Export Result From Project View Fraction information and peptide identification results can be exported to Excel or HTML format from the pop up menu in the Project View Right click on a project node a sample node or a fraction node and select the optio
83. ance 0 2 Da 6 7b RAW Spike 040806 2 RAW Spike3 040506 2 RAW Spikes 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 1 RAW Spike2 040806 1 RAW Spice 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 5 2 2 Export Summary and Detected Features 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 quantified proteins will appear in the top panel w
84. arian xms in the menu on the left hand side This will display the Varian instrument preferences on the right hand side Click Browse to tell PEAKS the location of the xm1rai exe file Varian xms Default xmlrai exe Location 3 8 PEAKS 5 3 Projects Projects created in any PEAKS 5 3 series software can be opened in PEAKS 6 To convert the project to a PEAKS 6 project open the project in the same way you would open any existing PEAKS project The project will be ih This project was created by an older version of PEAKS Do you want to convert it now Yes Mo thanks Choose Yes to convert the project and proceed The following Project Converter dialog will appear FY Project Convertor Converted Projects My PEAKS553 project 1 _version6_0 Project Location C PeaksSstudio6serverDB 07o 31 Loading Data to a PEAKS Project 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 is not altered Note For PEAKS 5 x projects early than 5 3 you need PEAKS 5 3 to convert these projects into PEAKS 5 3 projects first then use PEAKS 6 to convert the 5 3 projects to PEAKS 6 projects 4 Creating a New Project L 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 will appear FY New Pr
85. ation is filtered by the peptide spectrum match s 10lgP score Or one can simply specify the desired FDR false discovery rate by clicking the FDR button e The protein identification is filtered by the protein s 1OIgP score and the number of unique peptides the protein contains The de novo only peptides are those with confident de novo sequence tags but cannot be identified by other algorithms used for database search To report a de novo only peptide the TLC total local confidence and ALC average local confidence scores must be better than or equal to the specified threshold Meanwhile the spectrum s best database search result s score should be no greater than the specified 10lgP threshold TLC measures the approximate number of correct amino acids in the de novo sequence and ALC measures the approximate percentage of correct amino acids in the de novo sequence By default the 10logP threshold used for de novo only is locked to be the same as the 10lgP threshold used for filtering peptides To specify a different value first click the lock icon to unlock it After the filtration criteria are changed the Apply Filters will change to red Click it to apply the new criteria Overview PEAKS PTM 6 09 May 12 13 57 X Dom PLE i B Peptides 109P x 16 4 v Proteins 10lgP 20 and 3 unique peptides E a un gj DenovoOnly LC z 3 and ALC 50 and peptide 1019P 3 16 4 Apply Filters in
86. available options Select the output location and click the Export button to save the selected result components to the specified location HTML Report Text Formats Result summary summary html Save into D test PeaksExports Sample Project Identification DENC Car HTML Report Text Formats De novo peptides de novo peptides csv De novo peptides pepxml de novo peptides xml All de novo candidates all de novo candidates csv Save into D test PeaksExports sample Project Identification DENE Browse The export options are grouped into HTML Report and Text Formats based on the output format HTML Report This will generate a summary report in the specified location After the completion of exporting 1t will be opened in the default browser automatically The following exporting options are available 123 Exporting Data Reports and Printing e Result summary The Summary view page will be saved as summary htmi file in HTML format in the specified location Text Formats The following exporting options are available in various text formats De novo peptides The peptides identified by de novo sequencing will be saved in de novo peptides csv file in Comma Separated Values CSV format in the specified folder De novo peptides pepxml In addition to CSV format the peptides can be saved in pepXML format e All de novo candidates All de novo candidates for each
87. aw file convertor ABI wiff 2 Select General in the in the Preference dialog and click the Browse button below Default Project Folder to specify the default location 34 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 the TIC and all the scans The total ion chromatogram TIC is displayed on the left of the MS view The navigation buttons are circled in the figure To collapse the TIC chart click the left navigation button To navigate the survey scans use the up and down navigation buttons The survey scans can also be navigated by using the up and down arrow of the key
88. ble is almost the same as the peptide table in the Peptide View except that three additional columns are added Start the start position of the peptide in the protein End the end position inclusive of the peptide in the protein Checkbox this allows you to control which peptides appear within the Coverage Tab as blue bars Unique whether this peptide is unique to the current protein group Additionally the peptides from the protein and below the user specified score threshold are also displayed in the table but in a grey color Although their correctness is questionable they are worth examining once an interesting protein is confidently identified by other high confidence peptides 3 3 4 De novo Tags Tab The De novo Tags tab displays de novo tags from the De novo only View that can be loosely matched to the selected protein via five amino acid seed matches 1 e this displays de novo peptides that pass the confidence thresholds set in the filter pane yet are not confidently matched to peptides identified via any PEAKS database search tools for the protein in question 81 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 3 4 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 v
89. board Clicking on a specific position in TIC will display the corresponding survey scan The tandem scans associated with the current survey scan are shown in the bottom right panel 35 Data Visualization 2 TIC Intensity 9 5 j e 100 525 18 a m z e T TIC 665 21 550 20 c 426 14 Survey Scan co 50 e A e 791 15 cn Lm A miz o 500 1000 1500 2000 anne nuu 070828 O1 03 070824 DMSO 0h 2 RAW ms 1RT 7 7329 a Ji 1 1 2X 2Y ErrTol low intens label 252 T1C 2 64E7 449 12567 2 e Intensity 9 5 100 431 80 co e wD e Tandem Scan S 50 o o e M miz a 100 200 300 400 500 600 700 600 070828 O1 03 070824 DMSO Oh 2 RAW ms 2 RT 0 113 0 ow intens label SE ES 0 0095 119 9959 min Ma ylh 1 1 2x 2Y ErrTol A A gene ge ein The survey scans and tandem scans provide a few convenient way to zoom and navigate in the spectrum 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 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 Increase the peak intensity place the mouse pointer in the spectrum scroll the mouse wheel button 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
90. cid 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 The other PSMs can be examined by selecting the peptide See Section 3 4 2 Peptide Spectrum Match for details e 10lgP The peptide matching score Mass The theoretical mass of the peptide including the H2O but not the extra proton for the 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 e Spec Number of spectra assigned to the peptide Accession The accession number of the highest scoring protein containing this peptide 82 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER e PTM PTMs are indicated by colour coded icons e Found by the peptide is found by which PEAKS DB 3 4 2 Peptide Spectrum Match For each peptide the Peptide Spectrum Match shows the peptide spectrum matching details Scan F3 1708 m z 977 1248 z 3 RT 31 18 10lgP 79 77 ppm 2 3 1 mee enD D R a p r a x v 1 e p w op v z s sx r x e 11 H20 11 2 b23 H20 1262 65 ers Y7 b23 b7 ay 2554 21 b14 b22 804 28 b10 Y13 1667 71 b17 2541 14 b24 1116 51 1537 72 2024 84 2782 31 50 18 A 2126 07 12
91. ct a project sample or a fraction node Click the Data Refinement button a on the tool bar o Hlg C 4X SQW 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 to complete A new Data Refinement node will appear at the project tree Later analysis on this fraction will be based on the refined data PA Project View E js C Users bshan PeaksProjects Mew Project 3 A Sample 1 y data0109172008_TP10_25fm_BSA_OLRAW de DENOVO 2 30 Mar 11 11 14 i Le aTa 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 a The Data Refinement Parameters dialogue window will appear 46 Data Refinement FY Data Refinement Data Refinement Predefined parameters default Merge Scans Retention time window for raw files only Precursor m z error tolerance Merge CID and HCD scans together Correct Precursor Mass only j Mass and Charge states Min charge 1 4 Max charge Filter Scans Only keep scans satisfying Precursor mass between Retention time between inii min Quality value greater than suggest 0 65 Merge Scans Retention Time Window It defines the maximum retention time difference betwee
92. d Configuration From the Configuration window select Labeled Q Method from the left hand side menu to change the labeled Q method configuration R ITRAQ 4plex lil iTRAQ 8plex Quantification Method Detail Mame TRAQ Aplex Method Type Reporter Ion Quantification Modification Target N Terminal Modification C Terminal Modification Side Chain Modification at Modification Mass 144 10207 Label Options Name Reporter lon Mass Da 114 114 1107 115 115 1077 116 116 1111 117 117 1144 The built in labeled Q methods are listed in the methods list Select a method from the list to view the detail information in the Quantification Method Detail panel Methods with the R icon beside the name are reporter level methods Methods with the P icon beside the name are precursor level methods Name and Method Type Name and the type of quantification method The method type can be Reporter Ion Quantification or Precursor Ion Quantification Modification Target The modification target and mass of the unfragmented modification for Reporter Ion Quantification The modification targets can be N terminal C terminal or Side Chain Label Options List of quantifiable labels For reporter ion quantification methods label options contain name and reporter ion mass and for precursor ion quantification methods label options contain sample name modification and modification details Create a New Method To create anew labeled Q m
93. d in features csv file in Comma Separated Values CSV format To export summary of detected features press the Export button in the title bar of the Summary view panel The following export dialog will appear HTML Repart Text Formats Text Formats Result summary summary html Detected features features csv Save into D test PeaksExports LABEL_FREE_ OW Save into D MestlPeaksExports ABEL FREE 7 Select the output location and click the Export button to save the selected result components to the specified location 128 Exporting Data Reports and Printing 6 Export inChorus Result The inChorus exporting function contains the same exporting options as PEAKS DB Unlike PEAKS DB the exported results will contain inChorus scores and individual search engine scores for the supporting peptides of the protein identifications See Section 4 Export Database Search Result for details 129 Chapter 16 Advanced Configuration and Environment Preferences 1 PEAKS Environment Preferences This section describes the settings of the environment preferences including general raw file converter search engine and spectrum annotation configurations 2 ae To begin click the Preferences toolbar icon or select Preferences from the Window menu to open the Preferences dialog Select the preferences category from left to view the options available for that category Preferences
94. d that X Tandem only be used with small databases If used with a large database a taxon should be specified For example NCBI nr and SwissProt databases should be used with sub taxa selected when using X Tandem 141 Configuration and Preferences e OMSSA At the time of writing OMSSA cannot be used with databases that are not in NCBI nr or SwissProt format in a way that is available to inChorus Also a bug in OMSSA prevents database use when 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 does not work as it contains a space between My and Documents Using spaces in the database file name causes the same problem Once the databases have been downloaded and extracted save the database as ncbinr fas Or ncbi_nr fas rather than ncbi nr fas Mascot The database used by Mascot will have to be identical to the database configured in PEAKS in order for inChorus to parse Mascot results correctly 2 5 Instrument Configuration From the Configuration window select Instrument from the left hand side menu to change the instrument configuration Instrument List lt Built In gt Orbitrap Orbi Trap lt Built In gt Orbitrap Orbi Orbi lt Built In gt Triple TOF lt Built In gt Q TOF lt B
95. databases to be configured A small description of the database is displayed once selected If a standard database is already configured using this wizard then configured text in green will appear beside its name in the list It can be selected again to overwrite the configuration 21 Configuration Wizard Configure Instruments and Public Databases Configuration Wizard Select Databases E NCBI nr configured E UniProtKB Swiss Prot configured E UniProtKB TrEMBL 2 IPI IPI provides a top level guide to UniProt Vega Ensembl RefSeq and TAIR databases IPI human database provides minimally redundant yet maximally complete sets of human proteins The Configuration Wizard will download extract and configure the database Database Path D PeaksStudio6FastaDiB The Database Path displays the location where the configured database will be stored Click the Browse button to change the default database path Click Back to go back to the instrument selection panel Click Next to proceed to the download information panel 4 Download Information The download information panel displays all the requested instrument software and database downloads in two tables Downloads and Selected Instrument Software 22 Configuration Wizard Configure Instruments and Public Databases Configuration Wizard Download Information Downloads Thermo MSFileReader 251918 Sonrioaded at
96. date to the PEAKS defaults 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 the m z value on top of the annotated ions m z on Unannotated Select this to display the m z value on top of the peaks without ions sequence fragmentation Select this to display the 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 Set the intensity threshold for spectrum annotation to low 2 medium 5 or high 10 To apply this intensity threshold select the intensity threshold checkbox in the Spectrum Annotation view see Section 3 2 2 Spectrum Annotation 2 PEAKS Configuration This section describes the configuration of enzymes PTMs databases instruments and parameters To begin click the Configuration toolbar icon t or select Configuration from the Window menu 2 1 Enzyme Configuration PEAKS can use almost any enzyme or combination of enzymes in your analysis Select built in enzymes from the extensive list provided in PEAKS or define a new one From the Configuration window select Enzyme from the left hand side menu to change th
97. de view displays all the identified peptides and their intensities The interface 1s similar to the peptide table in a PEAKS DB result The intensities of the quantifiable peptides are displayed in the intensity columns with sample names incorporated into the header e g Heavy The peptide quantification ratios can be displayed instead of peptide intensity by selecting the proper option from the Display sample as drop down list at the top 1 12ofi2 y 9 Display sample as intensity v v scan serd QUA noresults E 3 Peptide Score 10lgP Mass ppm m z RT Scan FSpec Accession Light Heavy PTM c 1 VANPSGNLTETYVQDR 1762 8485 1 P21333 FLNA HUMAN 311991 844 715805 938 2 2 TGVELGK 6 02 PTHFTVNAK 6 02 100 0 62 55 1709 9502 8 9 855 9900 0 391 38 1 P21333 FLNA HUMAN 480824 062 622911 875 ss a 3 VEPGLGADNSVVR 99 9 45 91 1311 6782 3 3 1312 6899 1 225 102 1 P21333 FLNA HUMAN D WGTSGLVGR 99 9 42 26 931 4875 2 2 466 7500 0 893 72 1 Q69ZN7 MYOF MOUSE 152362 000 166322 641 a 5 YGGQPVPNFPSK 6 02 99 5 37 52 1295 6605 3 9 648 8400 0 289 27 1 P21333 FLNA HUMAN 1537743 500 1733465 000 s 6 DVDITDHHDNTYTVK 6 02 84 7 22 22 1789 8578 4 3 895 9400 0 296 28 1 P21333 FLNA HUMAN 294939 750 353419 406 s 7 DVDIIDHHDNTYTVK 84 0 20 96 1783 8376 0 3 595 6200 0 886 71 1 P21333 FLNA_HUMAN 967378 812 1157391 250 8 DATALDR 36 5 11 37 760 3715 67 3 761 4300 0 961 79 1 Q1IHA5 PANB
98. dence score filters 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 the Apply button The result will be updated in the Summary view and the De novo view accordingly StartPage X ij DENOVO 3 13 Jun 12 10 33 X Pe E ET Summary 59 Peptide De Novo Sequencing Note Whenever the score threshold is changed the Apply button will be highlighted in red to remind you that the change has NOT taken effect yet 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 top de novo peptides a pepXML file and all de novo peptides This provides the opportunity to supplement the results in a publication or put up the results on a website To export the filtered results Click the Export button at the top of the Summary View Different file outputs can be chosen from the resulting dialog 2 Click Browse and a file chooser will appear 3 Choose the location and directory name to put the exported files Click OK This will create a collection of files in the target directory which are also indexed by an html file Refer to Section 3 Export De Novo Result for details 6 Run Auto De Novo Sequencing on a Single Spec irum To perform auto de novo sequencing on a single spectrum select the spectrum in th
99. der eg 114 5 1 5820f582 v mM wscan search Q6 Q noresul 3 Peptide 109P Mass ppm m z RT Scan Spec Accession 114 115 116 117 PTM i 1 D 144 10 FNVGGYIQAVLOR 98 23 1709 8838 3 4 855 9520 62 65 4341 E E col 1 0 759 0 853 0 6 AE O CT O E E RETI amp 3 I 4144 10 EFELGDNAVFAGENFHHGDK 144 10 L E 2728 3293 3 1 910 4532 49 56 2981 eT VEAST 1 2 4 144 10 NGVFQEC 57 02 C 57 02 QAEDK 85 88 2034 8977 47 P02769 ALBU BOVIN 231 amp 5 A 144 10 TDGGAHGVINVSVSEAAIEASTR 2455 2405 2 5 819 4228 47 95 2790 PO0330 ADH1 YEAST 1 amp 6 m 144 10 PC 57 02 TEDYLSLILNR 84 46 1867 9274 2 6 934 9734 60 14 4103 P02769 ALBU BOVIN 5 993 Se an ERE POE 0 a s 1 526 vera iopaustPnivevsk 8358 16549355 32 828 4777 48 51 281 POZISSIABU BOVIN 5942 Note Select a peptide and zoom to the reporter ion region of the MS MS to examine the reporter ions 3 4 Filtering Quantification Result The Quantification result can be filtered based on the number of fold changes in proteins You can set the ap propriate values for the filters by changing the filtration parameter values from the drop down lists in the title bar of the Summary view panel and clicking on the Apply Filters button The result will be updated in the Summary view the Protein view and the Peptide view accordingly The intensity columns of the Protein
100. down list There are also options to delete the current set of parameters or to save current changes To examine the contents of another set of saved parameters select a predefined parameters set and the values will be displayed 3 Understanding PEAKS De Novo Sequencing Results 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 viewable 3 1 Summary View The Summary view performs three main functions Result filtration This is achieved by specifying the filtration rules in the area at the top of the summary view The filtration function is discussed in Section 4 Filtering De Novo Sequencing Results 2 Result exporting This is achieved by clicking the Export button at the top of the summary view The exporting function is discussed in Section 3 Export De Novo Result 3 Summary report Several statistical charts assist in obtaining an overall picture of the results and assessment of the result quality This is the main purpose 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 in
101. e 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 7 2 Manual De Novo Operations When the mouse cursor is placed in the Spectrum Annotation panel a green by default triangle 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 Intensiky 951 100 1585 79 13 5 71 1258 56 1150 64 50 1049 6 912 52 226 12 246 18 738 56 1031 40 ra 1489 76 T ell A Lal y Lid miz DO 00 go 200 1000 1200 1400 1600 Orbiample mzxXML msz2 mzzB02 90607 da ili 1 1 ZX ZY ErrTol Q 5Da intensity threshold 2 2RT 0 0729 TIC 1 79E7 1 260 5649 OLI 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 62 Peptide De Novo Sequencing Fe 1 Inbensity 7o 1585 79 1375 71 S 1049 6 226 12 245 18 749 46 1604 81 1459 76 1031 49 200 400 600 800 1000 Ak y 1400 ell zal es ey Hines Urnmsample mzXML me mz 802 90607 a wy 1 1 2X 2 ErrTol 0 5Da F intensity threshold A MEE
102. e License Request File Email confirm youremail yourcompany com Upinad License License Key YOUR LICENSE KEY Request File Important You will receive your license file via email Import License Activation Complete 2 Save a generated request file to a removable storage device e g a USB memory key 15 Manual Activation Gather Information Save License Request File Upload License Request File Import License Activation Complete Installation and Activation Click the button below to save the License Request File to directory e g an USB drive so that it can be later transfered to a computer that has internet access Save License Request File 3 From another computer with an Internet connection upload the license request file to BSI s license server as described in the following screen Manual Activation Gather Information Save License Request File Upload License Request File Import License Activation Complete The following steps should be done on the computer which has Internet connections Go to http www bioinfor com 1cs20 findex jsp Select the option I have the license request file I want to register the software and click next Click Browse button to select the license request file that has been copied to this computer type in the visual verification code and click next Now the software is being registered If you get a Operation completed
103. e MS MS view of the sample and click the right button of the mouse to display a pop up menu Select the PEAKS Auto DeNovo command from the pop up menu OrbiSample mzXML ln DENOVO 7 08 Mar 11 11 00 la MARL To Dua RA E 47 53 PEAKS Auto Denowo un 5 Correct Precursor Mass and Charge D Export As DTA T Export As PKL 7 Manual De Novo Sequencing PEAKS 6 provides a set of tools to help manually sequence a peptide using graphic cues from the spectrum Note Manual de novo sequencing does NOT support ETD spectra 7 1 Manual De Novo Graphical User Interface To create a new peptide candidate for manual de novo sequencing select the m z value in the Result Panel and right click to bring up a pop up menu 60 Peptide De Novo Sequencing Orbisample maXML_ x Mew Candidate for Manual De Novo tj 802 90507 E 695 34015 i Remove the selected Candidate 507 30252 E Config Error Tolerance in Manual De Nowo ie 547 59247 1 Config PTM in Manual De Novo fe 820 8849 RI H 798 0384 R da fe 582 81006 1 Redo eer 100 Add new sequence Can t Save amp gt 733 2819 Ri Select 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
104. e enzyme configuration Enzyme List lt Built In gt Pepsin pH 1 3 lt Built In gt Pepsin pH gt 2 lt Built In gt Proteinase K lt Built In gt Trypsin lt Built In gt Trypsin with DIP lt Built In gt None Enzyme Details Enzyme Name Trypsin Cleave Sites X all amino acids after RK and notbefore P or after and before or after and before or after and before new Addjupdate Delete Heb 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 135 Configuration and Preferences Note A built in enzyme cannot be deleted or edited Create a New Enzyme Provide the name of the new enzyme in the Enzyme Name field and specify how the custom enzyme will cleave the protein between two amino acids to create peptides in the Enzyme Details panel The letter X denotes any amino acid in this position while set brackets indicate any amino acid 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 Add multiple amino acids to indicate that cleavage happens before or after any of the stated amino acids For example after RK means after R or K not after R and K Click the Add Update button to save the changes The new enzyme will n
105. e how to create a PEAKS project from raw data and conduct data analysis Sections 4 5 4 6 4 1 Open an Existing Project The installation of PEAKS can be found at Chapter 2 Installation and Activation After installation and running PEAKS you can open the sample project by one of the following two ways see screenshot below 1 If this is a fresh installation click the Sample Project in the Recent Projects list of the Start Page 2 Click the open project button and browse to the directory where PEAKS6 was installed select SampleProject and click the open button in the file browser Overview F FJ PEAKS Studio e O A o File Tools Window Help BH i bET KX 00y A 2 F Project View Start Page X h gt gt gt Click to open a sample project lt lt lt 4 2 PEAKS Main GUI The main graphical user interface GUI of PEAKS is divided into several areas see screenshot below The project tree shows all the opened projects Each project may include multiple samples and each sample may include multiple fractions LC MS runs The analysis results are also displayed as result nodes under the project 2 The menu and toolbar Selecting a node project sample fraction or result in an opened project will highlight the common analysis tool icons available to the selected node 3 A result node in a project can be opened by double clicking the node All opened result nodes are shown here as differe
106. e mode only displays protein sequences and confident PTMs and mutations To show the MS MS data evidence for a specific position left click on the amino acid at the position or the PTM and mutation above the position All the identified peptides which cover this position will be shown as blue bars under the protein sequence Cursor over these blue bars some details of the identified peptide will be shown confident PTMs and mutations are shown in bold font Left click on the blue bar a window will pop up to show the spectrum matching information for that peptide Right click on the blue bar to show the pop up menu for some quick operations Remove peptide operation will hide the selected peptide to restore it check the corresponding checkbox in the peptide tab Sometimes there are also some grey bars shown below These grey bars are matched de novo only tags Left click these grey bars to check matching details Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER d 1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD 1 E e e s 101 d 116 144 154 d s o Al a A DDSPDLPKLK PDPNTLCDEF KADEKKEWGK KVLT sub A SSARQR Scan F3 523 m z 573 3395 z 2 RT 12 06 10lgP 224 06 ppm 2 7 by PEAKS PTM 214 Intensity 9 5 K v r v s s A R o n m 100 iso A AROR LRCASIQKFG ERALKAWSVA Y8 NH3 901 48 TI 1 spectrum 10lgP 24 06 1127 66 Seq KVLT sub A SSARQ
107. e percentage score is calculated in accordance with the empirical calculation used in Peptide Prophet Keller et al Anal Chem 2002 74 5383 92 The method of PeptideProphet M 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 Protein Score The protein score is also a percentage score It s calculated as follows The 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 94 PEAKS InChorus 1 280f28 y gt wscan search OQ O noresul E 5 Peptide Scan m z RT Mass ppm Spec Score PEAKS S SequestlE MascotiJl X Tandem Omssa a PTM ci 1 ELIGE 533 365 8648 24 04 1094 5720 0 6 2 99 96 HSK EA 273 40 92 o 7 31E 5 2 2 ASEALKPDSQK 194 391 8754 18 59 1172 6038 0 5 8 99 95 BI SIE S 56 42 2 43
108. e tecum heec E mme 59 3 23 45 PECUARIO dehinc ut Mure Ne pt ba a EE Ne A Qc ues A vals wares 59 3 2 0 SA o uitodibalfaafte cado uitam utu aene 59 4 Iilterme De Novo Sequencing Results i oii eate oe ds 59 Do XDOD DE NOVO RES e aber ossa ue ed o ad 60 6 Run Auto De Novo Sequencing on a Single Spectrum sss 60 T Manual De NOYOSSeQqUETCITIE ceno bias vat de 60 7 1 Manual De Novo Graphical User Intern ace eiit teak re ib Ee Lea rea ted 60 722 Manual De Novo Operations iS A ete imi qutbus LUE Dm SUI nS Ua eeeut 62 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM SPIDER eese 68 US CIVIC T 68 2 Set EEAISS Para eltets nai aaa 69 3 Understanding PEAKS Database Search Result ooococoocoonocconococcnnnoccnnanancnnnoconcncncnnnnnnns T2 3 J The Peptide and Proteini SCOre S at AR ESE SAA 12 CSV SU A AI E m TU UTEM 12 LOC VIEW CT 76 S 9 Lo AA Table oeeosest eoe ici JT 39 957 LONE e Dab a one EA ne Re camel Maen Dice di Ly 3 95 95 Peptides Da editae pde oai tale onmia ul t ofi oia bous e aded abe 81 3 94 De novo Tae s Labio iii ii 81 JAPE PUJE An ii ei cio da ios ei eee ms wala 82 Acs Peptide Fable cra A A bu E 82 34 2 Pephide specum Mati ls si 83 3 5 DENOVO ODIY VI ic ai 84 A EME Were ARS RC SUID a dados 84 SXBxport PEAKS Results Tor Publica Om A A A Men diae beds 85 6 Running PEAKS PTM and SPIDER Separately ott orte e a ds 86
109. earch tool san sexch AI 0 noresuls b scan seq contains RT PTM Am z Note To search with an approximate mass value type only the necessary number of digits after the decimal point 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 56 Peptide De Novo Sequencing Sequence DVGGLPK TLC 6 1 ALC 87 ppm 38 7 All candidates Intensity 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 600 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 which displays 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 28795 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 46 76 551 DVNLPK 75 38 7 VDGGLPRk 64 38 7 mear as 64 18 Y2 H20 226 16 b2 NH3 b2 H20 197 39
110. ed de novo sequencing is outlined below Details of each step can be found in later sections of this chapter La Select a project a sample or a fraction on the project tree Click the automatic de novo toolbar icon or select De novo from the Tools menu File Tools Window Help 5 H a Y X daw KO 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 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 FN Project View Mg D test Peaks Peaks Projects denovoProject3 Sample 1 OrhiSamalos zh iiy DENOVO 2 24 Mar 11 14 27 T L ILL 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 49 Peptide De Novo Sequencing liy DENOVO 3 04 Apr 11 14 01 X A Summary Denovo TLCz 5 y amd ALC
111. elated 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 this makes spiking unnecessary Label free quantification is based on the PEAKS DB results See Chapter 9 Peptide PTM and Mutation Identi fication PEAKS DB PEAKS PTM SPIDER The use of this function is outlined in the following overview l A Select a 4 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 Q parameters in the right panel of the window and click 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 S
112. elect 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 107 PEAKS Q Label Free Quantification Basic Options Mass Error Tolerance 0 2 Peptide Score Threshold 10logP Retention Time Range 3 0 in Protein Score Threshold 10logP 20 Upper Bound of Precursor Charge do normalization Parameter Table Fraction Add to EC Pme puni SE e Mme C IE BR CO M eee AAA HA M Rh E eme II E A 070828_01 040 pEMS 11D a ml III IS GN RN O RIA PES BPR SSA O NEO A or oo PEAKS TET AAA A O HA fr 009 Peas alar menm 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 PEAKS analyses with non centroided survey scans in LFQ experiments So set the mass error tolerance a little wider than the parent ion error tolerance in PEAKS DB Retention Time Range The maximum elution time range that is considered for the quantification of an iden tified peptide This also defines the search range for peptide feature pairing across samples Upper Bound Precursor Charge The precursor ion of an identified peptide may produce sibling ions of different charge states Only those sibling ions with charge le
113. em v2010 12 01 1 and OMSSA v2 1 8 PEAKS inChorus uses uniform FDR to combine multiple engines results For this step familiarity with PEAKS database search tools Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM SPIDER is recommended 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 phy Select a project node or a sample node Click the PEAKS inChorus button on the tool bar PU s sucio O File Tools Window Help ae LF A 2 If a search engine s result exists in the current project select it from the dropdown list of that search engine If the result is in a separate file select Import from the dropdown list Otherwise select New Run from the dropdown list of each engine to be used Specify the search parameters for each engine in the parameter dialogs that will pop up automatically Each engine s parameter setting interface in PEAKS is kept very similar to their native interface Please refer to third party softwares user manuals for how to use them For PEAKS database search tools refer to Section 2 Set PEAKS Parameters Important The results of the other search engines should be based on the same refined data node in order to do inChorus Mascot Please select Sequest Please select XiTandem Please select OMSSA Please select Engine Precursor Error Fragment
114. encoding may cause the activation to fail 3 If the computer is behind a firewall or has other internet connection problems the activation may fail Please follow the on screen instructions or refer to Section 4 4 Activate PEAKS manually 5 PEAKS Performance Configuration The PEAKS Performance Configuration tool can be accessed from the Windows Start Menu By default PEAKS automatically determines its performance parameters to take full advantage of the processors and memory avail able on the computer In most situations the Automatically Configure PEAKS Performance option should be used For advanced users the Manually Configure PEAKS Performance option will come in handy in situations where users want to start PEAKS using different JVMs or to change the size of JVM heap to their preferred configuration 18 Installation and Activation ah Performance Configuration Manually Configure PEAKS Performance ra mM rein o FEO ren C 1 Main Program JVM Heap Size MB This is the amount of memory assigned to the main PEAKS program In some computers more often on 32 bit systems if PEAKS fails to start lower this number e g 800 may help 2 Number of Computing Nodes to Start This option determines the number of concurrent processes PEAKS will use One PEAKS license will allow up to four computing nodes to be used The more nodes started the more memory PEAKS requires It is important not to start more nodes t
115. ers Database Mascot Database SampleWithDecoy Taa Local Database SampleDB Set View Tana Enzyme Enzyme Trypsin Alowupto 0 missed cleavages PTH Fixed Carboxymethyl C Modification Acetyl K gt Acetyl N term Acetyl Protein N term Amidated C term meum Amidated Protein C term Ammoniatoss N term C Biotin K Variable Deamidated NO Modification Oyidation M lt Biotin M term Carbamidomethvl 0 Display all modifications Error Tolerance Peptide Tol 20 ppm Pc 0 MS MS Tol 0 8 Da Peptide Charge 2 34 and 4 Monoisotopic Average Misc Instrument ESI TRAP v Reporttop AUTO hits OK Cancel 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 Database Search 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 Th
116. es for more details 3 2 3 lon Table The Ion Match tab at the bottom panel of the de novo view contains the Ion Table that shows the 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 as defined in the de novo sequencing parameters and the intensity of the ion must be at least 2 of the most intense ion The ion types displayed in the table are controlled by the same configuration as the spectrum annotation econ 322125 Septum BOISORBON b 1 2 230 08 O N 4 lass 19 x 18 438 16 1278 69 1260 56 1261 66 538 23 535 20 T i 3 pr ess mes sess om sss ese 1015 a3 1001 43 1002 40 Y 749 46 x 45 0x9 17 57 08 ae 1189 53 1171 51 1172 50 V 515 35 ipm A 416 29 1359 66 1341 62 1342 62 V 345 24 zu 17 DE ha 1 LX ear 239 1998 11 Clicking the header of a column in the Ion Table highlights the corresponding points on the error map and peaks in the spectrum annotation 58 Peptide De Novo Sequencing 3 2 4 Error Map The Error Map shows the mass errors of the annotated ions and is displayed
117. es 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 result See Chapter 9 Peptide PTM and Mutation Identi fication PEAKS DB PEAKS PTM SPIDER Ensure that you specified the isotope labels as PTMs when you performed the database search After database search is complete 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 Precursor Ion Quantification and specify the PEAKS quantification param eters in the dialog and click OK ra Wait for the analysis to finish A new quantification result node 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 ratio and standard deviation columns are 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 Precursor Ion Quantification eg SILAC
118. es that the wizard is going to make will be displayed Click the Finish button to commit to the changes to the configuration file The changes will be immediately available to PEAKS 24 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 node in the top left corner 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 on the toolbar The following New Project dialog will appear This is 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 enzyme fragmentation method and instrument type Clicking the Copy to whole project button will let the whole project share the same settings of enzyme instrument and fragmentation FY New Project Project Name My PEAKS 6 project Project Location D ltemp Data Files Sample Details EY My PEAKS 6 project E Jl Sample 1 Sample Name Sample 1 i c Enzyme Specify later Hi n
119. ethod click New button to open New Edit Quantification Method dialog where the quantification method details can be specified Specify the modification target modification mass and label options for a Reporter Ion Quantification method Use Add Label and Delete Current Line to add or remove a label Each label is defined by the sample name and reporter ion mass Use Add row and Delete Row to add or remove a label for a Precursor Ion Quantification method Each label is defined by sample name added mass target residue and labeling efficiency If one sample has multiple labels with different mass shifts a user can add multiple labels with the same sample name These labels will 139 Configuration and Preferences contribute to the same number in the ratio The modification for each label must be selected from the PTM lists which can be accessed by clicking the button in the Modifications column 2 4 Database Configuration To use the PEAKS DB function to search through a database to identify proteins PEAKS must have access to a protein or EST database in FASTA format the standard format for popular public sequence databases PEAKS can be configured to use existing databases on the system or download from servers Additionally taxonomy may be specified with certain databases From the Configuration window select Database from the left hand side menu to change the database config urati
120. f PSMs containing this PTM are listed The checkbox on the left controls whether to show this PTM or not Double clicking on the PTM name will show the detailed information about the PTM Right clicking on the PTM a pop up menu will show up to allow some quick operations Tools bar Tools bar is at the upper right corner of the protein sequence display area It has two icons on it 80 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER B8 78 de novo only tags sharing 6 AAs 35 confident PTM min ion intens 30 AAs per line 4 10AA gap AM PTM DE 57 02 Carbamidomethyla 1571 PIE 57 02 Carbamidomethyla Fi i 40 98 Deamidation NQ WEB 18 01 Dehydration 61 e Full screen Left click on this icon enlarges the coverage tab to full screen mode Click to exit the full screen mode Tool box PS Tool box contains the following tools e Copy template protein Copy the original database protein into the system clipboard e Copy mutated protein Copy the protein with detected mutations into the system clipboard e Save coverage tab as image e Coverage statistics e NCBI BLAST search e NCBI Entrez search e Multiple sequence alignment Multiple sequence alignment for selected proteins 3 3 3 Peptides Tab The Peptides tab displays the supporting peptides assigned to the protein The ta
121. formation are given here In the rest of this section we discuss the charts in the summary report are discussed 53 Peptide De Novo Sequencing Denove nc2 3 pland acce E y 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 3504 300 250 200 1504 1001 501 number of PSMs ppm 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 result Table 2 Result filtration parameters of MS Scans 4233 De Novo ALC 30 of MS MS Scans 9236 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
122. from the left hand side 98 PEAKS Q MS Level Quantification Precursor Ion Quantification Save as Basic Options Mass Error Tolerance 0 2 Da Upper Bound of Precursor Charge Reporter Ion Quantification l eg iTRAQ TMT Retention Time Range 1 0 min Peptide Score Threshold 10logP Precursor Ion Quantification E AM 2 eg SILAC Select Method Quantification Method Detail SILAC C 6 Name SILAC C 6 Method Type Precursor Ion Quantification 7 Label Free Mew Label Options Sample Mame Modification Modification Detail SILAC K6 6 0201d8 K 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 In a SILAC or ICAT experiment we are usually dealing with non centroided survey scans therefore the mass error tolerance should be set a little wider than the parent ion error tolerance used in the PEAKS DB search Upper Bound Precursor Charge The precursor ion of an identified peptide may produce sibling ions of different charge states Only those sibling ions with a charge less than the upper bound precursor charge will be considered for quantification of the identified peptide Retention Time Range The retention time range is the maximum elution time range that is considered for the quantification of an identified peptide
123. h After database search is complete 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 Reporter Ion Quantification and specify the PEAKS quantification pa rameters in the dialog box on the right and click OR rs 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 The quantification results with labels are displayed in a format that is similar to the PEAKS DB result A ratio is added to each quantifiable peptide and protein along with standard deviations for protein ratios 2 Setting Parameters The following parameter dialogue pops up when clicking the quantification tool bar icon Select Reporter Ion Quantification eg 1TRAQ from the left hand side under the Tools heading in the quantification window Enter the relevant MS MS level labeling quantification parameters on the right hand side of the window 102 PEAKS Q MS MS Level Reporter lon Quantification default Save as Basic Options Mass Error Tolerance 0 2 Da Peptide Score Threshold 10logP Reporter Ion Quantificati i Select Method Quantification Method Detail E Precursor Ion Quantification
124. han the computer can handle On 32 bit systems this number should always be set to one On 64 bit systems a safe estimation on the number of nodes can be calculated as min 4 RAM in GB 2 For example on the Windows 7 64 bit computer with 6GB RAM the maximum number of nodes that can be used is min 4 6 2 3 3 Start Client Separately This option provides the raw file reading abilities of a 32 bit JRE and the processing power of 64 bit system together Use the Browse button to point to the bin directory of an installed 32 bit Java Runtime Specify the Client JVM Heap Size MB to assign memory to PEAKS Client components Usually 1024 works fine 4 Start Compute Node Separately This option should be used on 64 bit systems when the Number of Computing Nodes to Start is greater than 1 Use the Browse button to point to the bin directory of an installed 64 bit Java Runtime Specify the Computing Node JVM Heap Size MB to assign memory to each node Usually a number greater than 1500 should be used Important The total amount of memory used by all the nodes computing nodes are Computing Node JVM Heap Size MB multiplied by the Number of Computing Nodes to Start 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 in
125. he score threshold for each search engine automatically to reach the target inChorus FDR Slight differences may exist between the final inChorus FDR and the target inChorus FDR Peptides Target Inchorus FOR 1 Edit filters Proteins Score 55 50 and 0 unique peptides De novo Only TIC 2 3 and ALC 50 Apply Filters Export Notes z m E E 3 un E D Click the Edit filters button in the Summary View to specify the peptide filtration rules A peptide is kept as long as one of the specified rules are satisfied Edit filters PEAKS 10lgP Sequest Xcorr Mascot Score x Tandem e value Omssa e value 96 PEAKS InChorus 4 Exporting inChorus Result The inChorus result exports contain the same export options as PEAKS DB Unlike PEAKS DB the exported results will contain the inChorus score and the search engine scores for the supporting peptides of the protein identifications Refer to Section 4 Export Database Search Result for details 977 Chapter 11 Precursor lon Quantification e g SILAC and ICAT 1 Overview Precursor ion quantification with isotope labels at the MS level is one of the three quantification modes that are supported by the optional PEAKS Q module 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 sampl
126. he search parameters the score threshold for peptides can be easily chosen by clicking the FDR button An FDR curve will pop up Move the cursor along the curve When the desired FDR is reached right click and select Copy score threshold or simply select a predefined FDR value 84 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER Score selection l lgP 25 9 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 number af peptide spectrum matches If the FDR estimation is turned off then an empirical threshold is needed Usually a score of 20 is a good choice At 10lgP 20 the equivalent P value is 0 01 Note P value and FDR are two very different concepts In PEAKS DB P value is defined as the probability that a false identification in the current search achieves the same or better matching score A 1 P value does not automatically correspond to a 1 FDR For more details please see http www bioinfor com peaks tutorials peaksdbscore html Proteins 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 least one unique peptide is needed for a protein group The thresholds here do not affect the peptide and de novo only v
127. i db protein amp val2 lt Accession ID gt Delimiter s Taxonomy Options end oe Follow these steps to configure a database 43 Adding a Sequence Database Select the database format from the FASTA Format Database drop down list or select Other if the desired format is not present If Other is selected you must enter custom parse rules In the Basic Options section enter a name for the database If the database FASTA file is already on the local system skip to step 6 Otherwise select Download A window will appear confirming the database chosen to be downloaded from the appropriate FTP or website Click OK to invoke the default FTP client software and download the database automatically Click Cancel to copy the URL to the system clipboard If Cancel was 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 or WinRar The desired result is a FASTA format text file fas or fasta file Move the database file into a directory that PEAKS can access Click Browse to inform PEAKS of the location of the database file If the selected database is an EST database check the box labeled EST da
128. icat Browse Save into b test Peaks60 PeaksExports sample Project Identificat Browse Export Cancel Export Cancel The export options are grouped into HTML Report and Text Formats based on the output format Select the items that you want to export from the available options Select the output location and click the Export button to save the selected result components to the specified location HTML Report This will generate single or multiple HTML reports in the specified location After the com pletion of result exporting the index file for the reports protein htm1 will be opened in the default browser automatically The following exporting options are available Export summary view The Summary view page will be saved as a summary html file in HTML format in the specified location Export protein coverage The coverage pane will be saved for each protein Export supporting peptides A list of supporting peptides will be saved for each protein Export best unique PSM The best unique PSM will be saved for each protein An individual protein will have its own HTML output file where the corresponding protein coverage supporting peptides and the best unique PSM are gathered Select Put all protein details in a single html to collect all protein reports in a single protein html file Text Formats The following exporting options are available for various text formats P
129. ie les ta les je lel 800 ten 15048 so on Bl Moise Level 42 Chapter 6 Adding a Sequence Database 1 Configuring Sequence Databases PEAKS has the ability to search through a database to identify proteins using the PEAKS DB function In order to use this function PEAKS must have access to a protein or EST database in FASTA format the standard format for popular public sequence databases PEAKS can be configured to use existing databases on the system or download from servers Additionally taxonomy may be specified with certain databases To add a sequence database click the T icon in the main toolbar and select Database from the left hand side This will open the database configuration dialog Click the New button to create a new sequence database entry or select a database from the Database List and click Add Update button to edit The database can be configured in the area below the Database Details ah Configuration Database List UniProt SwissProt MCBI nr Enzyme PTM Labeled Q Method Database Database Details FASTA format database NCBI nr 5 Export Decoy DE Basic Options Database name Validated Path or Download EST database Advanced Options Fasta Title Format Rule to parse accession id from FASTA title Mana Rule to parse description from FASTA title za Accession id URL http www ncbi nlm nih gov entrez viewer fcg
130. iew 3 4 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 1 10000f 1253 m scan sa QA O no results Peptide 10lgP Mass ppm m z RT 5can F5pec Accession PTM Found By i T 43 01 VMENFVAFVDKC 57 02 C 101 22 3350 4714 1 9 11178291 37 22 F1 2158 4 PO2769 ALBL BOVIN el PEAKSPT a C 457 02 C 57 02 HGDLLEC 57 0 3 EC 57 02 C 57 02 H 57 02 GDLL C 57 02 FLSHKDDSPDLPKLKPDPNTL 89 03 39127385 27 783 5529 3460 F3 1944 1 POZ7SS ALBU povn IBIS PEAKSDB 8453 1805 6768 L8 903 8441 23 12 FL1160 1 POZ SSIALBU EOVIN BEBE PEAKSPTM 8123 2540 3633 L2 981 1271 28 83 F31552 8 POZ SSIALEU EOVIN B PEAKSDE 5 57 02 ADDRADLAKYIC 57 02 D 79 77 2928 3594 2 3 977 1248 31 18 r3 1708 9 PO27S9IALBU EOVIN BB _PEAKSDB C 57 02 0 57 02 AADDKEAC 57 79 88 1926 7910 2 3 964 4005 24 21 Fi 1245 10 POZ7GSIALEU BOVIN MBE PEAKSDB DYLSLILNRLC 57 02 VLHEKTPVSE 78 76 2998 8003 1 5 833 794 45 55 F3 2611 1 POZSIABUL BOVIN B _PEAKSDB VEKEC 57 02 C 57 02HGD 57 0 76 88 2169 8989 L3 724 3059 20 78 Fi 1000 2 PO2769IALBU BOVIN BIBIBIB PEARSPTA 5 VGTRC 57 02 C 57 02 TKPESERMP 75 31 41640048 3 2 695 0058 4261 F2 2321 8 POZ SSIALBU EOV
131. iews 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 spectra only have database matches with 10IgP 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 empirical thresholds are needed A peptide 10lgP score of 8 is recommended This peptide 10IgP value for de novo only can be locked the same as the threshold for filtering peptides on the first line Or users can unlock this field to set a different value 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 5 Export PEAKS Results for Publication The Export button at the top of the Summary View allows exporting of the filtered results into multiple formats 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 Different file outputs can be chosen from the resulting dialog They are divided into two categories HTML Report Export all the figures together with peptides and protein in web page for
132. in N term PTMs New 10lgP score and FDR control for PTM and SPIDER results FDR control of the inChorus result Different search engines results can now be combined according to a unified FDR for each search engine e Improved precursor mass correction The mass correction function in the data refine step recognizes the real monoisotopic mass even if the instrument s raw data reported the isotope mass The algorithm is improved in the new version GUI Improvements Easier PTM selection interface The PTMs are separated in three common uncommon customized to make it easy to specify the PTMs to search for Additionally a Recent list includes all the PTMs recently used by the user De novo only view added to the inChorus result The de novo only view reports peptides found exclusively by de novo sequencing This useful view is now available to the inChorus result too mproved spectrum annotation view Mouse over an amino acid in the spectrum annotation will highlight the supporting fragment ions Setting an anchor peak will show the mass different between the current peak and the anchor peak 3 Overview Improved project creation interface The selection of instrument type and fragmentation mode is easier Users can specify a proteolysis enzyme for each sample at the project creation interface More searching functions to locate a specific de novo sequence result Now de novo sequencing results can be sor
133. infor com will be sent to you with the trial license key This key can then be used to fully activate PEAKS for evaluation purposes Important Each computer can only have one free trial Request trial license repetitively will not extend your trial automatically Please contact support bioinfor com to discuss a trial license extension 4 3 Use PEAKS as a viewer BSI has consolidated the PEAKS product line PEAKS Studio and Viewer are now one product Unlicensed PEAKS Studio can be used in viewer mode Researchers around the world are now able to take advantage of PEAKS most advanced user interface to share PEAKS results In the viewer mode all non analytical features such as read display raw data open navigate through existing PEAKS results exporting etc work the same way as the full PEAKS Studio version 4 4 Activate PEAKS manually In certain situations when the computer does not have an Internet connection or is behind a firewall that blocks the activation the activation process requires the assistance of another computer with an Internet connection or outside the firewall Manual activation can be accessed via the link on the bottom right corner of the wizard Manual activation consists of the following steps 1 Provide the license key and user information required to the license wizard on the computer that will be running PEAKS vL Gather Your Name Your Mame Information Email Address youremailtyourcompany com Sav
134. ing Quatititication Result ou oto cc 105 A Bxport Quantdireation Results nai ii a 106 ISS REARS Q LabelPre8 iS SS AA QUU E SUL LE 107 D OVervie VW abes ASS ENANA AAA AS agus AA eee DIU Be AUR 107 ZEUS Daran lef S odo ito aa AS es 107 3 Understandine the LEO Result iia iia 109 edhe aS UMN VIEN M E T UL S UU TM 109 3 2 PEOI V ICW aiai is A Mu Cathe enlaces aan auro ud n sud Dada 110 392 L Extracted Ton C MrOimato Mi e d et taie Cade ebat ie ode custo tis 111 SPP Heat Map T T TI 111 I2 MS ZA ds 112 Ded SOLOS SAS SA AA AAA A A ue vacate 112 a Per LEO Rea a 113 S Export Ouantilticadonm Results ida 113 07 Replicate Analysis DEC cie a a bnc desto dba d tes cda 113 6 1 Assign Replicate Number to a Sample nia A o ud dva 113 6 2 Run Rephlicate ANAIVSIS a a tai 115 6 3 Understand the Replicate Analysis Results eese 116 6 4 Export Replicate Analysis Result 5 222525 unma oo ee um qood e Sese um SU tau eSse Cun Sut dautess 118 12 5 WOFPKIIONO ioi der exceed eV aute ecd c cies ela RA NA daa af cubo cu AAA cds NENA 119 T Identification WoOPRITOW soiseid aenn enean ate besote oss b E besufta Dd estin fn bove fit uu 119 2 Quantification WOEKRTIOW nina ia LER tue bbb ove Cool va ees ev Miadwhacsbhincd Lal beue Uer eve Madden 120 3 MAC NOTUS W OKON 5c aseso otra io tob etd cl dd e 121 I5 Expottme Data Repotts and PEIBUITISE ooo re eet AA AT ebd e mlt Deve eMe Dil 122 E ouo ND CN ce cet ex
135. ion 0 02 Enzyme Specified by each sample Allow non specific cleavage at one end of the peptide Maximum missed cleavages per peptide ie PTM F Carbamidomethylation Set PTM A Oxidation M Switch type Maximum allowed variable PTM per peptide 3 Database o Select database Database UniProt_SwissProt 7 Paste sequence Taxa all species De Novo Tag Options Available de novo tags de novo with current parameter General Options Estimate FDR with decoy fusion E Find unspecified PTMs and common mutations with PEAKS PTM Advanced Setting Find more mutations with SPIDER 11 Chapter 2 Installation and Activation This section of the manual will guide users through the installation and registration of PEAKS 1 Package Contents The PEAKS package contains e This manual PEAKS Software Quick reference guide for PEAKS Quick reference sheet for mass spectrometry 2 System Requirements PEAKS runs and has been tested on Windows XP Vista and 7 The computer on which PEAKS is installed should meet the following hardware requirements Minimum A dual core processor 2GB RAM and 100GB free hard drive space this suggestion 1s mainly for viewing purposes only for data analysis we strongly suggest following the recommended requirements below Recommended A quad core processor 8GB RAM 500GB free hard drive space and 64 bit OS 3 Installation on a Windows Computer Important Please uninst
136. ion 15 0 ppm using monoisotopic mass Fragmention 0 5 Da Enzyme Specified by each sample Allow non specific cleavage at one end of the peptide Maximum missed cleavages per peptide 35 E Remove Switch type Switch type Maximum allowed variable PTM per peptide 3 Database Select database Database BamplebB View 3 Paste sequence Taxa all species Set View taxa De Novo Tag Options Available de novo tags de novo with current parameter General Options Estimate FDR with decoy fusion Find unspecified PTMs and common mutations with PEAKS PTM Find more mutations with SPIDER 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 fragment ions The parent ion error tolerance can be specified in either Daltons or ppm and using monoisotopic or average mass Enzyme Select enzyme used to digest the proteins Enzymes built into PEAKS can be chosen and new enzymes can be created Please refer to Section 2 2 Enzyme Specificity for further details If enzymes are specified when creating the project the option Specified by each sample can be selected which allows the search to use enzymes that were chosen for the samples during project creation Nonspecific cleavages specifies how many 0 1 or 2
137. ions It can identify all the PTMs and mutations compiled in the Unimod library as well as custom PTMs Note For more details check paper PeaksPTM Mass Spectrometry Based Identification of Peptides with Unspecified Modifications Journal of Proteomics Research 2011 10 7 2930 2936 SPIDER is a homology search tool dedicated to finding novel peptide sequences which are not present in the protein database Note For more details check paper SPIDER Software for Protein Identification from Sequence Tags Con taining De Novo Sequencing Error J Bioinform Comput Biol 2005 Jun 3 3 697 716 The entire PEAKS software follows the design concept of easy of use It is just a few clicks away from the raw data to the complete analysis report combining all of the search tools Select a project node or a sample node Click the PEAKS DB button on the tool bar e DHEA aAA 6Qw FN Project View a Peaksworkspace derbyServer serverDB MultEnzyme 3 p FL BSA Trypsin 1 RAW j Ji Lyst A F2 BSAL ysC 1 RAW gt Jl Glue z fs F3 BSA GluC 1 RAW 68 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 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 pop up dialog PEAKS PTM and SPIDER can also be enabled from this dialog box Most of the parameters are self explanatory and the default parameters provide a good s
138. it the link below http www bioinfor com peaks support formats peaks watersmicromass masslynx data html Converter Level Support massWolf exe Note If you want to use massWolf in PEAKS please make sure massWolf is installed correctly and works from command line on your computer If you have massWolf installed on your computer and prefer to use massWolf exe to convert the raw data AA define the convertor in preferences Click the Preferences toolbar icon 9 or select Preferences from the Window menu to open the Preferences window Clicking on Waters raw under the Raw file convertor section on the menu on the left hand side will show the preferences for massWolf Point to the location of massWolf exe on your computer and enable it Waters raw Enable the MassLynx Converter MassWolf Location D MassLynx ConverterlmassWolf exe Browse Options centroid nolockspray 3 3 Agilent Data Agilent Q TOF data can be loaded provided that MassHunter software is installed on the same computer as PEAKS Agilent Ion Trap data can be loaded provided that CompassXtract is installed on the same computer as PEAKS The spectral data will be contained in the yep baf or fid file If loading fid files select the top level folder to load them all at once 2 Loading Data to a PEAKS Project CompassXtract 3 1 is readily available on the Bruker Daltonics web site You may need to contact your Bruker
139. ith homologous proteins clustered together The ratio eg ratio of Sample 1 and Sample 2 appears in the Ratio column and the standard deviation eg standard deviation of Sample 1 and Sample 2 appears in the SD column 110 PEAKS Q Label Free O LABEL FREE 15 22 Mar 1109 46 X EH Quantification Result n 27824 CALX HLMA 2 57 563 453 0 8 xin precursor M 1 00 1 06 00 0 01 Accession ID Mass Score T Coverage Matched Description Rato D Marked E 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 the samples eg Sample 1 Sample 2 Click on the beside the Outlier folder to see the peptides that were not included in the protein 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 This displays the shape of the peptide features over the retention time range where they were identified Intensity 40 1 40 2 40 3 Retention Time mins Sample 1 Sam
140. ivation About BSI PEAKS Studio PEAKS Studio 6 0 build 20120529 Copyright 2000 2012 Bioinformatics Solutions Inc All 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 PEASDenwo E View end user license agreement du Warning This computer program is protected by copyright law S and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in E 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 Activate PEAKS with a trial or purchased license key or Section 4 4 Activate PEAKS manually for re registering PEAKS 4 6 Common Errors during Registration 1 The license key contains only English letters and numbers It is recommended to copy ctrl C paste ctrl V the license key whenever possible 2 The user information can only contain English characters letters digits and symbols Characters from a non English
141. j5 RAW Ion Source ESI nano spray Fragmentation Mode CID CAD IRMPD y and b ions MS Scan Mode FT ICR Orbitrap MS MS Scan Mode Linear Ion Trap PEAKS Q MS MS Level Show top proteins in each group w accession contains searah Qo no results E a Accession 10lgP Coverage Peptides Unique Avg M Description 114 115 116 117 SD114 SD115 SD116 SD117 Mark 5 150 Proteins Y E M P02769 ALBU BOVIN 344 3 77 6 2 Serum albumin OS Bos taurus GN ALB PE 1 SV P00489 PYGM_RABIT 341 97 Ill MAMI BRUNER MA i 590 61 61 97289 Glycogen phosphorylase musde form OS Oryctolagus c 1 52E9 1 046E9 4 656E8 6 483E8 0 00 0 19 0 01 0 11 v iz P00924 ENO1 YEAST 294 02 EE MAA EH INN NE NN E cocos 27 26 46816 Enolase 105S Saccharomyces cerevisiae GN ENO1 PE 5 318E8 6 972E8 1 452E8 3 547E8 0 00 0 36 0 09 0 31 y a P00330 ADHi YEAST 275 99 HIM NEN MOMIA som 36 21 36849 Alcohol dehydrogenase 1 OS Saccharomyces cerevisiae 2 105E8 2 722E8 6 606E7 1 726E8 0 00 1 44 0 14 1 06 y P00331 ADH2 YEAST 191 37 mmm EE gI 37 16 2 36732 Alcohol dehydrogenase 2 OS Saccharomyces cerevisiae 2 513E5 3 175E5 5 486E4 2 312E5 0 71 0 89 0 15 0 65 v P20369 ADH1 KLULA 119 64 E M E E 155 6 1 37261 Alcohol dehydrogenase 1 OS Kluyveromyces lactis GN 3
142. k Browse to tell PEAKS the location of the raw file converter Select the preferred mode of raw data to load This is a useful option for ABI 5600 instruments mzWiff Click Browse to tell PEAKS the location of the raw file converter Select Survey Spectrum Cen troiding if centroiding has been performed before loading the data into PEAKS Select Product Spectrum Cen troiding if centroiding has been performed on the product spectrum before loading it into PEAKS This is impor tant to insure PEAKS performs optimally MSX Click Browse to tell PEAKS the location of the raw file converter Select the preferred options to load the raw data 3 4 3 ABI 4700 4800 T2D files can be extracted and imported into PEAKS with a free tool created by BSI The PEAKS Config Wizard can download and install the AB 4X00 Extractor automatically see Section 2 Instrument Selection System Requirements This extractor can be installed on the same machine as the ABI 4700 Explorer and the Oracle database or another machine that has direct network access to the 4700 SERVER There cannot be a firewall or proxy between the computers Windows 7 or Windows XP is recommended for use of this tool Configuration Start the ABI 4700 converter tool Choose Settings from the File menu Configuration requires the following 20 Loading Data to a PEAKS Project e 4700 SERVER Name or IP Address input localhost if the Extractor i
143. l 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 119 Workflow FY Select Data Selected Data Sil D test Peaks60 PeaksProjects Sample Project inChorus 5 A D test Peaks60 PeaksProjects Sample Project Identification E 4l Sample 1 E Jl Protein Standard HCD fl D1008930 yeast SCX105 rak ft8E pc O1 RAW e t identification RAW B T jtest Peaks60 PeaksProjects Sample Project Identification ao IA iPRG2011 ETD EF de Protein Standard HCD 30100930 yeast SCX105 rak ft8E pc 01 RAW B T D ftest Peakse PeaksProjects 5ample Project PTMI ER SL PRG 2012 Ly IPRG2012 mzML mzML 2 ad D test Peaks60 PeaksProjects Sample Project Labelling Q E Jl iTRAQ f ITRAQSample RAW 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 run some differently than others then you must set up separate workflows Once the data is selected you can specify parameters for the identification analysis steps one by one by clicking the other buttons in the workflow dialogue Please refer to the chapters on each individual function if you require more details on setting up the
144. l be contained in the yep baf or fid file If loading fid files select the top level folder to load them all at once CompassXtract 3 1 is readily available on the Bruker Daltonics web site You may need to contact your Bruker representative to obtain CompassXtract 3 1 Instrument Preferences for Bruker Data To set Bruker data related preferences in PEAKS click the Pref erences toolbar icon i or select Preferences from the Window menu to open the Preferences window Click on Instrument and then Bruker yep baf fid in the menu on the left hand side This will show the Bruker instrument preferences on the right hand side Bruker yep bat fid Raw File Convertor Options Bruker file reader will export Raw data Line spectra Bruker fid file may contain several files do you want to merge them 0 yes no CompassXtract by default will export raw data If the attempt to load raw data results in no spectra then choose Line spectra A Bruker fid file may contain several samples By default these samples are not merged into one data set Select Yes 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 Preferences toolbar icon or selec
145. ll be displayed Note PTMs selected for the PEAKS DB are automatically added as preferred PTMs to the PEAKS PTM search and cannot be removed However you may add as many additional preferred modifications as desired 71 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER PTM SetPTM Remove Switch type Maximum allowed variable PTM per peptide 3H Filter Options Perform PTM search on spectra satisfying the following condition De novo ALC 96 score greater than 30 recommend 30 ES Find more mutations with SPIDER Select this option to enable a SPIDER search SPIDER performs a ho mology search on those spectra with good de novo hits but not identified by previous search tools SPIDER also searches against the FASTA database specified for the previous tools If SPIDER is enabled a SPIDER result node will be automatically generated after the search In the report both results from SPIDER and previous search tools will be displayed 3 Understanding PEAKS Database Search Result After PEAKS DB is complete several result nodes will be generated One is from the sub routine de novo sequencing when not using an existing de novo tag the others are all results from database search tools If more than one database search tool is enabled the results from previous search tools will automatically be merged into the last one Double click the last node to examine the analysis report The fin
146. ll ongoing downloads will be cancelled if the wizard is cancelled Click Next to finalize the configuration A warning message box will pop up in case any downloaded item is not installed 5 Commit the Changes After configuring the necessary downloaded vendor specific software and standard databases click the Next button to get to finalize the panel 23 Configuration Wizard Configure Instruments and Public Databases Configuration Wizard Configuring Instruments and Databases The following FASTA databases and or Instrument software are mstalled successtullv Thermo MSFileReader IPI The followmg FASTA databases and or Instrument software are downloaded but not installed ABI mzWiff A database or an instrument software will be configured for use with PEAKS only 1f you do so explicitly by clicking the Install button in the Download Information panel The Configuration Wizard could not proceed with configuring the following items as it required to download some files that can be available only from the Manufacturer representatives ABI Analyst AB SCIEX MS Data Converter Please contact the Manufacturer representatives to obtam the required files Press the Back button to return to downloads and selection panels where you can change your selection of databases or instrument software for configuration Click Finish button to commit the changes to configuration file A summary about the chang
147. llows the search to use enzymes that were chosen for the samples during their project s creation Note Semi versions of common enzymes can be created by allowing non specific cleavage at one or both ends of the peptide These semi versions are recommended since digestion enzymes often exhibit some degree of non specificity 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 the Enzyme Editor window Enzyme Name 7057 Chiave Sites X al amine acids 5 LELI You can provide the name of the new enzyme and define the custom cleavage rules sites 2 3 Fixed and Variable PTMs To select the PTMs for de novo sequencing click the Set PTM button to open the PTM Options window 51 Peptide De Novo Sequencing Mono mass Residue site Acetylation K 2 0106 Acetylation N term Amidation Ammonia loss C N term Beta methylthiolation Carbamidomethylation iTRAQ amp plex K N term 104 a Selected Variable PTM iTRAQ 8plex N term 304 a T Oxidation M IRAQ Splex protein n term 3 04 a QT Deamidation NQ nim a ooo The PTM Options list displays recently selected PTMs by default To view PTMs built into PEAKS select the Common or the Uncommon tab UNIMOD modifications are included in PEAKS and categorized under the Common or the Uncommon tab
148. log appears gu Quantification result to Excel File Select the Type of Results to EEE 3 Complete Protein List ia Details Omitted Export only Marked Protein 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 1s 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 the protein table in the software These additions include all of the supporting 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 5 2 2 Export Summary and Detected Features The Summary view can be exported in HTML format The detected features in the selected samples can be save
149. low the selected peak at 1260 5649 m z was designated as a y 10n 64 Peptide De Novo Sequencing B02 50807 2 E Manual De Neve Intensity a LOD 344 25 1241 55 1375 71 m 1150 64 5p 1049 6 x inm 1090 47 226 12 246 18 em 1031 49 160481 1359 66 1489 76 bi H20 bi a ii Llao Lil 7 miz 100 200 300 400 500 600 700 600 500 1000 1100 i200 1300 1400 1500 1600 1700 Ma y 11 2X 2r Errtol 0 5 Da V intensity threshold OrbiSample mzXML ms 2 mz 802 90607 z 2 RT 0 0729 TIC 1 797 1260 5649 62 7 5 0 5 o A5 T i 0 5 500 1000 1500 mjz 344 25 EEEE re IRE E1241 8 3 1344 25 Hia Intenaite 5 300 1000 1520 Note 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 color coded in the Spec trum 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 the residue and the mass remaining to be sequenced on either side of the residue All other panels will also reflect the changes 65 Peptide De Novo Sequencing 802 90507 2 j Manual De Novo Intensity 5653 344 25 A 1170 51 1375 71 y2 1150 64 yl
150. ly 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 90 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER Peptide Number Venn Diagram PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 400 600 300 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 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 PEAKS 12 27 Apr 11 19 54 PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr 11 19 14 4 PEAKS 12 27 Apr 11 19 54 7 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 Choose the image quality and filter the content desired for export m 9 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 Proteome Discover 1 3 Mascot v2 4 X Tand
151. mat It includes e Summary view statistical data summary page including figures Protein coverage the protein coverage figure with PTM legends e Supporting peptides the list of supporting peptides grouped by protein e Best unique PSMs the unique peptide spectrum match with the highest score for each protein 85 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER Put all protein details in a single html collecting all the above information into one single html report otherwise one reported html for each protein Text Formats Export all the results in csv or XML format for further processing Proteins the csv listing of selected proteins details e Supporting peptides the csv listing of supporting peptides peptides that support the identification of selected proteins DB search peptide spectrum matches the csv listing of all peptide spectrum matches De novo only peptides the csv listing of de novo tags from the de novo only view Proteins fasta the FASTA file of selected proteins Peptides mzidentml the mzIdentML file with all information for both proteins and supporting peptides Peptides pepxml the pepXML file with all information for peptides e De novo only peptides pepxml the pepXML file of de novo tags from the de novo only view 2 Click Browse and a file chooser will appear 3 Choose the location and directory name where you want to put the exported files Click OK Note
152. n Dante he Ira eco aU EE EE a taxam ous Cede tud o ceca 38 iii PEAKS 6 User Manual 25 Mark Feature 7 Uninark Beat oiseau ees ipae M oe omae cial aweeinuninas Mas orsa bed 39 AS SNOW M S24 Mde MS Zoco 40 AMO PID sean eite es n DO b vtr A SAA ASA ANA deve cut 40 Oss NOISE os dE e 41 0 Addirie d Seg ence Database ida lid 43 I Contisuras Sequence Database a di dilo 43 2 Databases to be Used in PEAKS inChorus Function esseseessssessesee mem emen 44 de Data REUNEN t Stu 46 TV OVEIVICW E 46 2 Data Refinement Batata e cues pt cun ncaa eb cett n imecat i ume cri Nr UM qutd 46 2 1 Saving the Parameters tor Future U Se Lei A A SAN A Dele oV 48 o Peptide De NoOYO SEqUETIGIDP ic 49 IS ciii TE 49 2 De NOVO Sequences Parame lers osos doi ono uto iio 50 2 T1 2 BEFOE TOlet all CO 3 aene A adita ue E uq 50 2 2 ENZO PECINICILY ti Risus uota lease ie ditta idu terc etis 51 2 95 cd and V arrable PTM Sida iia 51 O AAA o setate E icum olas ic cuente 53 2 5 5aving the Parameters tor Future D Se is uo SA oes teris bb euet ales ebd EVE 53 3 Understanding PEAKS De Novo Sequencing Results esses menn 53 SES IOS dA AI TTD 53 od dE Novo Peptide WCW rd in ci dra f Leo iu dde coli 54 22 As Peptide Table ca AA utu 55 32 2 S PEC ARNO LAO a S6 SS 1 A IE 58 A NUBE onion E eater cia nct Aon See ne e
153. n 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 NFLENVIR USD MUN n rue 05 231 38 4524 E 542 2884 28 4963 47 867 1511 82666 135 0541 49 84 E3182 412472 41 94 2565 50085 32 1928 161 86 690 4143 68 3736 49 89 785 980533 25 4232 71 49 151 3207 185348 530 3555 315186 J760 9249 46 4147 ERES 10 ED 11 13 al PEAKS 12 27 Apr 11 19 54 535 2538 718 8505 473 7518 bbb TRAE 3 1263 2 E Boneia 41 7395 ia fain 596 80834 17 3177 zl aln 567 5433 J33 1455 5 Tang t n i a z 2 505 8165 26 8705 m 7 523 7 545 9 944 6219556 39 11 E iu 5218543 33 0524 2 05 i 521 8541 37 9902 89 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 7 3 Protein Comparison The top proteins identified in the PEAKS DB results are displayed in the table The display setting score filter and coverage map function the same as in the peptide comparison frame The following information is 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 suppor
154. n list at the top of the window To examine the contents select a saved parameters file and the parameter values will be automatically displayed 48 Chapter 8 Peptide De Novo Sequencing 1 Overview De novo sequencing is not only the preferred method for identifying peptide sequences yet to be included in databases but also is a proven method to measure alongside database findings PEAKS is the most utilized tool for de novo sequencing in mass spectrometry labs 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 assist other PEAKS search tools including PEAKS DB for database search PEAKS PTM for unspecified PTM search and SPIDER for homology search to achieve in depth protein analysis 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 1s 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 local confidence for amino acid assignments in the sequence and TLC reflects the expected total number of correct amino acids in the sequence The use of automat
155. n two spectra to be merged Precursor m z Error Tolerance The maximum difference in m z between two spectra to be merged Merge CID and HCD scans together When merging scans the scans of different fragment type will not be merged If users want to enforce merging CID and HCD scans please check this check box Correct Precursor Mass only Correct the precursor s mass only Min Charge The minimum charge a precursor ion can be corrected to Max Charge The maximum charge a precursor ion can be corrected to Filter Scans Precursor Mass Range The precursor mass region to select scans for further analysis Retention Time Range The retention time region in minutes to select scans for further analysis Quality Threshold It defines the spectrum quality threshold to select scans for further analysis The recom mended value is 0 65 This is a percentage Note Data pre process centroiding deisotope deconvolution option is removed Now data pre process is enforced for all the data sets 47 Data Refinement Once all parameters are set press the OK button to initiate the data refinement process 2 1 Saving the Parameters for Future Use After setting up the desired parameters you can save them for future use Click the drop down list at the top right of the window select Save as and define a name for these preferences for future use reference when prompted Any parameters that are saved will be available in the drop dow
156. nction 79 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER 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 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 with decoy fusion 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 4F 800 oe A BOO MIR s mo s ie Le 400 number af P2Ms o_o O 10 20 30 40 50 60 70 80 90 100 110 PEAKS peptide score 10lgP A Decoy B Target Figure 2 b plots the precursor mass error verses score for all the PSMs This
157. nded that you quit all programs before Choose Install Folder continuing with this installation Choose Shortcut Folder l OPE Click the Mex button ta proceed to the next screen If vau want to ra Installation Surmrmnary nice m change something an a previous screen click the Previaus button Installing Install Cornmplate You may cancel this installation at any time by clicking the Cancel j f button Install amp nywhere by Macrovision 5 Follow the on screen instructions to finish the installation 4 Activation All users are required to go through a software activation process in order to use PEAKS A license wizard will appear to guide the activation process the first time PEAKS is launched 13 Installation and Activation FY Welcome to PEAKS Welcome to PEAKS Thank you for using PEAKS The most accurate sensitive easy to use software package for complete proteomics analysis Activate PEAKS with a trial or purchased license k By entering the license key the features of PEAKS will be activated and the software will be ready for use e Register to a free 30 trial license k IC By completing the online registration form a 30 day trial license key will be sent to you via email You will also gain access to free email and phone supports during the trial period m9 Use PEAKS as a viewer Vi p EAKS can be used as a viewer without activation Take advantage of the advanced user interface
158. ns Export Result to Excel or Export Result to Html The exported result contains fraction information PTM information and list of peptide identifications of the selected fractions ra Project View E RE ftest Peaks60 Pez ication JA Protein Standard Data Refine Be F1 identificat Replicate Analysis gt DATA RE a E DENOVO ef PEAKS 3 Peaks Database Search lle PEAKS P Spider Search WE SPIDER 5 i E M iPRG2D11ETD in Chorus Search E F3 D100930 N AW L amp DATA RE Export DTA file Jy DENOVO Export PKL file Mf PEAKS Export MGF file AE PEAKS PT SPIDER 7 Export Result to Excel Bl T D test Peaks60 Pea Export Result to Htm E LY D test Peaks60 Pea ua ERA RAQ Save A Copy As S i iTRAQSample co DATA RE Close Project fi DENOVO Heat Map Compare PEAKS 3 16 May 12 10 49 More function specific export options are available from the Summary view of the respective results The fol lowing sections describe in details about the function specific exporting options available in various formats 3 Export De Novo Result The PEAKS de novo sequencing result can be exported to csv html and pepxml formats All export functions are available through the Summary view panel 3 1 Export Summary and Peptides To export results press the Export button in the title bar of the Summary view panel The following export dialog will appear Select the items that you want to export from the
159. ns 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 7SE7 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 ZW y 7 HO After setting an ion the Spectrum Annotation panel the Spectrum Alignment and Error Map panel and the Ion Table panel will reflect the changes The peptide sequence candidate name as displayed in the Result panel and on the top of the Spectrum Annotation panel will also change to reflect the mass remaining to be sequenced on either side of the ion In the example be
160. nt tabs 4 Each opened result node provides several different views as different tabs In particular the summary view shows the result statistics The summary view is also the central place to filter and export the results 5 The information pane shows useful information such as the node properties and the progress of running tasks Overview File Tools Window Help MEG la bel 47 SQW IND V1 Project View Sg 10 PeaksProjects BSA3 BSA 3 iy DENOVO 4 09 May 12 13 57 PEAKS 5 09 May 12 13 57 PEAKS PTM 6 09 May 12 13 57 A SPIDER 8 09 May 12 13 57 PEAKS 16 09 May 12 15 43 PEAKS PTM 17 09 May 12 15 43 M SPIDER 18 09 May 12 15 43 PEAKS 23 13 May 12 12 02 PEAKS PTM 24 13 May 12 12 02 M SPIDER 25 13 May 12 12 02 M PEAKS 29 13 May 12 15 02 PEAKS PTM 30 13 May 12 15 02 M SPIDER 31 13 May 12 15 02 ig DENOVO 33 13 May 12 16 11 W PEAKS 34 13 May 12 16 11 PEAKS PTM 35 13 May 12 16 11 M SPIDER 36 13 May 12 16 11 B A Gluc Hy F1 BSA GluC 1 RAW E A LysC H F2 BSA LysC 1 RAW S A Trypsin H F3 BSA Trypsin 1 RAW of PEAKS 5 09 May 12 13 57 X PEAKS PTM 6 09 May 12 13 57 X e ii E Peptides i0lgP 2 116 4 w FOR Proteins i0lgp 20 w and 3 vw unique peptides De novo Only TLC 3 v and ALC 50 v and peptide 1019P lt 116 4 v l Apply Filters Export Notes 3 0 y 2 5 2
161. oad and configure them automatically The list of standard databases can be found in Section 3 Database Selection 2 Instrument Selection When you select Window Config Wizard the configuration wizard will run and display a welcome message Clicking the Next button shows the following instrument selection panel 20 Configuration Wizard Configure Instruments and Public Databases Configuration Wizard Thermo data can be loaded using Thermo MSFileReader library The config wizard wil download and install the MSFileReader autornatically AB SCIEX AB 5600 Instruments AB 4700 4800 Instruments AB SCIEX QTRAP Instruments AB SCIEX QSTAR Instruments Agilent Technologies E Agilent Ion Trap Instruments Agilent Q TOF Instruments Bruker Daltonics Bruker APEX micrOTOF HCT Ion Trap TOF TOF Instruments Shimadzu Corporation Shimadzu AXIMA CFR Instruments Waters Corporation Waters Micromass Q TOF Instruments Varian Incorporation Varian Mass Spectrometry Instruments The instrument selection panel lists all the supporting instruments and their vendor specific softwares Select the instruments as necessary A small description about the instrument is displayed once it is selected Click Next to proceed 3 Database Selection The database selection panel lists the standard databases The automatically downloadable databases are NCBI nr SwissProt TrEMBL and IPI human Select the
162. of previous versions of PEAKS SPIDER now defaults to what was previously called Homology Match 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 Filter The filter option asks for the minimum de novo tag score ALC for a spectrum to be used If the ALC is too small then the spectrum is unlikely to provide a significant hit 7 Comparison of PEAKS Results In PEAKS 6 we support comparisons of up to three PEAKS DB results including filtered results in one project To do such a comparison select those PEAKS DB nodes and right click Click on Compare Results and the comparison will be done automatically E D temp LFQ Heatmap Blank O LABEL FREE 13 27 Apr 11 21 25 J Sample 1 EM PanTumorSCX1 RAW Re DATA REFINE 2 27 Apr 11 15 50 22 e am DENOVO 6 27 Apr 11 17 05 Ur MEARS 10 27 Apr 11 19 144 a Il Sample 2 Compare Results EP PariTumorSCX2 RAW 8 DATA REFINE 1 27 Apr 11 Delete Result ig DENOVO 5 27 Apr 11 16 30 E APEAKS 9 27 Apr 11 19 00 7 1 Comparison Result After comparison is finished a comparison node will be added to the project as shown in the following picture 88 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER FA D temp LFQ Heatmap Blank Dg Compare run 9 10 12 20 May 11 16 45 O LABEL FREE 13 27 Apr 11 21
163. oject Project Name My PEAKS 6 project Project Location D ltemp Data Files Sample Details ne q pias Spee Sample Name Sample 1 E Replicate ni PIN Sample 1 OEE Slc Lc ta ES Specify later Instrument FTMS FT Trap Fragmentation CID Data File D Data OrbiSample RAW 2 Use the Project Name field to name your job Click Browse to select where to save the project This will appear in the Project Location text box Note Refer to Section 6 Changing the Default Project Location for changing the default save location for projects 3 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 type Fragmentation method and Enzyme name that was used to generate the experimental data from the drop down lists To apply the same instrument configuration to the whole project click on the Copy to whole project button 4 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 configuration 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 e
164. on The Database List on top lists all the configured databases in the system Select a database from the list to view the detailed information about the database in the Database Details panel Create a new database The database configuration parameters appear as follows FASTA format database NCBI nr m Validate Database Export Decoy DE Basic Options Database name Validated Path Browse or Download EST database Advanced Options Fasta Title Format Rule to parse accession id from FASTA title Gi l d Rule to parse description from FASTA title ELY Accession id URL http www ncbi nlm nih qov entrez viewer fcgi db 2protein amp val lt Accession ID gt Delimiter s f Taxonomy Options taxonid Browse Download taxdmp Browse Download Follow these steps to configure a database Select the database format 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 2 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 3 Click OK to invoke the default FTP client software and download the database automatically Click Cancel to copy the URL to the s
165. 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 cC CI Errar da I cC CI l i 200 400 600 600 000 1 200 1400 1600 miz 3 2 5 Spectrum Alignment The Spectrum Alignment is displayed under the Error Map presenting the entire spectrum It is used as a tool to help 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 A Se SH het te miz aut 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 55 100 802 91 50 miz 00 400 600 500 1000 1200 1400 1600 Ma lly 121 2x 21 ErrTol Z intensity threshold Orbisample maXML ms 1 RT 0 0106 scan 1 TIC 1 27E8 1607 9911 4 Filtering De Novo Sequencing Results PEAKS De Novo sequencing results can be filtered based on TLC Total Local Confidence and ALC Average Local Confi
166. onfiguration was invoked when specifying DB search parameters simply click OK 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 line To be able to perform PEAKS DB using a specific taxonomy corresponding files must be downloaded and then referenced by PEAKS in the Taxonomy Options panel Taxonomy files for NCBI nr database are gi taxid prot dmp gz and taxdmp zip for UniProt Swiss Prot they are speclist txt and taxdmp zip 1 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 OK Click Cancel to copy the URL to the system clipboard If Cancel was selected click OK on the dialog detailing the copy to the clipboard Next open a browser and paste the URL into the address bar When the file download window opens click Save 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 2 Now that the taxonomy files have been downloaded PEAKS must be given access to them by clicking the
167. oooocococconccconcnconcnnoncnncnnnnonos 20 Vs OCI VIC V c adonde 20 A A ie e dudes 20 S Database SeleC HOM ME AA A 21 4 Downlodd IntOLmatloll s oth iro re tula rd O Ia rica 2 Comite WAM OCS PCT T HS 23 4 Loadimne Data toa PEAKS Project aia tias ote rio D Fito iden dedicas ceca coda 25 ie vci TS 25 Ze SUPPONE d Data Horda net anar dira adi iria sde qd 26 3 Vendor Spec MC Isequitefriefite a alii 26 Sele PACINO AA E E M 21 2 2 Waters Micromass MassEvnx Dit aw Nn eR ETT ve dus MUEVE A ME du Ms 24 Suum rre 2 23 4 Applied BIOSVSCCIBS SOIOX Dita dt 28 SA COSTAR or OTRA P is eolica saad erenstauetaciuastebivaaetssans 28 34 23 Convertors DOT WEE iS AS fauces d Fond ee ou od 28 ASA AOA SOO ccce eco tact toons UA ar 29 3 S BUF Data ado dd aa 30 A mmm 30 I A 3l SIS PEAKS 9 PIOJECIS atra iod eodein d eret oA D Eire flat ero ruri o iaa 31 A MESS ou Rs die CCL E RO gu TEN 32 5 Addie Data to anm Bxistinp Pro elit ie eet E eric veniva ten AuSesadeu eL Que btpls 33 6 Chans Mathe Detault Project Location nr A oua DRE de pala te US das 33 Data ViDa za OM testa had Susi test Oa nutu ou tense Red FoU tae is 35 PONCIO W t RM r KE 35 IN qe m T 35 IE MS MS MIS edna anaes 36 A Heat MOD res oia dados 37 A Bim Usb Heal VIS a dla 38 22 Hie hle ht Feature Mde Peai us ooot ri
168. or base the search on an existing de novo sequencing result node Estimate the false discovery rate FDR with the decoy fusion method Decoy fusion is an enhanced target decoy method for result validation with FDR Decoy fusion appends a decoy sequence to each protein as the negative control for the search See BSI s web tutorial http www bioinfor com peaks tutorials fdr html for more details Including PEAKS PTM and SPIDER algorithms for the search By default PEAKS PTM performs a blind search for additional PTMs in the data Users can also limit the PEAKS PTM search on a large number of PTMs by clicking the Advanced Setting button SPIDER performs homology search based on de novo sequencing tags If selected the SPIDER algorithm will be conducted on every confident de novo tag ALC gt 30 whose spectrum is not identified by PEAKS DB with high confidence 10lgP lt 30 SPIDER will construct new peptide sequences by altering amino acids of database peptides For each spectrum the better sequence constructed by SPIDER or found by PEAKS DB will be used as the identified peptide SPIDER is good for cross species searches and for finding point mutations of the protein It makes no difference to invoke SPIDER through this workflow or by clicking the SPIDER icon in the toolbar 10 Overview WF PEAKS Search PEAKS Search Predefined parameters Error Tolerance Parent ion 15 0 ppm using monoisotopic mass Fragment
169. ow appear in the Enzyme List where it can be accessed later To delete a customized enzyme select the appropriate enzyme and click the Delete button Note For information on defining new enzymes on the fly for PEAKS de novo or PEAKS DB refer to 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 136 Configuration and Preferences Mame 4 hydroxynonenal HNE Acetylation K Acetylation M term Acetylation Protein M term Amidation Ammonia oss C N term Ammoniatoss Protein N term 0265 Sji Applied Biosystems original ICA Ij Applied Biosystems original ICA Beta methylthiolation Carbamidomethylation 57 0215 Carbamylation 43 0058 Built in PTMs The built in standard PTMs within PEAKS are listed in two separate PTM lists under Com mon and Uncommon tabs The Common list contains the most commonly used PTMs and the Uncommon list contains less frequently used PTMs Most recently used PTMs are listed in Recent tabs and the Customized tab lists all the user defined PTMs Double clicking on any of these PTMs will display the information about that PTMs in the PTM Info popup dialog The same information can be viewed by selecting a PTM from a list and by clicking the View button 137 Configuration and Preferences PTM name Ca
170. parameters Note PEAKS DB PEAKS PTM and SPIDER are now combined together see Chapter 9 Peptide PTM and Mutation Identification PEAKS DB PEAKS PTM SPIDER and are optional in Identification Work flow You can uncheck them if you do not want to perform those functions 2 Quantification Workflow The quantification workflow is similar to the identification workflow with an additional step for quantification where the quantification parameters to perform labeled or label free quantification can be defined 120 Workflow La Quantification Work Flow x Refine Data ia De Novo B ds Q Quantification 3 inChorus Workflow The inChorus workflow is similar to the identification workflow but offers the ability to specify inChorus param eters and invoke multiple search engines FY Inchorus Work Flow 121 Chapter 15 Exporting Data Reports and Printing PEAKS offers a rich collection of exporting functions to allow users to create reports and share the analysis results with collaborators colleagues and clients The supported formats include HTML Comma Separated Values CSV pepXML mzIndentML 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 c
171. perly Figure 3 a 1s 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 5 shows the number of peptides by number of missed cleavages for each sample which indicates the efficiency of the enzyme digestion 75 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER a 40 5 D 5 10 15 mass error ppm Missed Cleavages 0 1 Trypsin 237 114 LysC 227 100 Glut 309 119 3 3 Protein View The Protein View list all the proteins presented in the sample and characterize each protein at the amino acid level It has four components e Protein table List all the proteins presented in the sample e Coverage Characterize the protein sequence at the amino acid level All the PTMs and mutations happen on the protein sequence will be displayed together with the MS MS data supporting the inference e Peptides The peptides identified from this protein De novo Tags A subset of de novo tags from the De novo only tab that can be matched to the selected protein Show top proteins in each group w accession contains ses Qj G9 noresults a Accession 10lgP Coverage Pep
172. ple 2 3 2 2 Heat Map Select the 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 and repre sents low intensity in white If the peptide is identified in PEAKS DB there will be a star after the sample name 111 PEAKS Q Label Free Heat Map MS2 Annotation Sample 1 Feature intensity 1 46E8 Sample 2 Feature intensity 1 59E8 1001 0 1001 5 1002 0 1001 0 1001 5 1002 0 1002 5 Vertical Axis retention tme Horizontal Axis m z identified peptide Color Code 0 cR 100 3 2 3 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 DB search results spectrum annotation Please refer to Section 3 2 2 Spectrum Annotation for more details Heat Map XIc M52 Annotation Intensity 95 100 va Yii Yg b14 ya Y4 be D10 Vio b14 H20 00 1000 1500 m ENTRE 070828 O1 03 070824 DMSO Dh 2 RAW ms 2 Me ai 1 27 ErTol 0 503 W lowintens lab 5 1051 357 2 RT 40 06 con 4646 score 20 25 3 2 4 Isotope Select Isotope tab to view the isotope distribution detected in the samples 112 PEAKS Q Label Free Heat Map XIC MS2 Annotation Isotope Sample 1 Feature intensity 1 46E8 Sample 2 Feature intensity 1 59E8 Inten
173. protein coverage of their identified peptides in the window below The quantification ratios of those quantifiable proteins are displayed in the ratio columns with label names incorporated into the header e g Ratio Heavy The ratio is calculated from the unique peptides of the protein Proteins with no unique peptides will not be assigned a ratio The sample on which the ratio is based on can be changed from the ratio based on drop down menu at the top The normalization mode can also be selected in the Summary view SD represented 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 100 PEAKS Q MS Level Show top proteins in each group ratio based on Light F no normalization w accession contains search Q O no results Accession i E Proteins li P21333 FLNA HUMAN 4 Q69zN7 MYOF MOUSE Score 96 61 3 10lgP 42 26 Coverage Mass Description 280736 562 Filamin A OS Homo sapiens GN FLNA PE 1 SV 4 233321 766 Myoferlin OS Mus musculus GN Myof PE 1 SV 2 1 09 0 00 Ratio Light Ratio Heavy SD Light SD Heavy 0 00 Mark lt lt gt Q1IHAS PANB ACIBL 10 2 0 00 32172 795 3 methyl 2 oxobutanoate hydroxymethyltransferase OS 0 00 0 00 Q7WKR4 SYFB BORBR 9 4 0 00 87934 492 Phenylalanyl tRNA
174. rbamidomethylation PTM abbreviation Carbamidomethyl Mass Monoisotopic 57 0214655 Residues that can be modified C peptide protein H 3 C 2 N 1 O 1 Create a new PTM Click on the New button to display the New PTM dialog Provide the information about your PTM LAM OK PTM name PTM abbreviation Mass Monoisotopic Residues that can be modified peptide protein PTM Name This name will appear in the PTM list for future use after it is saved PTM abbreviation PTM expressed in shortened form Mass Monoisotopic The mass that the residue gains or losses as a result of the PTM 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 Formula The chemical formula of the PTM This should correspond to the mass listed above Rule This field can be used to enter a comment about the PTM to be used for your reference Click the OK button to save the changes The new PTM will now appear in the Customized PTM list where it can be accessed later To delete a customized PTM select the appropriate PTM from the list and click the Delete button Note For information on defining new PTMs on the fly for PEAKS de novo or PEAKS DB refer to section Section 2 3 Fixed and Variable PTMs 138 Configuration and Preferences 2 3 Labeled Q Metho
175. replicates To assign the replicate number in the quantification parameters window click the Assign replicates button below the Parameter Table on the right hand side This will open the Assign Replicate dialogue where the replicates can be defined 114 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 desired 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 The relative order of a sample in a replicate can be controlled by the Up and Down buttons located 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 bring up the Replicate Analysis window ds Jl RiS1 Peaks
176. rom the toolbar The original project window will open i AAA tSS FY Add Data to the Existing Project Project Name D ftemp My PEAKS 6 project Project Location D temp Data Files Ej PEAKS 6 project Sample Name Sample 1 Replicate Ea Orbisample mzXML i DATA REFINE 1 06 Jun 12 14 09 AVE Trypsin jg DENOVO 2 06 Jun 12 14 09 Inst i i rument ETMS FT la PEAKS 3 06 Jun 12 14 09 FTMS FT Trap i PEAKS PTM 4 06 Jun 12 14 09 SPIDER 5 06 Jun 12 14 09 Fragmentation HCD Copy to whole project OK Cancel 3 You can add more files to an existing sample using the Add data files button or create additional samples using the 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 Please make sure this folder is readable writable by PEAKS 33 Loading Data to a PEAKS Project 1 Click o from the toolbar The following Preference dialog pops up General Display Options RMI Connections Default Input File Directory D test Browse Derby Database Exam E Default Project Folder D NtestiPeaks60 PesksProjects R
177. roteins The list of protein identifications will be saved to proteins csv in Comma Separated Values CSV format e Supporting peptides A list of supporting peptides of each protein identification will be exported to pro tein peptides csv DB search peptide spectrum matches The peptide spectrum matches PSM with scores greater than the threshold will be exported to DB search psm csv 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 Proteins fasta A list of protein identifications will be saved in proteins fasta e Peptides mzidentml version 1 0 0 A list of peptide spectrum matches will be saved in peptides 1 0 O mzidin mzIdentML format version 1 0 0 Peptides pepxml A list of peptide spectrum matches will be saved in peptides xm1 in pepXML format 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 in pepXML format 126 Exporting Data Reports and Printing 4 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 3 2 Export
178. s 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 information 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 the CompassXtract Run time library is installed on the same computer as PEAKS The spectral data wil
179. s selected The normalization factor will be displayed in the text field 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 sam ples 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 109 PEAKS Q Label Free 1 Heatmap View ol a dues LE apunte Zi adi EL dur El adwrg apdues OL adweg p epdiuec gi a dues 1 tr pa i i logZiratio ALBU MOUSE BTGZ MOUSE FSD5 MOUSE PPAR MOUSE COTA OUSE LC7TLZ MOUSE TCPH MOUSE IGBP1 MOUSE CRUMT MOUSE TIEG3 MOUSE SERA MOUSE STATE_MOUSE 6 0 00 Dafuuit 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 Fractions Spiked 040806 2 RAW SpikeO 040806 2 RAW Spikel 04080 Mass error toler
180. 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 the MS View section 36 Data Visualization StartPage X p TEST 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 01 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 m 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 afo 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 24 888
181. se button to tell PEAKS where to find the search engine 1 3 3 OMSSA Settings Clicking on OMSSA Settings on the left hand side will display the OMSSA preferences Omssa Settings Default Omssa Fath omssa Browse 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 4 Spectrum Annotation Preferences Clicking on Spectrum Annotation on the left hand side will open the following window Spectrum Annotation H20 NH3 24 b wv vj vj C El P y v vi iV B i cH ium F internal precursor J Show Decimal Places 25 m z on fragmentation m z on unannotated sequence fragmentation 4 in place ion info Intensity Low Medium High Reset default The annotated spectrums in PEAKS results can be annotated by the selected 1on 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 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 134 Configuration and Preferences Note If you are upgrading from an earlier version of PEAKS or simply wish to reset the settings use the Reset default button to up
182. se of use when regularly performing quantification with the same parameters 3 Understanding the Result a Once completed the protein quantification result will be displayed in a quantification node in the Project View panel Double click on this node to open the result that contains three views Summary view Protein view and Peptide view The Summary view tab will appear by default 3 1 Summary View The MS MS labeled quantification results are summarized in one page in the Summary view In the heatmap proteins are clustered into a tree structure Proteins are clustered if they exhibit a similar expression trend across samples Move the mouse onto the tree in order to select a cluster and left click to show the variation trend chart for that cluster 103 1 Heatmap View Q4PD66 CCPR2_USTMA F00489 PYGM RABIT Q05905 YL301 YEAST P00331 ADH2 YEAST P00330 ADH1 YEAST QS80WC3 TNC18 MOUSE Q7MTV8 ENO PORGI P23254 TKT1 YEAST F00924 ENO1 YEAST QG6D5V3 PDXH ERWCT FP00761 TRYP PIG Q9KIV1 HGBB HAEIN P02789 ALBU BOVIN Q3SZR3 A1AG BOVIN P12763 FETUA BOVIN Q29443 TRFE BOVIN P49065 ALBU RABIT Q3MHNS VTDB BOVIN Ca Is PEAKS Q MS MS Level log2 ratio 5 0 0 0 5 0 Default group 1 Cell colour represents the log ratio to the Control Sample Control Sample is marked with 2 Notes 3 Result Statistics Table 1 Statistics of data and unfiltered result of MS Scans 11982 of MS MS Scans 22175
183. search speed sensitivity and accuracy You can choose to perform a fresh new de novo sequencing with current parameter setting or select from the existing de novo sequencing results if there are any Estimate FDR with decoy fusion Select this option to enable PEAKS database search tools to validate the search results with an enhanced target decoy method A few important statistical charts in the analysis report will depend on this Uncheck this only if you want to do your own result validation Find unspecified PTMs and common mutations with PEAKS PTM Select this option to enable a PEAKS PTM search after PEAKS DB PEAKS PTM searches those spectra with good de novo hits but not identified by PEAKS DB The default setting for PEAKS PTM is to search for all PTMs and mutations in the Unimod database Advanced Settings allows users to only search for a list of preferred PTMs from the Unimod database or their own customized PTMs Although PEAKS PTM allows any number of variable PTMs to be searched limiting the number of PTMs does improve the searching speed and accuracy In advanced settings users can also specify the maximum number of variables per peptide which is recommended to be less than 4 and define the threshold for what is a good de novo hit by specifying the de novo ALC If PEAKS PTM search is enabled a PEAKS PTM result node will automatically be generated after the search In the PEAKS PTM report both results from PEAKS DB and PEAKS PTM wi
184. significant improvement in the final analysis 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 problems in 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 attempts to correct the charge information Low quality spectrum removal this function attempts to remove the junk spectra This will save some analysis time Use this function with caution as it may also remove a small portion of identifiable spectra Centroiding and charge deconvolution and isotope deconvolution centroiding the peaks and deconvolution of the multiple charge ions 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 You can run the data refinement function separately by selecting a fraction sample or project on the project tree All the fraction s under the selected node will be refined The use of this function is outlined in the following 1 Sele
185. sity So 100 i a SN Ho RT 40 05 RT 39 04 1001 34 1002 35 1002 35 4 Filter LFQ Result PEAKS Q 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 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 11 05 35 X Peptides with fold changez 2 Export Editmotes Select Samples Summa 5 Export Quantification Result PEAKS label free quantification results can be exported to Excel xls or HTML html format The summary page and the detected features can also be exported in various supported formats Refer to Section 5 2 Export Label Free Quantification Results for details 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 validate the quality of proteomics data based on sound statistical principles are needed in this field In PEAKS comparison functions are provided at three levels Assess the reproducibility of MS data from technical replicates Perform comparative analysis of peptides and proteins Assess the reproducibilit
186. ss 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 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 108 PEAKS Q Label Free 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 ease of use when regularly performing quantification with the same parameters All the parameters in quantification will be saved excep
187. stall specific software libraries to support the instrument See Section 3 Vendor Specific Requirements for this requirement To conduct database search for protein identification a protein or EST sequence database must be configured See Chapter 6 Adding a Sequence Database If you are eager to try PEAKS now leave these two configuration steps aside for a while and try out the 15 minute walkthrough to get familiar with PEAKS GUI and basic operations See Section 4 Quick Walkthrough 19 Chapter 3 Configuration Wizard Configure Instruments and Public Databases 1 Overview The configuration wizard guides you through some easy to follow steps to configure PEAKS for instrument raw data support and database searching The configuration wizard can also be invoked from the menu Window Config Wizard PEAKS supports different instrument vendors raw data formats A list of supported formats can be found in Section 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 The configuration wizard helps you to select the proper instrument and install the appropriate vendor software A sequence database must be configured in PEAKS to identify peptides and proteins with the MS MS spectra by database searching The configuration wizard helps you to select the appropriate database from a list of standard sequence databases downl
188. synthetase beta chain OS Bordetella B 0 00 0 00 B6YR21 ACP AZOPC 9 3 0 00 9311 368 Acyl carrier protein OS Azobacteroides pseudotrichony 0 00 0 00 Q8WZ42 TITIN HUMAN 8 9 0 00 pr pu pu pu o pur pu pu pu pa 3816081 250 Tin OS Homo sapiens GN TTN PE 1 SV 2 0 00 0 00 iii Coverage Peptides Tool Box gt sp P21333 FLNA_HUMAN Filamin A OS Homo sapiens GN FLNA PE 1 SV 4 lt show position QGDVSIGIKC APGVVGPAEA DIDFDIIRND NDTFTVKYTP RGAGSYTIMV LFADQATPTS PIRVKVEPSH DASKVKAE 891 900 936 5 GLSRTGVELG KPTHFTVNAK AAGKGKLDVQ FSGLTKGDAV RDVDIIDHHD NTYTVKYTPV QQGPVGVNVT YGGDPIPF A M FSVAVSPSLD LSKIKVSGLG EKVDVGKDQE FTVKSKGAGG QGKVASKIVG PSGAAVPCKV EPGLGADNSV VRFLPREE SS show peptides show de novo tags show 10AA gap lt s S 80 gt AA per line PTM B SILAC K6 lt s YEVEVTYDGV PVPGSPFPLE AVAPTKPSKV KAFGPGLQGG SAGSPARFTI DTKGAGTGGL GLTVEGPCEA QLECLDNC _ TCSVSYVPTE PGDYNINILF ADTHIPGSPF KAHVVPCFDA SKVKCSGPGL ERATAGEVGQ FQVDCSSAGS AELTIEIC 1248 s AGLPAEVYIQ DHGDGTHTIT YIPLCPGAYT VTIKYGGQPV PNEPSKLOVE PAVDTSGVQC YGPGIEGQGV FREATTEE 2x 51 DARALTQTGG PHVKARVANP SGNLTETYVQ DRGDGMYKVE YTPYEEGLHS VDVTYDGSPV PSSPFQVPVT EGCDPSRY pm re d 3 3 Peptide View The pepti
189. t Preferences from the Window menu to open the Preferences win dow Click on Instrument and then Shimadzu AXIMA run in the menu on the left hand side This will show the Shimadzu instrument preferences on the right hand side Click Browse to tell PEAKS the location of the Shimadzu run2xml exe file 30 Loading Data to a PEAKS Project Shimadzu AXIMA run Shimadzu run2xml exe File Location 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 data files are 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 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 or select Preferences from the Window menu to open the Preferences window Click on Instrument and then V
190. t te haat 122 Ze TAX POLL Result From Project VICW dida 123 3 Export DE NOVO RESULTS cast Ud Let 123 3 Jl EXPO suniman and Peptides 2e Pc oi acre AAA NA euo err 123 E LIMA SCS ia 124 A Export Database Searah Resultado 125 4 1 Export summary Proteins and Peptides te o EH RH ba tb edu Res 125 AL ER POT Images os oca epe a a boot ASAS A AS un arb en Dude 127 2 Export Quan caon Results d etaed oct 127 23 1 Export Labeled Quantification Results uo ee Maie aie oid toit n ea toot ita bien Loud 127 5 2 Export Label Pree Quantification Results 5 ooi te qot ape sucre eda oue gute E su dU 127 PEAKS 6 User Manual 22 T Export ResUl r Exec kor EEDNID uiu hte onc eb ree cian 127 522 2 Export Summary and Detected Features xus io qoe i et sao Su ortae oer sd qut 128 O Export inc horus Result weaves aha waldo Meehan dte ue tab bust Ful NS 129 LO Contisuracion and PEEFOIEDDES stated 130 L PEAKS Environment Preterendes vidad wee baad edad m ioi one Nave decide t uds 130 TT encral etorri do m ts e uia 130 LAST Display Options LS AS eds eu Ni 131 I 2 RIVET COMME CUONS ula 131 iS Derby Data Base ridad dato 131 1 2 Raw Pile Converter Pree rnEE Socr edades 132 I2 ABLA WALI SAS ANA AN E AA A AS 132 1 22 BrUker YE py Daly FIG las 132 123 Mmadz AXIMA AUD dis 132 1 2 4 Wat an AOS ti SS n 132 1 275 M METS A LAW AAA A AAA Ss 133 1 5 Staten Enetie lt PrererenCes it tos ui 133
191. t 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 the LFQ Result Once completed the label free quantification result will be displayed in the quantification node in the project tree 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 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 5 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 Y Normalization Settings Linormalze P epbde Ratios 9 Automatically Normalize Peptide Ratios Manualy Normalize Peptide Ratios 1 0 0 7 If you select Unormalize Peptide Ratios the protein ratio will be calculated from peptide ratios without nor malization PEAKS will normalize the result when Automatically Normalize Peptide Ratios 1
192. ta e mzML e DTA file or a directory of DTA files e MGF e PKL e PEAKS 5 3 projects See Section 3 8 PEAKS 5 3 Projects Library Level Support The instrument vendor s software library is required to be installed on the same computer as PEAKS PEAKS will call the software library to read the data directly e RAW file Thermo Fisher Scientific instruments See Section 3 1 Thermo Data e D directory Agilent instruments See Section 3 3 Agilent Data e LIFT or D directory Bruker instruments See Section 3 5 Bruker Data RAW directory Waters QTOF instruments See Section 3 2 Waters Micromass MassLynx Data Convertor Level Support Third party convertors are required Users need to install the required convertors correctly 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 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 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 pro
193. tabase 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 section 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 Click the Add Update button to add the configured database The database name will appear in the Database List 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 line To be able to run PEAKS DB using a specific taxonomy corresponding files must be downloaded and then referenced by PEAKS in the Taxonomy Options section Taxonomy files for NCBI nr database are gi taxid prot dmp gz and taxdmp zip for UniProt Swiss Prot they are speclist txt and taxdmp zip 1 To download the taxonid file click the Download button A window will appear confirming the
194. tarting point for the analysis Click OK when ready Note If data is not yet refined you also need to specify the data refinement parameters first then click next Refer to Chapter 7 Data Refinement 3 Wait for the analysis to finish A new result node will appear in the Project Tree or several result nodes if PEAKS PTM or SPIDER are enabled Double click the last node to examine the analysis report StartPage X E us SPIDER 7 30 May 12 18 50 X ry Project View Si D Peaksworkspace derbyServer serverDB In depth Protein Analysis jg DENOVO 4 30 May 12 18 50 p PEAKS 5 30 May 12 18 50 D US peave oo 2n h4 q Show top proteins in each group rc T Accession Proteins ll P02769 ALBU BOVIN 473 04 T i 18 5 EME ASP IDER 7 30 May 12 18 50 i E J Trypsin Ei FEBSA Trypsin 1 RAW Lo DATA REFINE 1 30 May 12 18 50 E J Lysc E F2 BSAL ysC L RAW L 4 DATA REFINE 3 30 May 12 18 50 EH Jl Gluc Ely F3 BSA GluC 1 RAW i j DATA REFINE 2 30 May 12 18 50 H Azvaz4 ALBU MACFA H PO2758 ALBLI HUMAN De novo only Peptide Protein Summary Coverage Peptides Denovo Tags 2 Set PEAKS Parameters After selecting a data node in the Project Tree click the PEAKS DB toolbar icon ti The PEAKS DB parameters dialog will appear 69 Peptide PTM and Muta tion Identification PEAKS DB PEAKS PTM SPIDER PEAKS Search Predefined parameters default Error Tolerance Parent
195. tdes Unique PTM Avg Mass Description Mark H E O Proteins v i il PO2769 ALBU_BOVIN 469 20 NEM 7 438 214 69294 Serum albumin OS Bos taurus GN ALB PE 1 SY 4 A 3 D P14639 ALBU SHEEP 37 63 IEEE NH 75 155 3 BeamaBcHe rm 69188 vi je x gt PO8835 ALBU PIG 287 37 1 EIL NE PH E 67 1 Bdaced mem 69692 vj e P49064 ALBU FELCA 279 69 NW MN i MILE 31 66 4 gH t 9 9 Q28522 ALBU_MACMU 258 4 NNNM 1 MMM 1M1 310 52 0 Bacon 67881 vi o O QSWHS ALBU PONAB 245 03 IRR E LUN RINT 33 46 4 escamnhnmuBeEm 69367 Serum albumin OS Pongo abeli GN ALBPE 2SV 1 Y 2 O P49822 ALBU_CANFA 259 28 MII MMMM 29 47 4 Bascarnnnnam 68605 Serum albumin OS Canis familiaris GN ALB PE 1S Y 8 0 P35747 ALBU HORSE 24588 Fi MEI 1 21 31 1 BudENZEBmI PL 68599 Serum albumin OS Equus caballus GN ALBPE 1S Y P49065 ALBU RABIT 251 56 EINE P E Eos 36 2 giansBen P J 68910 Serum albumin OS Oryctolagus cuniculus GN ALB Y QSXLE4 ALBU EQUAS 221 40 fat toma T i 19 25 2 eaaet 68539 albumin OS Equus asinus GN ALBPE 2SV 1 Y Coverage Peptides Denovo Tags sp P02769 ALBU BOVIN Serum albumin OS Bos taurus GN ALB PE 1 SV 4 Eu F 9 outline coverage 1 Y de novo only tags sharing 6 5 Ads 3 5 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCY Y confident PT min ion intens 80 5 AAs per ine Y 10AA gap 101 11 144 1 P
196. ted and searched with the amino acid sequence Display absolute intensity in PEAKS Q Users now can choose to display absolute intensities or ratios in PEAKS Q results For the ratio display users can choose which sample the ratio is based on More statistics in the summary view The PTM profile table is improved A protein FDR value is added One click specification of common FDR values e g 1 in the FDR selection pane Better Community Support A new configuration wizard to assist the download and installation of public protein databases and raw file readers convertors Major accuracy and sensitivity improvement on the analysis of AB SCIEX TripleTOF data Proteome Discover support Now PEAKS can load the pepXML result file generated from Thermo s Proteome Discover software Support mzIdentML result format Now PEAKS can export mzIdentML file for downstream analysis such as Scaffold PTM Export high resolution images of the spectrum annotation and protein coverage view Export to website or single webpage format for easy sharing of results 4 Quick Walkthrough In this section we present a quick walkthrough of a typical data analysis and result visualization process By using the sample project included in PEAKS installation we first introduce the main GUI of PEAKS and showcase how to filter and visualize the analysis result Sections 4 1 4 4 This will help understand what can be accomplished with PEAKS After that we demonstrat
197. tide score 10lgP A Decoy B Target B Decoy Target 4 4 Result Visualization Besides the summary view there are three other views protein peptide and de novo only for visualizing the results in different ways The protein view contains a list of proteins passing the filtration The proteins identified with the same set or a subset of peptides are grouped together The peptide shows all the peptide identifications passing the filtration The multiple spectra that identified the same peptide sequence are grouped together e The de novo only view shows all the peptides identified exclusively by de novo sequencing Here we only focus our attention on the new protein coverage view in PEAKS 6 Click the protein view tab and select one protein The following protein coverage will show at the bottom of the protein view The protein coverage view maps all peptide identifications of the selected protein onto the protein sequence It enables the effortless examination of every PTM and mutation on each amino acid Some most commonly used operations on this protein coverage view are listed in the following see screenshot below 1 Each blue bar indicates an identified peptide sequence A gray bar indicates a de novo only tag match Peptide identifications with the same amino acid sequence and the same interesting PTMs are grouped to gether and displayed as a single bar A PTM is interesting if it s checked in the displa
198. ting 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 Protein JD Cover Map PEAKS 9 27 Apr 11 19 00 PEAKS 10 27 Apr Ts 14 PEAKS 12 27 Apr if 19 54 P42656 RAD24 SCHPO a POC7JSILRFN1 RAT n 1 1 Q684U9 PRDX1 MYOLU Boe 125 49 16 6 5 26 00 33 17 131 11 16 b 5 26 09 33 17 139 03 25 7 30 43 37 19 Q9NY65 ITBAS HUMAN gum 82 2 p 5 17 13 13301 6 3 bo 7 5 1L8 112 14 5 3 7 5 111 8 A2A3NGIPIPSL HUMAN goo E 1 1 1 2 1 97 Q7SBUSIVID21 NEUCR DeO 0 n 1 1 0 62 0 69 Q85QHS ADT2 BOVIN HEN 53 54 3 1 1 2 38 4 35 182 4 2 1 4 76 7 38 51 5 3 1 1 2 38 14 36 P10096 G3P_BOVIN geo 59 15 2 1 1 2 78 4 2 118 76 6 2 2 5 56 10 51 QSZKHAINDEL 1 CHICK 00w 5143 2 1 1 2 22 3 5 P30084 ECHM_HUMAN gee 47 77 2 2 2 6 06 13 45 37 47 1 1 1 3 03 6 21 1987 2 2 2 5 05 112 76 P35998 PRS7_HUMAN Oo 148 99 2 2 2 3 33 14 85 ALIMBLIRNEC_YERES osc 0 1 1 1 1 18 1 58 P24724 HSP90 THEPA NEN 56 02 2 1 p 0 92 11 94 5779 1 1 p 0 92 1 94 183 52 3 2 p 183 3 33 Q8PD231CH60 XANCP CIC 0 2 2 2 3 23 5 68 A4WWS9 DNAK RHOSS LIC 65 32 5 1 p L3 2 52 ATEDF3 IRS4 SCLS1 Ma a 0 il 1 1 0 97 2 28 QSCGPOIH2836 MOUSE goo 189 94 10 2 1 7 14 19 05 P09103 PDIA1 MOUS
199. uilt In gt TOF TOF lt Built In gt Ion Trap Instrument Details Instrument Name Orbitrap Orbi Trap Basic Options Ion Source ESI nano spray mi MS Precursor Scan FT CR Orbitrap Msn Product Scan Linear Ion Trap Advanced Options Precursor mass search type Monoiostopic Average Parent mass error tolerance 15 0 ppm mi Fragment mass error tolerance 0 5 Da Built in Instruments The names of the built in instruments are provided in the Instrument List Select an instrument to view the detailed instrument information in the Instrument details panel below Note The details of a built in instrument cannot be deleted or edited Create a new instrument 1 Click the New button and provide a name for the instrument in the Instrument Details panel 2 Next fill in the details in the Basic Options panel 3 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 4 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 142 Configuration and Preferences Use the MSn Product Scan drop down list to select the
200. vide 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 26 Loading Data to a PEAKS Project 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 The PEAKS Config Wizard can download and install MSFileReader automatically see Section 2 Instrument Selection MSFileReader is publicly available and can be found at the following link http sjsupport thermofinnigan com peg file MSFileReader zip Converting with MSFileReader will only work if there are English only characters in the file path 3 2 Waters Micromass MassLynx Data Two ways can be used to load Waters s raw data e Library Level Support wolf exe RAW data from Waters instruments can be imported provided that MassLynx 4 1 software 1s installed on the same computer as PEAKS MassLynx 4 0 users can download a different version of wolf exe Command line can be used to convert raw files to mzXML with wolf exe The file Peaks installation directory wolf exe can be replaced with the program compatible with MassLynx 4 0 For links to different versions of Wolf vis
201. whose corresponding spectra only have low confidence database matches High confidence de novo sequences mean the TLC and ALC score of the sequence passes the corresponding user specified score threshold A low confidence database match means the peptide 10lgP 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 on how to use it 4 Filter PEAKS 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 change a score threshold the Apply button changes color to remind you to apply the filter by clicking it StartPage X 9 SPIDER 7 30 May 12 18 50 X De novo Only TLC 2 3 and ALC 50 and peptide 10gP FY 10 2 Apply Filters Export Notes Peptides 10lgP z 10 2 FDR Proteins 10l0P 20 land 0 unique peptides tein Summary Peptides The threshold here will affect both Peptide and Protein Views and therefore has to be chosen with caution for the peptide view only peptides with 10lgP score above the threshold will be kept in the table For the protein view the number of supporting and unique peptides is based on the filtered peptide results If the Estimate FDR with decoy fusion option was turned on in t
202. xport 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 Quantification LFO 32 Loading Data to a PEAKS Project 5 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 LFQ 6 To delete a sample or data file select the appropriate node sample or data file and click the Delete button 7 To change the order of the samples within a project or data files within a sample using the Up and Down buttons 8 Click the OK button once all data files and samples are added to the project 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 i m To open a saved project select Open Project or Open Recent Project command from the file menu or from the toolbar 2 To add data to an existing project choose the project from the Project View panel and select the Add Data PE command from the file menu or use the add data icon G f
203. y changes Searched Tags Selected Tags EN wl r 1 D is L T YA WF Mr MM LT YAV EN Undoing an edit If an error has occurred during 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 the pop 66 Peptide De Novo Sequencing up menu to return to the previous peptide sequence This button can be used multiple times to return to previously made edits Manual De Nowe 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 When correcting an error made during sequencing if the Undo button is selected too many times right click the mouse and select the Redo command from the pop up menu with the peptide candidate still selected in the Result panel This button can be clicked multiple times to return to later stages in the edit Error Tolerance To set the mass error tolerance in manual de novo sequencing with the peptide candidate selected in the Result panel right click the mouse and select the Config Error Tolerance in Manua De Novo command from the pop up menu to open a dialog where the error tolerance can be set PTM Configuration To identify post translational modifications PTM
204. y 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 a replicate number in two ways in the New Project window when adding a sample to a project and in the quantification window when setting the label free quantification parameters 113 PEAKS Q Label Free Project Name IfaProject2 Project Location D test Peaks54 PeaksProjects ij lfgProject2 Sample Name Sample 4 S A R151 Sample 1 1 070828 O1 02 070824 DMSO Dh 1 m Enzyme Specify later Add data les and SL R152 Sample 2 5 070828 O1 02 070827 drug Oh 1 R Instrument FTMS FT Trap E a R251 Sample 3 Fragmentation CID 070828 O1 03 070824 DMSO 0h 2 O GREER 15 070828 O1 03 070827 drug Dh 2 R 1 eM 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 beside 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
205. y option see item 5 Overview 2 PTMs and mutations are highlighted with colored icons and white letter boxes Highly confident PTM and mutations are displayed on top of the protein sequence A PTM or mutation is regarded as confident if the two fragment ions at both sides of the modified residue have relative intensity higher than the user specified threshold in the display option see item 5 3 Click a peptide to show the spectrum annotation 4 Mouse over an amino acid to show the supporting fragment ion peaks 5 Options to control the coverage view display The coverage outline choice turns on off the peptide bars The de novo only tag specifies the minimum number of consecutive amino acid matches between a de novo only sequence and the protein before it can be displayed as the gray bar e The confident PTM specifies the minimum fragment ion relative intensity in one of the MS MS spectra before a PTM location is regarded as confident and displayed on top of the protein sequence The checkboxes in the PTM list specifies which PTMs are interesting Click the color boxes to change a color Double click a PTM name to see the PTM detail 6 The full screen button and tool box button Full screen provides a larger view of the coverage The tool box provides some common tools such as exporting the coverage pane as a high resolution image file Coverage Peptides Denovo Tags gt sp P02769
206. ystem clipboard If Cancel was selected click OK on the dialog detailing the copy to the clipboard Next open a browser and paste the URL into the address bar When the file download window opens click Save 4 Once the database has been downloaded check to see if it is compressed If so extract the file using a program such as WinZip or WinRar The desired result is a FASTA format text file a fas or a fasta file 5 Move the database file into a directory that PEAKS can access 140 Configuration and Preferences 6 Click Browse to inform PEAKS about the location of the database file 7 If the selected database is an EST database check the box labeled EST database If not ensure that it is blank 8 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 9 If the configuration dialog was invoked from the toolbar click the Add Update button and then OK If the c

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