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PEAKS Studio Manual 4.0 - Bioinformatics Solutions Inc.

1. Accession Mass Score Coverage Query matched Marked Description PEAKS DB Search o SN 1sp P00 97 158 100 62 85 75 1spiP00489 PHS2_RABIT GLYCOGEN PHO H 1spja29 77 753 100 48 15 34 1sp Q 29443 TRFE_BOVIN SEROTRANSFER SN 1sp P02 25 107 100 63 84 14 1sp P02666 CASB_BOVIN BETA CASEIN PR SN 1sp P02 69 294 100 23 89 15 gt 1sp POZ769 ALBU BOVIN SERUM ALBUMIN SN V00494 1H 69 296 26 7 88 4 Human messenger RNA for serum albumin ALBU HUM ano 1 Dl 02768 homo 3 A09561_ NM U22961 Filename ca20 Jun 06_1 29 02 _12mix IPI NR custom_reportxls M12523_ Format xls x AF190169 Export only marked proteins AF13007 Full protein and peptide report homologous proteins grouped Si A00279_ X A15293_ Protein and peptide report for currently highlighted homolog group Complete protein list peptide details omitted A30884_ Current marked proteins and corresponding peptides SN 1sp P00 1Sp P02 E A 1Sp P00 4 028 608 100 62 93 12 O 1spiP00921 CAH2_BOVIN CARBONIC ANHY gt This section of the report behaves like an index listing each protein found in the sample Very similar proteins containing the same set or a subset of the matched peptides are grouped together To see the full list of proteins within each grouping click the sign In the example above the Bovine Serum
2. max missed cleavages 3 Edit Enzymes New database Edit database PTM selected for search Taxonomy selection Fixed Carbamidomethylation Mycobacterium var mOxidation Escherichia coli Fungi max variable PTM per peptide Add Remove PTM Set Taxa Deconvolute the data for analysis use for non deconvoluted data Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search 8 have already run de novo don t run it again Run de novo using different parameters than the above Run de novo using the same parameters as above default O Parent mass error tolerance determines how much random and systematic experimental error on the parent precursor ion PEAKS will account for in its analysis Select a tolerance from the dropdown list Fragment mass error tolerance determines how much random and systematic experimental error on the fragment daughter ion PEAKS will account for in its analysis Select a tolerance from the dropdown list Instrument choose the type of spectrometer that produced our data Choose from a dropdown list Enzyme choose from a dropdown list of enzymes that we used to digest our protein sample Click the Edit Enzymes button to edit the enzymes defined in this list or to add to it Report top set how many de novo seq
3. Pa Save As Cancel ers After entering the settings as shown click the Save As button to save these parameters for future use When prompted type OrbiStandard and press enter Click OK to commence analysis Analysis will be initialized most of this time is spent creating a partial database which only has to be done once this takes one or two minutes After this de novo sequencing will commence For this sample it takes just over a minute after which PEAKS database search will proceed In total the process takes 16 The numbers under the Mass heading represent the mass of the protein displayed The Coverage numbers represent the percentage of the proteins sequence covered by the matching peptides less than two minutes for this sample depending on the system s processing speed and memory The PEAKS auto de novo algorithm derives sequence candidates for each of the eleven spectra in our example data file These sequence candidate results for all eleven spectra in the example are then used for the database search component of PEAKS Studio 4 0 PEAKS uses a unique sequence tag plus peptide fragment fingerprinting approach to protein identification After the search is finished the protein identification results will appear on screen The Peptide View is displayed by default The display shows each spectrum for which PEAKS found a matching peptide
4. 36 Width and Height together these determine the size of the output image measured in pixels Format select an image file format from the drop down list Bitmap JPEG and Graphics Interchange Format are supported Filename type in the textbox or browse to a file a to enter the file name of the image that will be created Export selected area the default image output is the full spectrum as shown in the spectrum alignment frame checking this box will allow us to print one of the other items For example if we have zoomed in on a portion of the spectrum and wish to print that zoomed in view we click the export button and select the Export selected area checkbox then the current spectrum window radio button 37 Print Image Dialogue m Paper Size kee El Source Automatically Select y m Orientation Margins inches Portrait Left Right E C Landscape Top o Bottom o The default print output is the full spectrum as shown in the spectrum alignment frame If we wish to print something else we must use the export image functions and then print the image from another application Cancel Printer Toolbars Main window toolbar S Open data file button This allows us to open a raw data file built by our mass spectrometer or a PEAKS data file in ANZ format that also contains peptide analysis data The file should be in PKL DTA MGF or ANZ format 5
5. Bioinformatics Solutions Inc PEAKS Studio 4 0 User Manual www BioinformaticsSolutions com BIOINFORMATICS SOLUTIONS INC PEAKS Studio 4 0 User s Manual Bioinformatics Solutions Inc 145 Columbia St West Suite 2B Waterloo Ontario Canada N2L 3L2 Phone 519 885 8288 Fax 519 885 9075 Written by Iain Rogers Please contact the author for questions or suggestions for improvement INTRODUCTION A OU 3 INTRODUCTION TO PEAK STUDIO A Di a A IES tier eben A 3 HOW TO USE THIS USER S MANUAL ary GETTING STARTED WITH PEAKS STUDIO 4 0 ccsssssssssssssscesssssssscssscsssesssssescsssesesssssssessssessssessnssssesssssssessssasesseseasessessssssessssassssessassesessessssessnee 7 WHAT WE AVL NEED ie Eae IA OSCE Sc EO AREA AAA O RS Ui Ee 7 Package contents 7 System requirements Instrumentation 47 INSTALLATION ANO 8 REGISTERING PEAKS voca ia 9 DATABASE CONFIGURATION A A I 9 FEATURES WALKTHROUGH wecirscccccisccesccsorseseassvsnscstensnsesssexesestsstesessenestsoatsessescstasssoatenecesceursossnseqeesscessnosensereeseseseass eseneessnctvnssdessyuesseesdees essyseseeacseese 15 BEGIN THE WALKTHROUGH neehoor riiai iii EEE EEEE EEE E SEV E E A E E A a a ea aa 15 GRAPHICAL USER INTERFA CEivicsscecsvcsacsvesscntsnessonnshenceadedusssatsdnesdatcontsuatscnvscatesnesenedsvesdecsseesveocsvaccevessacedadvuscesesuestsensvesdssnseussssucvveussvcevenssbenveaasvenssente 22 WINDOWS DIALOGUES FRAMES AND REPORTS hee r i e
6. Close data file button Close the selected data file Press this after Orientation paper orientation is shown in the picture at the top Change this by clicking the Portrait or Landscape radio buttons Paper Set the paper size and soutce by selecting from the appropriate dropdown list Printer button pressing this will bring up another dialogue where we can select from a list of printers installed on our machine Ok button this will commence printing selecting a data file in the Peptide Data Frame Save data file button Save any changes made to the file a will appear next to any file that has been changed The file will be saved in the ANZ format Press this after selecting a data file in the Peptide Data Frame E Save all files button Save all files Any changes to files will be saved in the ANZ format 38 e 2 l Copy button Copy selected spectrum data Cut button Cut selected spectrum data Paste button Paste spectrum data into the selected data file Manual merge spectra button After selecting more than one spectrum in the peptide data tree this button becomes enabled Right click it to merge these spectra into a s ingle MS MS spectrum and remove the old ones Data Refinement button Merge scans of the same peptide remove noise spectra preprocess within each MS MS spectrum and recover peptide charge state The data refinement options
7. We can toggle whether or not we d like to see the positions of the y ions and b ions and the proposed residues in sequence between them on the alignment view by pressing the y ion alignment M and bion alignment M E icons in the main processing window toolbar To view another peptide candidate as determined by auto de novo click on another peptide in the Peptide Candidates Frame and under PEAKS Auto De novo The information in the Ion Table will change as will the tags on the spectrum to reflect the selected peptide candidate s sequence Editing sequencing results preparation We cannot change the results provided by PEAKS auto do novo or PEAKS database search However we can make a copy of any sequence and edit it using manual de novo techniques To copy a sequence for editing 1 Select a peptide sequence candidate from within the Peptide Candidates Frame We can only select one peptide sequence candidate at a time 2 Right click the mouse button while holding the mouse over that sequence A popup menu will appear 67 3 We can select the popup menu item Copy for manual de novo In this case the sequence will be automatically placed under the Manual De nove heading A Manual De nove heading will be created if there wasn t one there already 4 Now we select our newly copied sequence under the Manual De nove heading to display this sequence in the Ion Table Frame Spectrum View Frame and Spectr
8. istry URL to get detailed information with accessionid htto srs ebi ac uk srsbinicgi biniwgetz e UNIPROT AccessioniID Delimiter Taxonomy Options aA A _Aa ____ ________________ _ taxid Browse taxdmp Browse Previous Next Finish Cancel e PEAKS will ask us to enter the database nickname This is a nickname that we chose to represent the database we are configuring It doesn t matter what name we enter but we must enter at least one character e The Path textbox shows where the database is located It will be blank so we must tell PEAKS where the database is Type the location of the file into the textbox or we can browse to find the file on our system We must sure to select the FASTA database not the compressed file of the same name see step 5 12 Database header format is important for protein ID result reports If parsed correctly accession numbers and protein names will be shown in full If we chose one of the public standard databases in step 3 its format style will be displayed in the advanced options box The selected database format is shown in the dropdown list Accession number information and the way PEAKS parses the database headers i e the parsing rules are shown in the textboxes below If our database is an EST database containing DNA sequences check the EST database checkbox If we ch
9. To export protein identification results including protein and corresponding peptide information to an MS Excell file right click in the protein view PEAKS will then ask us which portion of the results we d like to export The resulting xls file contains a collapsible list 87 About Bioinformatics Solutions Inc BSI provides advanced sofovare tools for anabssis of biological data Bioinformatics Solutions Inc develops advanced algorithms based on innovative ideas and research providing solutions to fundamental bioinformatics problems This small adaptable group is committed to serving the needs of pharmaceutical biotechnological and academic scientists and to the progression of drug discovery research The company founded in 2000 in Waterloo Canada comprises a select group of talented award winning and intelligent developers scientists and sales people At BSI groundbreaking research and customer focus go hand in hand on our journey towards excellent software solutions We value an intellectual space that fosters learning and an understanding of current scientific knowledge With an understanding of theory we can focus our talents on providing solutions to difficult otherwise unsolved problems that have resulted in research bottlenecks At BSI we are not satisfied with a solution that goes only partway to solving these problems our solutions must offer something more than existing software The BSI team recognizes that
10. a LE The spectra are grouped sorted by index number Since a spectrum may match to more than one peptide there may be more than one entry per spectrum The list is sort able click the heading on each column to experiment with sorting by score by mass etc Work Flow Report Filename PEAKS Orbi_Lactoglobulin pkl done Click the Protein View tab PEAKS Studio 4 0 presents a list of proteins that it believes to be the best match for the sample The top section is an index listing them by accession number ranked in descending order from highest score on downward Accession Mass Score Coverage Query matched Marked Description PEAKS DB Search LJ X gil229460 18 367 100 61 73 18 gil28972720 78 207 29 0 84 1 y lactoglobulin beta mKIAA1321 protein Mus musculus The correct protein Lactoglobulin beta is shown at the top of the list and with high score Since one cannot distinguish between the different forms of Lactoglobulin Beta PEAKS Studio 4 0 groups them all together thus avoiding cluttering the report Click the plus sign next to gi 229460 for a listing of other possible lactoglobulin The peptides matching these homologues will be the same set or a subset of Cytochrome c matches Collapse this list of homologues by clicking the minus sign next to gi 229460 The listing as shown above is simply an index We will find this useful in the future when dea
11. 9 673 3 1 672 37 GLDIQK 672 38 0 0028 19 9 14 gi 229460 Y 10 6744 1 673 41 IPAVFK 673 41 0 0002 76 78 83 gil229460 Y 10 6744 1167341 LPAVFK 673 41 0 0002 76 24 29 gil 2659199 Y 12 697 7 3 2 090 LDAINENKYLYLDTDYKK 2 090 0 0022 100 84 101 gil229460 Y Y 12 697 7 3 2 090 IDALNENKYLYLDTDYKK 2 090 0 0022 100 84 101 gil2194089 Y Y 13 771 7 3 2 312 VYVEELKPTPEGDLEILLQK 2 312 0 0044 100 41 60 gi 229460 Y Y 14 818 3 2 1 634 TPEVDDEALEKFDK 1 634 0 0067 100 125 138 gil229460 Y Y 16 9031 3 2 706 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 0 0007 99 15 40 gil229460 Y Y 17 903 5 1 902 56 TKIPAVFK 902 55 0 0013 3 76 83 gil229460 Y 18 933 5 1932 53 LIVTQTMK 932 53 0 0012 34 11 8 gil229460 Y 18 1933 5 1 932 53 INTQTMK 932 53 0 0012 34 1 8 gil 2079 Y 20 1065 1 1 064 VLVLDTDYK 1 064 0 0035 5 92 100 gil229460 Y 21 1157 2 2 312 VYVEELKPTPEGDLEILLQK 2 312 0 0085 100 41 60 gi 229460 Y Y 22 1354 2 2 706 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 0 0037 90 15 40 gil229460 Y PEAKS displays the same Peptide View as before Now X Tandem search results are considered as well Where X Tandem agrees with a PEAKS assignment there s a checkmark in the Peaks column and a checkmark in the X Tandem column Also the score on this peptide is increased in this way we increase confidence in the assignment Since the
12. AEKNFDLK For spectrum 2 there were three possible peptides matching for number 3 there were two and so on Each peptide is given a score and the protein it matches is displayed for reference under Accession No If the report was generated after inChorus protein ID each search program that agreed on the peptide is given a checkmark under its column This list is sort able Click on a column header to sort the list using that columns values For example sort by score Clicking again on the same column header will toggle ascending descending sort When sorting the grouping by MS MS scan number will be retained except when sorting by Accession No Clicking on this report will highlight the spectrum in the Peptide Data tree on the left Select multiple spectra by clicking and dragging using shift click or ctrl click In this way the highest scoring peptides may be selected and isolated for further analysis Protein view is accessed by clicking on the Protein View tab It collects all the peptide identifications together summarizes which proteins were present in the sample and groups homologous proteins together The same information is displayed in the peptide view as in this protein view however the results are organized to best enable us to evaluate at the protein level 79 Try the features walkthrough in Chapter 3 for more help with viewing Protein ID results Peptide View Protein View Search parameters
13. Albumin node has been expanded to reveal several similar proteins Clicking in the same place now a sign will collapse the list This view is helpful when building a summary that can be sent to a customer collaborator Simply right click to export to an MS Excel file We can export interesting parts of the report or a whole summary Mark proteins of interest by clicking their checkboxes and export protein and peptide information for those Or highlight a homologue group and export proteins and peptides in that group Or just export the whole report Whenever we click on a protein the Matched Peptides panel bottom changes to display the spectra and peptides that were found to be supporting evidence for that protein It may be necessary to scroll this panel down to see the complete list Clicking a peptide in this list brings up the Main Processing Window for the corresponding spectrum and displays the ions that were found in support of this peptide Scrolling the Matched Peptides panel further down reveals the complete sequence of the highlighted protein with the matched peptides highlighted in red Where an EST database was used the translated sequence is shown with all six reading frames concatenated Manually Manipulating Data Files A note on preserving data results integrity Protein ID and de novo sequencing results obtained for a given dataset prior to use of the tools in this section may become invalid since some spec
14. Copying spectrum data will duplicate the spectra when pasted To cut copy spectrum data Select a spectrum by clicking on its name select multiple spectra by holding down the control key and clicking on any number of spectrum in the Peptide Data Frame Right click on one of the selected spectra A small pop menu will appear Select Cut or Copy OR Click the Copy button or Cut button e in the main toolbar Copied Cut items will remain on the clipboard until replaced by another copied cut item Warning unless pasted a cut item will be lost as subsequent cut copied items will displace it from the clipboard Pasting Spectrum Data After having copied or cut spectrum data we would like to paste it into another data file or the same data file To paste spectrum data 1 Select the data file into which we wish to paste the spectrum or spectra by clicking on its name in the Peptide Data Frame We may only choose to paste into one data file at a time If we know which proteins to look for we can create our own FASTA database to search against 2 Right click on one of the selected spectra A small popup menu will appear Select Paste from the popup menu OR Click the Paste button Bl in the main toolbar The pasted spectra will appear in the Peptide Data under the data file into which we pasted Selecting unmatched de novo results When working with unknown organisms a
15. and place the files we copied here But we re not finished we must also register these files with Windows 3 We can find a file on our system called regsvr32 exe using the Find or Search tool in our windows start menu It is probably in C WINDOWS System32 If it s not there substitute the correct location in step 4 4 Open a command prompt or the Run tool from the start menu and type the following C WINDOWS System32 regsvr32 C MassLynx DACServer dll All on one line with one space in the middle as shown Press the enter key If successful windows will pop up a success message Please check the license The libraries mentioned in this section are part of the MassLynx software distributed by Waters corp Please check the MassLynx license agreement or contact a Waters representative to make sure it is okay to copy and use the libraries in this way Importing Data from the ABI 4700 or ABI 4800 BSI has created a converter to extract the data from an ABI Oracle database If we require this separate free tool we must ask a BSI representative Once installed we can start up the ABI 4700 Data Extractor from the Start menu System Requirements This extractor can be installed on the same machine as ABI 4700 Explorer and the Oracle database we will call this machine the 4700 SERVER in the following instructions or another machine that has direct network access no firewall no proxy required to the 4700 SE
16. anew database iii sro sndo corda AANE toa cap dunas ado sa ven duda da de a s da dada da ad da ada 44 Remove a Database Edit a Database Moving Updating a Database s IMPORTING AND EXPORTING PEAKS PROPERTIES sczsvsvesessssseseavsavisavsaveseswsavesesvsasadegasavescaaeascesvaavsaeaieasedesvnaseueavussedeguusaeusaves pounquvssesusiwanoveavvaresessvasousaeeanes CONFIGURING THE JON TABLE 225 sc0sccceucessassusessscavdeccescdacesdausesovaansissutassecsse asus ssbaihnescatocuesedecsuscseacduscstas usessigcesessubeedevsaedasevsuidesevsancasedbandosatsancissnbasessaxesuass PEAKS ENVIRONMENT PREFERENCE CONFIGURATION NH AY ERO TAG Batre OEE eR CECE eS EE Ida a EE ARS CRT COTTIER RGE TOTTI RORTT RECT ERT CRETE CORTE Colors EAEE EE Sena Rest igh CURTE RES CAN Susi ROS EE SRA GTA VR si a CRU a CV Gh Ng a vive igh Ae ah Ca igh Ga da a Rb Sata e LAS av ote 50 Manual de novo Removing Saved Parameters PEAKS STUDIO USAGE 0 0 ssscccsssscssscedsacssssevisconndetncossescneesedsessocsasscsnnsdsssswrsaserossedsorestacdsvesendosssevsssesapsedaavoseseveveseesbiveondestnapdedseesoesieesousc de acscoseswssdsdesnseeen 53 LOADING DATA INTO PEAKS STUDIO 4 0 ri sitas 54 Opening data files siena pea Es studs Aa asi ONER AAA ear AA AA AA alo ua Sete Tha seep ati 54 Loading a directory full of DTA fles oirnn st a e deb dede dan aa E N a ok Geeta si 55 Loading Thermo RAW data kes ccacccccseccesssun tenets aara ns tons se
17. customized PTM at any time and the built in PTM will reappear Creating a New PTM To create a new PTM we open the PEAKS Properties dialogue ensure that the PTM tab is selected and click the new button To create a new PIM on the fly while setting up PEAKS auto de novo or PEAKS Protein ID click the new PTM button while selecting PIMs The PTM Editing dialogue will appear 42 PN PTM Editing Abbreviation tst Mass Monoisotopic 57 992905 Neutral Loss Mass Monoisotopic Formula CNO2 Residues that can be modified Non location specific AC N terminus DEF C terminus GHI Middle Only L Rule This is not a real modification It s for demonstration purposes only Figure 1 Create new PIM Now we type information pertaining to our PTM in the appropriate boxes see above section on Interface for a more in depth explanation of these fields At a minimum we must enter a name a mass and one residue that may be modified Enter the mass of the modification either by typing in its monoisotopic mass difference directly or by entering its empirical formula It is unnecessary to do both each will override the other Click the Ok button to save changes and create our new PTM or click the Cancel button to exit discarding changes After clicking the Ok button we return to the PEAKS Properties dialogue to find that our new PTM is listed at the top of the PT Ms lis
18. from 1 4 from the drop down list in the middle 3 Click the Add with charge button For example configure the ion table to display y ions by selecting y from the list on the left and 2 from the dropdown charge list Remove ion types from the ion table i e remove columns from the table by selecting one of mote items in the list on the right and clicking the Remove button PEAKS Environment Preference Configuration One of PEAKS Studio 4 0 preferences PEAKS Environmental Preference allows us to customize PEAKS Studio 4 0 to our needs PEAKS Environmental Preferences include Environment Color and Manual de novo and Parameters To edit PEAKS Studio 4 0 Environment Preferences Click the EE icon in the toolbar Or from the Edit menu select Configuration then Environment Preference The Environment Preferences dialogue will then appear This dialogue box has four tabs Environment Color Manual de novo and Parameters Clicking a tab will allow us to edit the Environmental Preferences corresponding to that tab 48 Environment Preferences Environment Color ManualDe Novo Parameters Default Data Input Directory ca 8 Previous directory User directory Browse Default Data Output Directory CA 7 e Previous directory User directory Browse Default Configuration File Directory CiDocuments and Settingsiirogersi peaks Clo
19. it then clicking Open The data file will appear in the left hand frame Make sure OrbiOrbi pkl Le the data file is selected In the Tools menu select Protein Identification The protein identification options dialogue will appear 15 Enter the settings as shown Settings can be changed by clicking on the drop down list and selecting one of the options D Work Flow 132 gt Merge Spectra 584 auto De Novo 42 inChorus protein ID E Mass Calculator 792 92 Database Search Options Protein Identification Parameters Saved parameters Iotof standard Parent Mass Error Tolerance 0 01 Fragment Mass Error Tolerance 0 01 Instrument FT Orbitrap CID SORI Database to search Enzyme Semi Trypsin NR max missed cleavages 3 Edit Enzymes New database Edit database PTM selected for search Taxonomy selection Mammalia max variable PTM per peptide Add Remove PTM Set Taxa Deconvolute the data for analysis use for non deconvoluted data Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search O Ihave already run de novo don t run it again Run de novo using different parameters than the above 18 Run de novo using the same parameters as above default
20. on the spectrum we open the Environment Preferences dialogue and ensure that the Color tab is selected Environment Preferences Environment Color Manual De Novo Parameters Please choose color for Spectrum_Peak Freezed_Peak w Current_Peak Red Y_Alignment B_Alignment Y Jon K EEE SS rio y 230 Choose the object whose color we d like to change from the list at the left Then we click on the slider bar type in a number 0 to 255 in the textbox or scroll up and down on the arrows next to the textbox to select how much of the corresponding color we d like to apply Choose an amount for all three colors In the example above we ve chosen pure red 255 to represent a spectrum peak After we ve chosen colors we may click the Ok button to exit and save changes Manual de novo We may wish to sequence a peptide manually using spectrum data PEAKS Studio 4 0 provides us with a set of tools to help us do so We may need to tweak these tools to adjust for error tolerance and to customize the working environment To adjust Manual de novo options open the Environment Preferences dialogue and ensure that the Manual de novo tab is selected Environment Preferences Environment Color Manual De Novo Parameters E search parameter Maximum tag length 3 ES Maximurn return ie random machine error Default m
21. open the PEAKS Properties dialogue ensure that the Database tab is selected select a database from the list of databases and press the Remove button This will not permanently remove it from our system it may be reloaded follow procedure for configuring a new database at any time We can t edit the database itself from within PEAKS Studio 4 0 Edit a Database This tool allows us to change the name that PEAKS Studio 4 0 associates with a database taxonomy files and the header parsing rules for that database To edit a database we open the PEAKS Properties dialogue ensure that the Database tab is selected select a database from the list of databases and press the Edit button Moving Updating a Database If we choose to move a database to another directory or delete it entirely we should tell PEAKS We must remove the database from the list and re load it Until we do so the database name will appear in red in the list of databases and any protein identification using that database will fail Y PEAKS Properties Enzyme list PTM Library Database List of databases NR Dilirogersidatabasesinr_20051 120 fas SPROT Diirogersidatabasesisprotfas Set as default Cancel If we choose to update the database perhaps by downloading the latest database file and overwriting the old database file PEAKS will show the datab
22. peptide will not be modified more than a few times 61 and as such limit the number of variable modifications that can occur on each peptide Auto De novo Sequencing To begin auto de novo sequence derivation we 1 In the Peptide Data Frame select the data file s containing the spectra that we wish to sequence by Auto de novo We can also select an individual spectrum or a few spectra within a data file auto de novo will proceed on only the spectra selected 2 Click the Ed Automatic De novo toolbar icon Or Select Auto De novo from the Tools menu Or Right click on the selected spectra or data files and select Auto De novo from the popup menu The Auto de novo Parameters dialogue window will appear We should begin by using the suggested error values then try some slightly higher or lower ones to find the best result De Novo Options De Novo Sequencing Parameters Saved parameters lain1 Parent Mass Error Tolerance Fragment Mass Error Tolerance 0 1 Instrument Quadrupole TOF Enzyme Trypsin Report up to peptides 5 Edit Enzymes PTM selected for search Fixed Carbamidomethylation Var AceAib Var Acetylation Var Deamidation Var Phosphorylation Add Remove PTM Y Deconvolute the data for analysis use for non deconvoluted data Py Save As Ok Cancel 3 If we wish to change any of
23. problems with a database may not appear until we run a search While PEAKS is quite tolerant of format errors in databases other search engines called from the inChorus tool may not be If there is an error in the search it will be reported in a summary screen after the work has finished If there is a problem check the best practices outlined in this section If the problem persists it is possible that the database download was corrupted try downloading again Please contact technical support for help 14 See the section entitled Merging Spectra for more help Features Walkthrough Lets familianize oursehes with PEAKS his section of the manual will walk us through most of the basic functionality of PEAKS Studio 4 0 After completing this section we will have seen how easy it is to load and view a data file perform de nove sequencing and database search protein identification Begin the walkthrough Run PEAKS Studio 4 0 then download and configure the NCBI nr database The procedures for doing so are outlined in the previous section The demo sample data should load automatically on startup under the heading OrbiOrbi pkl If it is not loaded open the data file by clicking the sil icon on the toolbar in the upper left corner of the PEAKS window or selecting Open from the File menu Sample data is located in the C Program Files PEAKS StudioA data folder Load the file OrbiOrbi pkl by clicking on
24. toolbar select Save from the File menu or right click on the data file and select save from the popup menu This will save the processed spectra in ANZ format and of the same name as the data file we opened To change the name of the ANZ file choose Save as from the File menu or right click on the data file and select Save as from the popup menu We may then change the file name To save all currently opened data files select Save all from the File menu To export data to a PKL file we select the data file not an individual spectrum to export Then from the File menu select Export then Export PKL File The spectrum data will be saved in PKL format but all sequencing and protein data will be lost To export peptide sequencing results to a FASTA format file select the data file not an individual spectrum to export Then from the File menu select Export then Export Peptide Sequence The sequencing data will be saved in FASTA format but will not retain any spectrum data To export peptide sequencing results to an HTML file select the data file not an individual spectrum to export Then from the File menu select Export then Export HTML File Peaks will then ask us which results we would like to export We can choose from any de novo sequencing or protein ID run we have done Each will be listed with the parameter set we used
25. two tools take different approaches we may discover that PEAKS finds some peptides that X Tandem misses and vise verse Where this is the case only one checkmark will be displayed and the score is penalized slightly in some cases Sometimes we can find a good hit that the other search engine would have missed In this way we increase coverage Click the Protein View tab to see a summary of PEAKS and X Tandem s results at the protein level Thus concludes our walkthrough of PEAKS Studio 4 0 s basic features 21 Graphical User Interface A reference section to help us find our way around his chapter deals with interface elements It is meant to be used as a reference so we can look up certain interface elements when we get stuck For instructions on how to use PEAKS Studio to perform certain tasks the section entitled Using PEAKS Studio will be more instructive The first part of this chapter describes windows dialogues frames and reports This tells us what certain dialogue boxes windows and frames do and how to read them The second part of this chapter deals with toolbars Toolbars are a very useful way to quickly get at the functions we use most Windows Dialogues Frames and Reports PEAKS Studio 4 0 main window Comprises PEAKS Studio El Peptide data frame left This displays a listing of parent ions by m z and charge Clicking on one will bring up the its MS MS spectrum The colored d
26. whatsoever 4 Limitation of Warranty THE LICENSED SOFTWARE IS PROVIDED AS IS WITHOUT ANY WARRANTIES OR CONDITIONS OF ANY KIND INCLUDING BUT NOT LIMITED TO WARRANTIES OR CONDITIONS OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE LICENSEE ASSUMES THE ENTIRE RISK AS TO THE RESULTS AND PERFORMANCE OF THE LICENSED SOFTWARE 5 Limitation of Liability IN NO EVENT WILL LICENSOR OR ITS SUPPLIERS BE LIABLE TO LICENSEE FOR ANY INDIRECT INCIDENTAL SPECIAL OR CONSEQUENTIAL DAMAGES WHATSOEVER EVEN IF THE LICENSOR OR ITS SUPPLIERS HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGE OR CLAIM OR IT IS FORESEEABLE LICENSOR S MAXIMUM AGGREGATE LIABILITY TO LICENSEE SHALL NOT EXCEED THE AMOUNT PAID BY LICENSEE FOR THE SOFTWARE THE LIMITATIONS OF THIS SECTION SHALL APPLY WHETHER OR NOT THE ALLEGED BREACH OR DEFAULT IS A BREACH OF A FUNDAMENTAL CONDITION OR TERM 6 Termination This Agreement is effective until terminated This Agreement will terminate immediately without notice if you fail to comply with any provision of this Agreement Upon termination you must destroy all copies of the Software Provisions 2 5 6 7 and 10 shall survive any termination of this Agreement 7 Export Controls The Software is subject at all times to all applicable export control laws and regulations in force from time to time You agree to comply strictly with all such laws and regulations and acknowledge that you have the responsibility to obtain all necessary
27. 6 14 47 33_NR inChorus protein ID results og Bd Peptide View Protein View Search parameters Index miz z mass Peptide Mr Calc Delta Mass Score Start End Accession No Peaks XTandem 1 452 2 2 902 55 TKIPAVFK 902 55 0 0035 68 76 83 gil229460 Y 2 458 7 2 915 46 LDAINENK 915 46 0 0035 71 84 91 gil229460 Y 2 458 7 2 915 46 IDALNENK 915 46 0 0035 71 84 91 gil2194089 Y 3 467 2 2 932 53 LIVTQTMK 932 53 0 0053 35 1 8 gi 229460 Y 3 467 2 2 932 53 INTQTMK 932 53 0 0053 35 1 8 gil72079 Y 4 533 2 2 1 064 WLYLDTDYK 1 064 0 0048 85 92 100 gil229460 Y 5 545 9 3 1 634 TPEVDDEALEKFDK 1 634 0 0050 100 125 138 gi 229460 Y Y 7 597 3 2 1 192 VLYLDTDYKK SIE PE 0 0074 100 92 101 gil229460 Y Y 8 623 2 2 1 244 TPEVDDEALEK 1 244 0 0023 100 125 135 gi 229460 Y Y g 673 3 1 672 37 GDLQK 672 38 0 0028 50 619 624 gil28972720 Y 9 673 3 1 672 37 LGDLGK 672 38 0 0028 50 136 141 052782259 Y 9 673 3 1 672 37 LGDIQK 672 38 0 0028 50 736 741 gil63586345 Y
28. AA TIO AS A AAA A AAA AAA A A AA AL Redoing ad edit assisi iesse SUGGESTING A SEQUENCE TO SEE HOW IT FITS THE DATA PROTEIN IDENTIFICATION ccs cccesseceeseeeeeseeeeeeneeeeaeees Se PEAKS proteiniidentification serasa toa SOSA esas Ete UNO C DIAR cuca Ami NES IN CNOTUS PFOLCINACENUPICALION ssieshsieassdviucessanyicudssenvsiasseationtscesusnssadssreslerontgessronciasaconesteusbnededarvacsedesinetebeonsesentguecdsdenverdouedteciatesndipiarasinestoronigaderonccasecite VIEWING PROTEIN IDENTIFICATION RESULTS as MANUALLY MANIPULATING DATA FILES eseese ree Ei Eeee teei er EEE EEEE EEA EEE ESNEA acid Editing Precursor information sssiiisprieirrsiri ittie ENTER ETE ECIS ESETRE OSTNE REENA CRS RCE TN ETENE R EEEREN ESETRE RE EREEREER EEES Manually merging MS MS scans of the same peptide z Cutting and Copyne Spectrum DAA rn EAR A i AA EAS AAA A NN SELECTING UNMATCHED DE NOVO RESULTS RUNNING PROTEIN IDENTIFICATION ON SELECT SPECTRA USING THE MASS CALCULATOR ooccccoccnoncconcoonnconcconcconoos 3 CREATING HIGH THROUGHPUT WORKFLOW viii eds 86 SAVING RESULTS pci aaa ANC 87 ABOUT BIOINFORMATICS SOLUTIONS INC cscsssssssssssssssscsssssssssssssessssssscsessessssesssssssesssesscseseassesesssssssessssassssossassesessesssessssesscsesensesseseassssessees 89 PEAKS SOFTWARE LICENSE scscssssssssscssscsssssssssessccsssessssssssssssassssessssssscssssasscsessassssesssssssessssasscseseassesessssssessssassssssase
29. EAKS Studio When using the mass calculator remember to start ET Mass Calculator Edit Help with water NAYYEICLSK lt H20 gt H gt lt Acetyl gt Mono Mass 1104 5759 average Mass 1105 3322 Clear Water Proton i PTM Num Back D 4 hydroxynonenal HNE a H pplied Biosystems original ICAT TM dO plied Biosystems original ICAT TM d8 M pplied Biosystems cleavable ICAT TM light Lene pplied Biosystems cleavable ICAT TM heavy R W pplied Biosystems ITRAG TM multiplexed quantitation chemistry crylamide adduct We can click any of these buttons multiple times to repeatedly add that mass Amino acids are represented by their single letter symbols Clicking on an amino acids button will add it to the sequence above and add its mass to the mass of the peptide Note that the peptide s monoisotopic and average masses are both computed Add a Proton by clicking the Proton button It will be represented by an H in the peptide above To compute the mass of the peptide as if it had been modified select a PIM from the list and press the PTM button to apply them to the peptide If the PTM we wish to add does not appear in the list we may wish to enter it s mass manually To add a mass numerically click the Num button and enter a numeric value in the dialogue box that appears Press the OK button on t
30. KS creates temporary databases on our behalf To avoid this best practices suggest we put all our databases in a folder c peaksdatabases The folder c my documentsl databases wouldn t work because it contains a space between my and documents Using spaces in the database file name causes the same problem So after we download and extract our database we should call the database file ncbinr fas or ncbi nr fas rather than ncbi nr fas 5 The database we downloaded may be in a compressed file perhaps a zip or a gz file We must find the file and use a decompression utility such as WinZip 11 The taxonomy options are only available if the NCBI nr database is selected and the Apply button has been pressed or NCBI nr was selected ona previous screen or WinRar to extract its contents The file inside the compressed file will be a FASTA format text file a fas or a fasta file 6 Return to the Import Database Wizard and click the Next button This screen will allow us to configure the database Click on the hyperlink next to each field for more information Import database wizard Database Nickname Path Browse EST database Advanced Options Fasta header format set up Use built in database format UniProt y Apply Rule to parse accessionid string from FASTA title Jas Rule to parse description string from FASTA title
31. KS on If we are connected to the internet registration will be completed automatically If all is well a dialogue will show Registration Successful and PEAKS will load Re registering PEAKS may be necessary if our license has expired or if we wish to update the license We will need to obtain a new registration key from BSI Once we have obtained this new key select Register Peaks from the Help menu The License Upgrade dialogue box will appear cautioning us that we are about to update the license Press the Ok button to continue Follow the on screen instructions Database File Edit Tools Windows Help Configuration aja Rio In addition to de novo Peptide Data sequencing of peptides PEAKS Studio 4 0 also has the ability to search through a database search to identify Microsoft Internet Explorer for example is an FTP client We may use Internet explorer and the provided URL to download a database The next section provides a walkthrough of PEAKS Studio 4 0 s main functionality using the NCBI nr database proteins But in order to use this function PEAKS must have access to a protein or EST database in FASTA format or an EST database of DNA sequences We can point PEAKS to an existing database on our system or download one Additionally we can associate taxonomy with certain databases This is database configuration We can use PEAKS without the database search PEAKS wil
32. LFCMENS AEPEOSLVCO CLYRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI Above 18 of the original 22 spectra indicated a peptide sequence matching with Lactoglobulin Beta Each peptide match shows a high confidence strong evidence for having found the correct protein We can also see exactly where the peptide fits into the protein sequence with the matching sequences highlighted in red at the bottom As mentioned above the peptide sequence results are based on a database search guided by an initial de novo sequencing analysis Let s see how the de novo sequencing was able to help Click on the 467 2729 hyperlink This will bring up the main processing window for spectrum n 467 2729 2 Look in the BE 467 2729 2 Main processing window top right frame to see the EEA de novo and database results Peptide Candidates E PEAKS Auto De Novo 0 01 0 01 TEMP Color coding shows positional confidence peels 49 2 scores By the letters coded sq Ee in red we can see that the LLVAASTMK lt 1 PEAKS auto de novo EE PEAKS DB Search NR 0 01 0 01 TEMP analysis returned with age ss ps IIVTQTMK 33 4 gt 90 confidence the partial peptide sequence LLVXXTMK but was not as sute of the middle two Color Code gt 90 80 90 60 80 lt 60 18 residues The PEAKS DB Search was able to confirm this result returning the peptide LLVTQTMK Selecting another spectrum from the Peptide D
33. LO bd NN dd Search parameters This tab displays the protein identification parameters that were used to guide the search that generated these results 33 The main processing window is used to perform manual de novo sequencing and to examine the results of auto de novo sequencing Main Processing Window mme 11 Peptide Candidates PEAKS Auto De Novo 0 1 Trypsin with DVVEK lt 1 VLTEK lt 1 VDPMK lt 1 LTVEK lt 1 PEAKS DB Search sprot 0 1 Trypsin VDVEK 99 72 08 100 03 147 11 sd AM ro SS ee 72 08 187 88 04 D 472 473 Es v 357 358 415 102 E 258 259 129 130 258 14 pros oor 0 00 276 16 314 13 b3 37522 442 18 490 25 E ba ya ok oms amom o inue oy awe o 500 b lax Main Processing Window Toolbar quick access to many processing functions See Toolbars section below Peptide Candidates Frame top left PEAKS shows peptide sequence candidates ranked by score for the selected spectrum Peptide sequences are grouped by the headings Auto de novo Manual de novo user defined result type and database search results depending on how they were derived For de novo results positional confidence is color coded on each residue More specific positional confidence appears when the mouse is held over a sequence th
34. PEAKS We may make new databases available to PEAKS from here 27 Enzyme Editor Dialogue Enzyme Definition Saved enzymes Chymotrypsin Digestion Rules Cleavage site residues at the end of a peptide FLMVVY P start of a new peptide use set brackets around 2 residue to denote any residues at the end of a peptide start of a new peptide amino acid except the ones enclosed in these fesidues at the end of a peptide start of a new peptide bracket Use Xto 3 E E denote any residue residues at the end of a peptide start of a new peptide Specificity Parameters example of what peptides can be returned Find peptides that satisfy the above at both ends Find peptides that satisfy the above only at C terminus and or peptides that satisfy the above only at N terminus Shorthand notation for the above digest FLMWYIKP y semi O Save Enzyme Cancel Digestion Rules This is how we specify where our enzyme will cleave the protein between two amino acids to create peptides Use set brackets around a residue to denote any amino acid except the ones enclosed in these brackets Use X to denote any residue Listing several amino acids in one box means any one of these residues Specificity Parameters Peptides can break down such that only one end is a cleavage site Check the boxes to tell PEAKS to search for only for p
35. PEAKS and XCalibur are installed on the same computer or dta format and concatenated dta formats with the ability to load an entire folder full of dta s Waters Micromass instrument s data in RAW format provided that PEAKS and MassLynx are installed on the same computer or pkl files Applied Biosystems instrument s data in wiff format provided that PEAKS the Infochromics converter plug in and Analyst are installed on the same computer PEAKS has the ability to read directly from the 4700 4800 Oracle database All other instrument s data as can be converted into mzXML pkl dta or mef Installation If we already have PEAKS installed on our system we must uninstall it before proceeding 1 Close all programs that are currently running and end all non system tasks 2 Insert the PEAKS Studio 4 0 disc into the CD ROM drive This is the BSI disk which lists its contents as PEAKS Software PEAKS Movies PEAKS Tutorial 3 Auto run should automatically load the installation software If it does not find the CD ROM drive and open it to access the disc Click on the exe file 4 A menu screen will appear with the title PEAKS Studio 4 0 Select Install Peaks Studio 5 The installation utility will begin the install Wait while it does so Choose English as the language for installation instructions When the PEAKS Studio 4 0 installation dialogue appears click the Next button 6 Read t
36. RVER Windows 2000 or Windows XP is recommended for use with this tool Configuration Before using the ABI 4700 Data Extractor we should configure it To do so we can choose Settings from the File menu Configuration needs the following 4700 SERVER Name or IP Address input localhost if the Extractor is tunning on the 4700 SERVER 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 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 we do not need to change this one either Data extraction procedure 1 Load Spot Set List from the database Do it via menu File Load Spot Set List The extractor will export the peak list of a spot set into a PKL file 57 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 run There is a radio button before each Job Run only the MS MS job run can be selected for export because we need the precursor information Select a Job Run and click Convert to do the extraction 4 Choose a filename to save After clicking the Convert button the us
37. a spectrum this is reflected by how close together can two PEAKS be and still be told apart Mass accuracy this refers to the accuracy of the spectrometer and its resulting data On a spectrum this is reflected by how close the PEAKS are to the actual masses of the ions they represent ESI Electrospray lonization A method for ionizing a sample into the mass spectrometer MALDI Matrix Assisted Laser Desorption lonization A method for ionizing a sample into the mass spectrometer This has a characteristic effect of only producing singly charged ions PTM Post Translational Modification A protein just translated and hence newly formed may differ from its final form as a result of interaction with the cellular environment or the experimental environment As they interact chemically with the environment residues may gain or lose molecules This change is referred to as a post translational modification Since PTM changes the mass of residues it must be accounted for when sequencing peptides by mass spectrometry Built in PTM PEAKS comes equipped with a library of possible post translational modifications These can be incorporated into a de novo analysis at the click of a button Customized PTM If the post translational modification we are looking for is not in the PEAKS PTM set we may create our own entry or modify an existing one This will appear as a customized PTM in the set Enzyme The residues PEAKS can find in dif
38. achine error 0 1 When sequencing a peptide using the manual de novo tools we can get PEAKS to help us by searching to the left or right of a selected peak and returning a set of possible sequence tags see Manual De novo section in the next chapter We can choose how many search results we d like to see and we can choose how long number of amino acid residues we d like these tags to be at a maximum To choose how long tags will be we click on the Maximum tag length dropdown list box and making a selection Choose the number of search results displayed by clicking on the Maximum return dropdown list and making a selection Changing the default machine error sets the amount of error PEAKS will tolerate when tagging a residue For example we have a mass difference of 113 19 between two y ions that we have labeled We are fairly confident that this should be tagged L Leucine with actual mass of 113 08 but PEAKS is not labeling it for us This may be because 113 19 is too far out of PEAKS error tolerance for the mass of L We can tweak the settings until we get the desired result To do so type a value for error larger numbers indicate greater tolerance into the Default machine error textbox After having made all desired changes click the Ok button to save changes and exit the dialogue box Click the Cancel button to exit discarding changes 51 Removing Saved Parameters After mont
39. ase information in light gray A light grey colour could also mean that the database does not have header information Y PEAKS Properties Enzyme list PTM Library Database List of databases NR Diirogersidatabasesinr_20051120 fas SPROT Dilirogersidatabasesisprotfas Set as default Cancel 45 Importing and Exporting PEAKS Properties We may wish to use PEAKS Studio 4 0 on another system However if we have a large number of user defined PTM and Enzyme PTMs sets it could take a great deal of our time to re input those This is where importing and exporting of PEAKS properties is useful The export function will save PEAKS Properties information in a XML file The import function can read a PEAKS properties XML file and overwrite local mae ula lle eel E PEAKS Auto De Novo 0 2 Unknown enzyme with L2custorng Copy sequence to clipboard Ctrl C PEAKS Properties with the ca A n A custom Copy for manual de novo information from XML file If we wish L2custonl cd f View Sequence parameter to use our PEAKS properties on a eee tonn als PEAKS DB Search Original View colleague s system we must remember VDVSAY 53 8 GAKGEAG 41 to export our colleague s properties to a separate file so that it will not be lost and can be imported later aim dy Delete g A note on shari
40. asks but also how they may be used to perform less obvious operations PEAKS demo data can be found in the DATA sub directory located in the PEAKS directory Loading data into PEAKS Studio 4 0 PEAKS Studio 4 0 can be used to process data from any MS MS instrument provided the data is accessible or can be converted to an accessible format PEAKS handles data files in the following formats a PRL DTA MGF ANZ the zip compressed XML based file format associated with PEAKS XML format files using the mzXML schema RAW files from Thermo Electron instruments RAW files from Waters QTOF instruments XML format files from Waters ProteinLynx software DAT files created by BSPs ABI converter software Opening data files In order to do any data processing we must first load our spectrum data into PEAKS Studio 4 0 To open a data file click the E icon on the toolbar in the upper left corner of the PEAKS window or select Open from the File menu Edit View Tools Windows Help Load Directory Load raw data files Load 4700 Data EJ Close Save Ctrl S E Save As E Save All Ctri Shift S CI x Look In C Data x fa Es multiple spectra phosphorylated single spectra E SampleData anz E SampleData pkl File Name Open Files of Type ann anz dta mgf mzxml pkl xml bt Spectrum Data File hg Cancel Select a f
41. ata frame left e g 545 928 3 will allow us to view the results from that spectrum without having to return to the protein identification result Click on the time and date stamp beneath the filename to return to the report Let s try another kind of search This time we ll use inChorus database searching this technology unique to PEAKS allows us to launch other search engines that will help improve the results The best confirmation of results comes from using two or more methods to confirm the peptide matches Select OrbiOrbi pkl from the Peptide Data frame left and choose inChorus protein ID from the Tools menu The inChorus Database search dialogue appears InChorus Database Search Peaks Database Search Engine 7 Peaks database Search EX Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines lv Tandem Search EL Options Omssa Search F Options Spider Search Options port Database Search Results Mascot Result dat MI Browse _ Sequest Result summary html gt Browse Cancel Make sure that Peaks database Search and X Tandem Search are selected Notice that there are three Options icons on the right They correspond to each search engine Click the Peaks database Search options button The options pane is similar to the one we ve seen alread
42. ation pertaining to that spectrum Manually merging MS MS scans of the same peptide If we ve done several MS MS scans of the same peptide we may want to reduce the amount of data to process and at the same time improve the data quality by merging all of a peptides MS MS scans together Often we choose to automatically merge appropriate spectra from the whole data file using the Data Refine tool see above But this can also be done manually Changes made to the original spectrum after duplication will not affect the duplicated spectrum To manually merge spectra after opening a data file 1 Select those spectra we wish to merge together from the Peptide Data Tree left using shift click and ctrl click 2 Next right click in the Peptide Data panel and choose Merge Spectra from the popup window that appears OR click the E Manual Merge Spectra toolbar button 3 A dialogue will appear asking what should be the correct value for the precursor mass and charge After reviewing and or correcting the value press ok The spectra will be merged Cutting and Copying Spectrum Data If we wish to move spectrum data from one data file to another we may do so by copying and pasting it see below for pasting instructions Also we may wish to make a copy of the spectrum in the same data file in order to re sequence an individual spectrum using different preferences Cutting spectrum data will remove it completely until pasted
43. bundant but uninteresting proteins like keratin can get in the way We may find it more convenient to eliminate them from the analysis To do so we must first identify which peptides belong to those proteins To do so run PEAKS Studio 4 0 s protein identification tool The proteins identified in the sample will be shown in the protein ID report In the Protein ID Result report peptide view first sort by accession number by clicking on the header for that column Next scroll down to an abundant but uninteresting protein Click and drag downwards to select those spectra matching to that protein F 01 Nov 05 15 50 40_NCBI nr index _miz_ z mass __ Peptide mrCalc DeltaMass Score Start End Accession No Peaks v vi 1 4827 2 963 38 LCLKEFK 963 51 0 1246 0 0 402 408 gil76650345 ui k ba Click and drag x They appear highlighted in the peptide data tree on the left We may then chose run auto de novo or protein id on these spectra or on everything but these spectra Read on Running protein identification on select spectra When searching our dataset against a particular database Peaks may not have found a hit for certain spectra If these are good data we may wish to try searching them against a more general database Before we do so we must create a new data set with 83 these good spectra that did not match This is essential so that we can organize our data well and becaus
44. cted PTM selected for search Fixed Carbamidomethylation Var Deamidation Var Oxidation Var Phosphorylation Add Remove PTM Generally the more variable PTM we turn on the more ambiguous will be the results To add modifications to this list click the Add Remove PIM button The Modification dialogue appears The entire PTM library i e all lt built in gt and user defined modifications that are available to PEAKS are displayed in the list on the left We ll choose the modifications we need from this list To add a new modification to the PTM library click the New PTM button To edit a modification in the PIM library select it from the list on the left and press the Edit PTM button To remove a modification from the PTM library select it from the list on the left and press the Remove PTM library If you remove edit a PTM it will be removed or changed from in any saved parameter set that refers to it The lists on the right show what PT M will be enabled for the search Use the Select as Fixed gt Select as Variable gt and lt Unselect buttons to move them in and out of these lists Press the Ok button when finished and the changes we made will be reflected on the protein ID options dialogue Modification PTMs that can be selected lt BuiltIn 4 hydroxynonenal HNE lt Built In gt Acrylamide adduct lt Built In gt Amidation lt BuiltIn Applied Biosystem
45. dialogue will allow us to choose and to set parameters for each of these refinement tools Automatic De novo button perform auto de novo for a selected data file spectrum or list of data files Press this after selecting one or more data files or spectra in the Peptide Data Frame An auto de novo options dialogue will allow us to set parameters before we begin Protein Identification button perform protein identification a selected data file Press this after selecting one or more data files or spectra in the Peptide Data Frame A protein identification options dialogue will allow us to set parameters before we begin Protein Identification button perform protein identification a selected data file Press this after selecting one or more data files or spectra in the Peptide Data Frame A protein identification options dialogue will allow us to set parameters before we begin Environment Preference Configuration button configure the environment spectrum color coding and manual de nove parameters PEAKS Properties Configuration button define PIM Enzymes and add FASTA protein or EST databases EI Import Database Wizard button help user download and configure database 39 Main Processing Window Toolbar ial y ion Alignment button toggle show hide the location of PEAKS corresponding to y ions and the corresponding proposed peptides between them a b ion Alignment button button
46. e Apply button Press the Cancel button at any time to exit the search discarding changes Undoing an edit If we have made an error in our sequencing it is possible to undo the change With the Peptide candidate still selected in the Peptide Candidates Frame click the previous peptide E button to return to the previous peptide sequence We can click this button multiple times to return to successively earlier stages in our edit Redoing an edit If we have undone one too many changes we can redo that change by clicking the next peptide 8 button We can click this button multiple times to proceed to successively later stages in our edit 71 Suggesting a sequence to see how it fits the data If the data is ambiguous PEAKS Studio 4 0 may not have displayed a particular candidate that we wish to evaluate after auto de novo or protein ID We may enter this sequence and have PEAKS Studio 4 0 find if there is any evidence for it in the data For instance PEAKS may give the sequence RMYNVHGC phosphorylationS K for a particular spectrum and we may wish to see if there s any evidence for the phosphorylation being on the Tyrosine As such we may type in our own version of the sequence and have PEAKS find ions that might support our hypothesis To do so open the spectra in the Main Processing Window and right click on Peptide Candidates in the Peptide Candidates Frame Then from the pop up menu that appears choos
47. e New Candidate for Manual De Novo PEAKS Studio File Edit View Tools Windows Help leio esse ejes 5 88 minto fuja joe aei 7 E PEAKS Auto EDL LAY RR ea E DELL aS KT I e EPMLAYLK lt 1 LEDLAYLK lt 1 FPLLAYLK lt 1 x PEAKS DB Search SwissProt 0 1 0 1 Trypsin with Cam EDLIAYLK 99 Pa A new node will appear with the heading Manual De Novo and beneath it will be the mass of the residues yet to be sequenced in square brackets Right click on this heading In the pop up menu that appears choose New Candidate with user input sequence and the Sequence Input dialogue box will appear 72 Short forms for the modifications may also be used 182 7 S hfa lale ajajo pen Peptide Candidates Sequence Parameter Config 945 38 E PEAKS Auto De AA PTM Set Config EDLLAYLK 4 DELLAYLK 3 New Candidate for Manual De Novo EPMLAYLK EEE E PEAKS DB Searfcr EDLIAYLK 99 z r ma maa cc b Pa dz 4 se e h WISSPTOLU T OT Trypsin Y ITP PT Sequence Input x Please input sequence We can now enter our proposed sequence The total mass of the residues modifications and un sequenced masses should equal the total mass of the peptide minus water We might find the mass calculator tool Tools menu useful in this regard Enter sequences in the format MPELAYLK 228 09JELAYLK DE 226 168 AYLK EDLLA phosphorylationY LK DE 226 168 A phosphory
48. e Thus we need to download it from the Internet and tell PEAKS where the database is located PEAKS provides the Database manager as a tool to help us do this To see a list of databases available to PEAKS Studio 4 0 load the PEAKS Properties dialogue and click the Database tab From here we can edit a database s properties load a new database or remove a database F PEAKS Properties Enzyme list l PTM Library Database List of databases NR Diirogersidatabasesinr 20051120 fas SPROT Diirogersidatabasesisprotfas Set as default gt Cancel Load Configure a new database For an in depth look at configuring a database see the Database Configuration section in Chapter 2 To configure a new database we open the PEAKS Properties dialogue ensure that the Database tab is selected and press the New button Now we open up our web browser to find a database to download Find one download it and unpack it If taxonomy is available for this database download those files too Return to PEAKS Studio 4 0 and find the file on our system where we unpacked it Name the database and select the header format to use or we can define our own If taxonomy is available for the database find those files too Click Ok The new database will now appear listed by our chosen name in the list of databases Remove a Database To remove a database we
49. e AS TA ANE a SEARLS ENC EISEN INSITE SERGI ae Canes Meee Oe 56 Importing MASSING RAW GIG total ateos RETEA estes ean ies LOCO ENEA MOLE VE E EEEa 56 Importing Data from the ABI 4700 or ABI 4800 cscsscssseeseeseeseeseeseeseesecssceseeseeseesecsecsecsaeeseeseesecsecsecceeseeaeeseesecsecaceaeeseesecsecaeceeeeaeeaeeaeeaeeeesneeeneeaeeaees 57 System Requirements I Configuration Data extraction procedure REFINING DATA BEFORE ANALYSIS orton e e EEEE A NERE TEATRE RAEE A 58 USING PEAKS STUDIO WITH MODIFICATIONS PTM sscsssesseeseeseeseeseeseesecsecsseeseesecsecsecscesseeaeesecsecsecseseaeeaeeaecsecaeeceseeaeesecsecaecaeeaeeseeaecaeseeeaeeaeeaeeaeeatees 60 AUTO DE NOVO SEQUENCING a VIEWING AUTO DE NOVO RESULTS ar 66 EDITING SEQUENCING RESULTS PREPARATION cassssanasesssuarensrnasisisasssaciiraaeeabeeuaeniaiGienae eatin SRA CGA RO EESK USE E RR rasa apa 67 MANUAL DE NOVO SEQUENCING Creating a fresh spectrum for SCQUENCING a a aa AS raa aa EE RE raa 69 Manual De novo Operations e o eiat e EEE ga send dus datas baveccessescucsssceuscstsesssesdevrscissveranssuvwasestseseutevteseacedsavcusiss SpucuksasoucavsivovessesductdevSe i 69 Selecting a peak Measuring distance along the m z scale Measure the m z difference between two PEAKS i Deselect a peal NN Zoom inon Part of he PEC ira it Add remove ions to from a peak Using sequence TA invicta lio A AAA AAA dE aaa aan Roo A AE A
50. e Peaks will only run Protein ID on all the spectra in a data node To create a new data node 1 Make a new Data node by 2 Select the relevant spectra 3 Click the new node and press right clicking on the peptide data using lt shift gt click and the paste button Pressing the node The new node appears as lt tri gt click Then press the next to Data will expand it Datat cut button and reveal the pasted spectra a File Edit Tools Windows File Edit Tools Windows eael lk Now we ve essentially removed the already matched peptides from our dataset We can now run protein identification on Datal or on the remaining spectra in our original dataset We can save that dataset in a new file or any of the other functions that apply to regular nodes Make sure the new node is selected before running protein ID or any other function on it Using the Mass Calculator ak Bay inane a tool to help us determine the 3 File PL Windows Help molecular weight of a peptide To access the mass calculator open the Tools menu and click Mass Calculator The mass calculator will appear auto De Novo Protein Identification 1479 7 1 We can also load the mass calculator outside of Peaks and separately To access the mass e 0 1639 8 1 calculator without having to load A PA PEAKS click on the mass calculator s icon in the start menu It will appear in the same program group as P
51. ent of the mouse This is the Position Bar and it is used as a cursor for all manual de novo operations The cursot s position on the m z scale is enumerated on the top of the Position Bar Selecting a peak To select a peak click on it An orange by default bar called Freeze Bar indicates the selected peak Alternatively an ion peak can be selected by clicking on its corresponding cell in the Ion Table Measuring distance along the m z scale Once a peak is selected with the Freeze Bar moving the mouse left or right will display the Position Bar along with a value that represents the m z difference as an absolute value between the selected peak orange and the Position Bar blue In the example below the distance between the selected peak and the position bar is 51 02 Daltons 374 14 277 44 b2 293 11 541 25 3422 pr 294 14 341 2199 NHS 5122 e2 enzo a b2H o ROV Y mo bero Measure the m z difference between two PEAKS Select a peak orange line by default with the Freeze Bar and move the mouse to the left or right Hold the Position Bar above another peak The number above the Position Baris the difference between the two PEAKS Deselect a peak Double click anywhere in the Spectrum View Frame Zoom in on part of the spectrum In the Spectrum View Frame or the Spectrum Alignment Frame click and drag the mouse horizontally The selected area will be shown in the Spectrum View Frame Add remove ions
52. eptide Match Reports bottom section PEAKS presents each protein candidate with a peptide match list beneath it Each peptide that matched the protein sequence is shown in order by spectrum The confidence that the correct peptide sequence was found is displayed next to each peptide sequence At the bottom of this list the complete protein sequence is shown with matching Protein View peptides highlighted in red showing two proteins in the index and beginning the full report 32 05 Jun 06 14 47 33_NR inChorus protein ID results Peptide View ProteinView Search parameters Accession Mass Score Coverage Query matched Marked Description PEAKS DB Search NR 0 01 0 01 TEMP jax 10 i 7 100 ES gil28972720 78 207 29 mkKIAA1321 protein Mus mus X gil62648518 57 150 29 PREDICTED similar to MKIA X gil52782259 85 124 29 phosphofructokinase muscle X gil63586345 261 103 29 PREDICTED similar to MKIA NCBI BLAST search of gil229460 Links to retrieve entries containing this sequence from NCBI Entrez AccessioniD Description gil229460 lactoglobulin beta Peptides List Mz Charge Mr calc Score 452 28497 902 5589 68 TKIPAVFK 458 73856 915 4661 nu LDAINENK 467 2729 932 5366 35 LIVTOTMK 533 2925 1 064 5752 85 VLYLDTDYK S45 92804 1 634 7673 TPEVDDEALEKFDK 597 3386 1 192 6702 VLYLDTDYEK 623 2947 1 244 5771 TPEVDDEALEK 673 38513 672 3807 GLDIQK 674 4233 673 4163 IPAVFK Ro Sl
53. eptides that have proper cleavage sites on both ends or to require that only one end be a proper cleavage site Shorthand notation Advanced users may specify their enzyme cleaveage in shorthand notation but it is not required Saving Loading Enzymes After setting up an enzyme we can save it for future use Click the Save Parameters button and choose a name for future reference if prompted Don t worry we can t accidently overwrite the defaults Any enzyme we save will be available in the drop down list at the top of the window To see what s inside just select one and the enzymes digest rules boxes will be populated This dialogue allows us to create or edit a PTM PTM Selector Dialogue Modification PTMs that can be selected Selected Fixed PTMs lt Built In gt 4 hydroxynonenal HNE Carbamidomethyation lt Built In Acrylamide adduct lt BuiltIn Applied Biosystems cleavable ICAT TM lt Built In gt Applied Biosystems cleavable ICAT TM lt Built In gt Applied Biosystems TRAQ TM multiple lt BuiltIn gt Applied Biosystems original ICAT TM i lt Built In gt Applied Biosystems original ICAT TM lt Built In gt Beta methylthiolation lt BuiltIn gt Biotinyl iodoacetamidyl 3 6 dioxaoctane lt Built In gt Biotinylation Select As Variable gt lt BuiltIn gt C Mannosylation lt Built In gt Carbamidomethylation lt Unselect lt BuiltIn Carbamylation Edit PTM Selected Variable PTMs lt Built In gt C
54. er needs to input a file name And the peak lists of the selected job run will be exported Refining data before analysis Since mass spectrometry data often contains noise and redundant data it makes sense to purify the data before analysis This will increase the quality of the results while saving time spent on database searching and or de novo sequencing MS MS spectra that are purely noise can be removed from the data peptide charge information can be verified and recovered multiple low quality scans of the same peptide can be merged into one scan with intense signal peaks and the MS MS scans themselves can be centroided filtered for noise and deconvoluted To begin refinement of data from a whole MS MS tun we 1 In the Peptide Data Frame select the data file s containing the data that we wish to tefine 2 Click the Data Refine toolbar icon Or Select Data Refine from the Tools menu Or Right click on the selected data file and select Data Refine from the popup menu The Data refinement options dialogue appears Data refinement options Instrument type lon Trap v Merge scans ofthe same peptide yes Retention time window for raw files only miz tolerance Correct precursor charges yes Remove low quality MS MS scans O yes a Preprocsss MS Ms scans O no already done yes Cancel 3 Choose the data refinement tool
55. escription of the Ion Table itself 30 The Basic and Advanced lon tables differ only in the number of ions they can display The Basic table displays up to six ions P4 advanced Ion Table Settings x Please choose ion types charge combination and add selections to ion table columns list lon Types lon Table Columns Immonium Immonium a E gt a a H20 b a NH3 b H20 b c b H20 y b NH3 y H20 c z c H20 z c NH3 y 2 x apply Cancel Ion Types list A listing of all the types of ions PEAKS Studio considers in its analysis Ton Table Columns list A list of the columns that will appear in the ion table each representing a type of ion The columns will contain the masses at which the particular type of ion was found if at all Charge list box unlabeled Each type of ion can be added up to 4 times depending on the charge we specify Add with charge button After selecting an ion type from the Ion Types list and a charge from the list box clicking this button will add that ion to the Ion Table Columns list Protein Identification Result Window The protein identification result window contains the results from one protein identification run on one data set It is organized into three tabs peptide view protein view and search parameters Peptide View The peptide view summarizes the results for each MS MS spectrum All peptides that match to eac
56. ext button NCBI nr bd UniProt Human Mouse and Rat IPI Human Mouse and Rat Previous Next Finish Cancel 10 we wish to use click the provided link to begin downloading A dialogue box will appear with instructions on downloading using file transfer protocol FTP It does not matter where we put the download file but we must remember where it is A note on downloading databases The links in the Wizard may be outdated because the owners of those download locations may change their URL periodically If this is the case remove all but the domain name and browse from there ftp ftp ebi ac uk pub databases MassSpecDB msdb fasta z becomes ftp ftp ebi ac uk Best practices configuring databases for use with XITandem At the time of this writing X Tandem had trouble searching through large databases and would crash It is therefore suggested that X Tandem only be used with small databases or if used with a large database a taxon should be specified The NCBI nr and Swiss Prot databases are ideal for this purpose Best practices configuring databases for use with OMSSA At the time of this writing we could not use OMSSA with databases that were not in NCBI format or Swiss Prot format and have those results available to inChorus Also a bug in OMSSA prevents us from easily using databases with OMSSA when they are stored in a folder that contains a space in its path This creates problems when PEA
57. ferent positions in the sequence This is based on information about the enzyme used to digest our protein sample PTM set A listing of all possible built in and custom entered post translational modifications that PEAKS can use as a part of its analysis Enzyme PTM set Combined the enzyme information and post translational modification information provide PEAKS with the relevant parameters of the experiment sample This will be applied to the corresponding data set when PEAKS performs its de novo analysis It is a required parameter Fixed modification selecting a post translational modification as a fixed modification tells PEAKS that this modification is applied to all occurrences of the residue s that the PTM can act on Variable modification sclecting a post translational modification as a variable modification tells PEAKS that this modification may or may not be applied to any given occurrence of the residue s that the PTM can act on FASTA Fast All A standard sequence database file format used for protein identification PEAKS can identify proteins from any FASTA format database of proteins PKL The file format associated with Micromass instruments DTA The file format associated with SEQUEST software MGF The file format associated with Mascot software BSI Bioinformatics Solutions Inc The makers of PEAKS and other fine bioinformatics software ANZ file a PEAKS zip compressed XML based Annotated spectrum fi
58. ften returns better results if the auto de novo analysis is run with no variable PIM perhaps one or two if necessary but with the correct enzyme and fixed PTM Modifications should be then turned on for the database search 75 function m z tolerance can also be adjusted separately for each phase to allow us to tweak the results Report up to Type a number here to specify the number of proteins including all homologs but counting them as one entry to include in the result report PEAKS will report up to this many Database to search Select from this dropdown list one of the FASTA databases that we ve set up in PEAKS If the database we d like to search is not in this list click the Load new database button Taxonomy selection This list displays the taxa we ve chosen for our search If the database selected has taxon information available we can click the aptly labeled Add Remove Taxa button Otherwise the whole database will be searched The selections correspond to established hierarchy i e selecting Mamalia will search all of horse cow tat mouse human etc Preprocess before auto de novo PEAKS Studio has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data Check this box to turn preprocessing on Notes on pre processing BSI highly recommends using PEAKS to preprocess all data as opposed to using instrument vendor software if the data is to be used b
59. h spectrum are displayed By default the peptides are grouped by spectrum but the list is sort able by any of the columns in the table Selecting one or more click and drag or use shift click items in this list selects those spectra in the peptide data tree left 31 01 Nov 05 12 33 21_NCBI nr ta Peptide View N E Index miz z mass Peptide Mr Caalc Delta Mass Score Start End Accession No Peaks showing peptides Mi TGPNLHGLFGR 0 0295 H 1176690040 rai grouped by ail 3 spectrum E p 633 39 LFVQK 633 385 0 0078 97 22 gi 76690040 TGQAPGFSYTDANK 0 0776 i gi 76690040 KTGQAPGFSYTDANK 1583 7 0 0275 gi 76690040 MMALSDNLPAGYTHK 1583 78 0 0055 gil55628230 EHWOREEQQRLEEEGR 0 2407 gil 3975307 QKEFLRDTPQRHCEHTK 0 2957 gil73964720 Protein View The protein view is most useful as a summaty of what proteins were present in a sample and the peptides matched to them It has two sections Index top section PEAKS presents a list of proteins that it believes to be the best match for the sample This index lists them by accession number ranked in descending order by score Very similar proteins i e ones that contain most of or all of the sequences identified by PEAKS are grouped together only the first entry in this group is shown here Show the whole group by clicking the sign In the example below lactoglobulin beta is the top ranked protein candidate P
60. he dialogue and the mass will be added to the sequence To remove a mass that we ve just added to the peptide press Undo Creating a high throughput workflow In some situations we may have many data sets that we wish to process all at once and in the same way PEAKS Studio 4 0 allows us to do this kind of work and with minimal effort on our part By setting up a workflow we can start a batch process of several data files and not worry about it until it is finished It is important to note that all the files we load will be processed in exactly the same way using exactly the same parameters If we want to do some differently than others we must set up another workflow Work Flow Step 1 Load Files Import data phosB mzXML ecolit 1 mzXML phosB mzXML Input file list ecoli11 mzXML Step 2 Data Refine Data Refine Step 3 Auto De Novo by PEAKS v Auto De Novo Step 4 InChorus Database Search EE Options 2 Options Peaks Database Search Engine 7 Peaks database Search EX Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines 7 Tandem Search C Omssa Search C Spider Search Import Database Search Results C Mascot Result dat Sequest Result summary html Step 5 Save Result phosB mzXML save as phosB anz EM Options AM Options Opt
61. he license agreement If we agree to it we change the radio button at the bottom to select I accept the terms of the License Agreement and click Next 7 Next we choose the folder directory in which we d like to install PEAKS Studio 4 0 Press the Choose button to browse our system and make a selection or type a folder name in the textbox Click Next 8 Choose where we d like to place icons for PEAKS Studio 4 0 The default will put these icons in the programs section of our start menu Click Next 9 Review the choices we have made We can click Previous if we d like to make any changes or click Next if those choices are correct 10 PEAKS Studio 4 0 will now install on our system We may cancel at any time by pressing the Cancel button in the lower left corner 11 When installation is complete click Done The PEAKS Studio 4 0 menu screen should still be open One may view movies and materials from here To access this menu again we simply insert the disc in our CD ROM drive Registering PEAKS The first time we run PEAKS wc will be told that the product is not registered Press the Ok button and a dialogue will appear Enter the registration key that came with the product whether it be a key for the full version or time limited trial version We must also enter our name the name of our organization and the MAC address of the machine we are going to use PEA
62. hs of use our list of parameters saved for use with PEAKS Protein ID or PEAKS auto de novo may become cluttered with infrequently used parameter sets It makes sense to clear them out from time to time To do so open the Environment Preferences dialogue and ensure that the Parameters tab is selected A list is shown for each tool that has savable parameter sets Select one or more using shift click and ctrl click and click the adjacent Remove button to remove it from the list Environment Preferences Environment Color Manual De Novo Parameters De novo Parameters TOF J sample LTQ Trypsin John s special Remove Database Search Parameters qtof standard OrbiStandard PEAKS Studio Usage A task based guide to processing our data with PEAKS Studio 4 0 his chapter deals with usage It is broken up into tasks that a typical user might perform It assumes we can identify parts of the Graphical User Interface and that we are familiar with how PEAKS Studio 4 0 can be configured The preceding two chapters provide in depth help on these subjects and should be used as a reference Such detail has occasionally been omitted from this chapter in the interest of succinctness The four cores of PEAKS technology are the data refinement manual de novo sequencing automatic de novo sequencing and protein identification tools Surrounding Help is provided for these t
63. ile in ANZ pkl mef dta or txt format Click the Open button The data file we just opened appears in the Peptide Data Frame on the left It is represented by its file name Each spectrum contained in the data file is represented by its precursor ion information m z value followed by the charge of the precursor ion that generated the spectrum Loading a directory full of DTA files DTA spectrum data files can be opened by the same procedure as listed above However as we know some DTA files contain the data for only one spectrum As such we may find it useful to import a whole directory containing DTA MS MS spectrum data files for a whole MS run at once and consider it as one MS run PEAKS Studio 4 0 provides a tool for doing so Under the File menu click Load Directoty Now browse to the directory we wish to load Do not select a file within the directory rather select Edit Tools Windows Help the directory itself Press the Open button a ELA After loading the spectra we can choose sort the spectrum by the source filename or by the precursor m z value of spectrum To do so right click the parent node on the Peptide Data and choose to sort Load data files from directory Save Ctrl S Save As E Save All The data file we just opened appears in the Peptide Data Frame on the left It is represented by the folder name Each spectrum contained in the data Some versions
64. ions Mi Browse 8 Browse ecoli11 mzXML save as fecoli 1 anz Cancel Step1 Load files click the browse button to open a file chooser From the chooser select several files by shift click or ctrl click and pressing the OK button Load more files by pressing browse again or remove them from the list by right clicking on them Step2 Data Refine choose how to filter and correct data for maximum utility Step3 Auto de novo choose whether or not to do auto de novo sequencing Note that PEAKS database search requires some de novo sequencing results Step4 inChorus protein identification choose which protein identification programs with which to run the data PEAKS database seastch is Step5 Save results saved automatically into an ANZ file with the same name as the data file All files will be placed in one folder Typing in the textboxes or clicking each file s button changes the name and or save location Saving Results Saving results will preserve our work for later use Saving files in PEAKS s ANZ format will preserve spectrum data manual de novo sequence information automatic de novo sequence information protein identification results and information about any PTM that were found in sequence To save the results of our analysis we first select the data file we wish to save in the Peptide Data Frame To save click the E icon in the main window
65. is shows the confidence in each of its parts 34 The ions displayed in both modes can be edited See the section in configuring PEAKS Studio 4 0 To switch views choose Alignment by from the View menw Ion Table Frame top right the Ion Table shows the proposed ions with their corresponding masses i e the mass of the b1 ion is shown in the top right corner The default Ion Table will display b a immonium yH2O yNH3 and y ions in basic mode it will display b b H20 a c immonium y y H20 z z and y 2 ions in advanced mode To switch from basic mode to advanced mode choose Show ion table from the View menu The Ion Table Frame also contains an error plot it may be necessary to scroll down to see the error plot The error plot shows the confidence each ion is assigned The most confident results lie on the centerline Clicking a cell or column in the Ion Table highlights the corresponding points on the error plot and corresponding PEAKS on the spectrum Spectrum View Frame middle Shows a graphical representation of the spectrum Peak masses are labeled as are the peaks associated with identified ions We can zoom in on the spectrum by clicking and dragging over an atea Spectrum Alignment Frame bottom Shows a graphical representation of the spectrum This view always shows the whole spectrum and is used as a tool to help us navigate the spectrum view frame A blue bar along the horizon
66. itrullination moOxidation lt BuiltIn Deamidation New PTM lt BuiltIn Dimethylation lt Built In Farnesylation Remove PTM lt BuiltIn gt Flavin adenine dinucleotide lt BuiltIn gt Formylation lt Built In Gamma carboxyglutamic acid lt Built In Geranyl geranyl lt BuiltIn Glucosylation Glycation lt Built In Guanidination Built In Homoserine Select As Fixed gt Cancel Here we can from a list of available post translational modifications We can choose any PTM as Fixed PTM or Varied PTM to tell PEAKS that it may or may not occur To make this selection click on a PTM in the list at left and then click the Select As Fixed gt or the Select as Varied gt button If a PTM is already selected as a fixed PTM it cannot be selected as varied PTM and vise versa If we change our mind about a PTM after having selected it it is still possible to unselect it Click the erroneous PTM from the list of Selected Fixed PIM or Selected Varied PTM and then click the lt Unselect button to remove it from either list of Selected PTM PEAKS software ships with some pre defined PTMs These are listed as lt Built In gt If we want to create a new PTM we can click New PTM to create a new one The Editing a PTM and Creating a New PTM sections below describes how this is done This dialogue allows us to create or edit a PTM PTM Editing Dia
67. l perform de novo sequencing only WARNING Downloading a database can take a long time 8 hours depending on connection speed To configure a database p PEAKS Studio 1 Load PEAKS Studio 4 0 If File View Tools Windows Help we have not yet configured ali Boom e es 2 6 e a database the wizard will E P Ro y El Paste tomatically o a Configuration PEAKS Properties ae Ps ll i Environment Preference 2 In the edi menu select o Ene e Configuration then pa ao Import Database Wizard O PNI ae Per ae 3 The Import Database Wizard will load and ask us to select a database to download from the dropdown list If we already have a database we wish to use we can select Other database from the dropdown list and skip to step6 Click Next 4 Having selected a database the Import Database Wizard will provide us with some information about that database If this is in fact the database Import database wizard PEAKS Studio can do protein identification with any FASTA format sequence database This wizard will help you download database and configure it Ifthe database you want to download is in the following list please select it and click Next button Ifyou already have FASTA format database in local harddisk or the database is notin the list please choose Other database from the list and click N
68. lationY LK Then press the OK button The sequence we just entered will appear under the Manual De Novo heading and when selected the ions that PEAKS has found to match the proposed sequence appear on the spectrum spectrum alignment view and ion table 73 Protein Identification PEAKS introduces an amalgamative approach to protein identification called inChorus With inChorus protein identification technology we can use PEAKS together with several other protein identification methods This will deliver more protein coverage and more confidence in results than any one method on its own An integral part of the inChorus search PEAKS own protein identification method is unique an improvement on and the ideal compliment to existing tools The unique approach is a combination of sequence tag searching and fragment ion mass matching The following two sections deal with usage of PEAKS protein identification on its own and usage of inChorus protein identification PEAKS protein identification PEAKS Protein ID search engine is a hybrid approach that uses sequence tag information to filter the protein or EST database before fragment ion fingerprinting So to get useful protein identification results we must first perform de novo sequencing on the spectrum data If we already have sequence information for this data we may use this existing sequence information manual or auto de novo sequences to filter the database If we do
69. le format ANZ files preserve all the information from the PEAKS session ANN data file within the ANZ file a folder contains ANN data files that store the MS MS information and peptide information of one spectrum ANN index file within the ANZ file is one compressed file used to organize the data the ANN index file links to a directory containing multiple ANN data files Getting started with PEAKS Studio 4 0 Everything we need to know from the beginning and step by step his section of the manual will guide us through the process of installation and configuration of PEAKS Studio 4 0 If we run into any problems we can refer to the frequently asked questions section of this manual If problems persist contact technical support What we will need Package contents The PEAKS Studio 4 0 package should contain This manual PEAKS Studio 4 0 System requirements PEAKS Studio 4 0 will run on most platforms with the following requirements Equivalent or superior processing power to a Pentium at 500 MHz At least 512 MB of memory RAM 1024MB is recommended JAVA Virtual Machine 1 5 or better Instrumentation PEAKS Studio 4 0 will work with data from any type of tandem mass spectrometer designed for proteomics work mzXML is a standard data format from the Sashimi Project It is an XML based format PEAKS will accept data in the following formats Thermo Electron instrument s data in RAW format provided that
70. licenses to export re export or import as may be required 8 Assignment Customer may assign Customer s rights under this Agreement to another party if the other party agrees to accept the terms of this Agreement and Customer either transfer all copies of the Program and the Documentation whether in printed or machine readable form including the original to the other party or Customer destroy any copies not transferred Before such a transfer Customer must deliver a hard copy of this Agreement to the recipient 9 Maintenance and Support BSI will provide technical support for a period of thirty 30 days from the date the Software is shipped to Licensee Further maintenance and support is available to subscribers of BSI s Maintenance plan at BSI s then current rates Technical support is available by phone fax and email between the hours of 9 am and 5 pm Eastern Time excluding statutory holidays 10 Governing Law This Agreement shall be governed by and construed in accordance with the laws in force in the Province of Ontario and the laws of Canada applicable therein without giving effect to conflict of law provisions and without giving effect to United Nations Convention on contracts for the International Sale of Goods 91 Reference PEAKS Paper Please use the following reference when publishing a study that involved the use of PEAKS Bin Ma Kaizhong Zhang Christopher Hendrie Chengzhi Liang Ming Li Amanda Doherty Kirb
71. ling with complex mixtures Clicking any protein s gi number will display the peptides matched to that protein in the bottom pane 17 NCBI BLAST search of gil229460 Links to retrieve entries containing this sequence from NCBI Entrez AccessioniD Description gil229460 lactoglobulin beta Peptides List Mz Charge Mr calc Start End Score Search Engines Peptide 452 28497 2 902 5589 76 83 67 p TKIPAVFK 458 73856 2 915 4661 34 91 69 p LDAINENK 467 2729 2 932 5366 1 8 33 p LIVTQTHK 533 2925 2 1 064 5752 92 100 94 p VLYLDTDYK 545 92804 3 1 634 7673 125 138 99 p TPEVDDEALEKFDK 597 3386 2 1 192 6702 92 101 99 p VLVLDTDYKK 623 2947 2 1 244 5771 125 135 99 p TPEVDDEALEK 673 38513 1 672 3807 9 14 19 p GLDIQK 674 4233 1 673 4163 78 83 74 p IPAVFK 697 7167 3 2 090 1260 34 101 98 p LDAINENKYLVLDTDYEK 771 75635 3 2 312 2517 4 60 99 p VYVEELKPTPEGDLEILLOK 818 3876 2 1 634 7673 125 138 99 p TPEVDDEALEKFDK 903 1304 3 2 706 3687 15 40 65 p VAGTUYSLAMAASDISLLDAQSAPLR 903 5675 1 902 5589 76 33 3 p TKIPAVFK 933 5426 1 932 5366 1 8 34 p LIVTOTHK 1065 586 1 1 064 5752 92 100 5 p VLYLDTDYK 1157 1289 2 2 312 2517 41 60 99 p VYVEELKPTPEGDLEILLOK 1354 1898 2 2 706 3637 15 40 90 p VAGTWYSLAMAASDISLLDAQSAPLR Matched peptides shown in Red 1 LIVTOTMEGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLOK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYL
72. logue VS PTM Editing Name testPTM Abbreviation tst Mass Monoisotopic 57 992905 Neutral Loss Mass Monoisotopic Formula CNO2 Residues that can be modified Non location specific AC N terminus DEF C terminus GHI Middle Only L Rule This is not a real modification It s for demonstration purposes only OK Cancel Name This will appear in the PTM list Abbreviation This will appear in the auto de novo results if it is found Mass monoisotopic The mass that the residue gains or loses as a result of the PTM Enter this numerically here or enter the chemical formula below Neutral Loss Mass The mass that the modified residue loses as a result of fragmentation E g 28 would signify a loss of 28 Daltons Formula The chemical formula of the PTM This will automatically enter the mass Residues that can be modified Enter residues that can be modified anywhere residues that can only be modified if they are at the N terminus and residues that can only be modified at the C terminus and residues that can only be modified if they are not on either terminus Rule user entered a comment for our reference lon Table Settings Dialogue There are two such dialogues one each for the Advanced Ion Table and the Basic Ion table The two dialogues are identical but for the table they effect See the below section entitled Main Processing Window for a d
73. m or abbreviation please consult the glossary found in this section The glossary will tell us what a particular term means but it will not tell us how it applies to PEAKS usage Scope PEAKS users are assumed to be familiar with computer usage and the operating system environment As such it is beyond the scope of this manual to instruct the user on the use of windows dialogue boxes menus file storage etc Please refer to the operating system s manual or computer help books for such information Similarly PEAKS users are expected to be familiar with mass spectrometry standard operating practices and data Terminology and Abbreviations Glossary m z mass to chatge ratio Deconvolution rearrangement of the spectrum to show each monoisotopic peak as if it were singly charged Thus to reposition them on the scale PEAKS multiplies the m z of ion s that were doubly charged by two Note that the deconvolved scale PEAKS shows is at 1 a ions an N terminal fragment holding at least one charge similar to b ions and c ions This is a prefix fragment of the peptide The a ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues subtract the mass of Carbon Monoxide b ions an N terminal fragment holding at least one charge similar to a ions and c ions This is a prefix fragment of the peptide The b ion s mass will be the sum of the masses of the N terminal g
74. n designed for use on ion trap data Preprocess MS MS scans Deconvolution de isotoping centroiding and noise filtering within the MS MS data Data is always saved in the ANZ file along with the PEAKS results Preprocessing can save hard disk space or upload time But make sure to have the original data available in case we need to refer to it later A note on preserving data results integrity Protein ID and de novo sequencing results obtained for a given dataset prior to use of this tool may become invalid since some spectra are removed merged corrected and the data results relationship may be broken PEAKS Studio 4 0 will warn us when this may occur and prompt us to save a separate copy Using Peaks Studio with modifications PTM PEAKS Studio 4 0 provides the most flexible handing of post translational modifications of any software built for de novo sequencing and protein ID Users are free to create their own modifications see the Creating a New PTM section and search for any combination and any number of modifications Modifications can be considered as part of auto de novo sequencing or protein identification The search is set up the same way for both tools The options screen for each tool has an area titled PTM selected for search Any modifications to be considered during the search will be shown here and labeled as Fixed or Var When we first load PEAKS Studio the box will be blank meaning no PTM are sele
75. next to the proposed sequence the auto de novo confidence score is shown Positional confidences that is confidence that the correct residue in each position has been identified are readily available 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 a low confidence less than 60 For more detailed positional confidence we can place our mouse over ne al s e Tiina LOA a_ imm Seq H20 NH3 Ye 4 mme ula ale ajaj Peptide Candidates PEAKS Auto De Novo 0 1 Trypsin with Cam PEAKS DB Search sprot 0 1 Trypsin w VDVEK 99 DVVEK lt 1 VLTEK lt 1 VDPMK lt 1 p LTVEK lt 1 E PEAKS DB Search Swiss Prot 0 1 Trypsin with Cam VDVEK 99 Color Code 90 80 90 60 80 lt 60 eZ Z Go gt e te e e o tes 100 03 14 28 10 ai fe y Color Code 90 80 90 60 80 lt 60 eo M7 weet 2 the sequence of interest A Position Confidence Table will appear showing the confidence that each tag subsequence is correct In the Ion Table frame select a cell from the Ion Table each cell represents an ion This will highlight its position on an error plot scroll the Ion Table frame down if the error plot is not visible A point cl
76. ng sequences with PTM Sequence data and protein identification results for a given spectrum are stored in an ANZ file Any modifications that were found in the sequence are also included As such user defined modifications will still show up if the file is viewed on another machine It is not necessary to import all PEAKS properties to view these modifications Also user defined modifications can be extracted from an ANZ file and added to the local PEAKS properties To export PEAKS properties to a file open the PEAKS Properties dialogue and press the Export button Type in a file name and press the Save button To import PEAKS properties from an file open the PEAKS Properties dialogue and press the Import button Select a file or type in a file name and press the Open button This must be a PEAKS configuration file in XML format To import a user defined PTM from another user s ANZ file we open the ANZ file and find a sequence containing the user defined modification Right click on that sequence to bring up the popup menu Click the View Modifications menu item This brings up a dialogue box named Modifications Select the PTM of interest from the dropdown list in this example Lab 2 custom PTM and click the import modification button ll Modifications Lab 2 custom PTM snowmoancaton Importadas 5 46 Configuring the ion table The ion table displayed in the top right of
77. not have existing sequence information or if we wish to refine our database search by providing brand new sequence information we can ask PEAKS to perform auto de novo before searching the database Brand new results will not overwrite any existing sequence data that we have 1 In the Peptide Data Frame we select the data file s that we wish PEAKS to use to identify our protein s This can be done by clicking on a data file s name in the peptide data frame 2 Click the Protein identification toolbar icon E Or Select PEAKS Protein ID from the tools menu The Protein Identification Parameters dialogue window will appear 3 If we wish to change any of the protein identification search parameters we do so now Parent mass error tolerance determine how much random and systematic experimental error on the parent precursor ion PEAKS will allow for in its analysis Select a tolerance from the dropdown list or type in a value New PEAKS users should try setting this a little higher than past experience would suggest Fragment mass error tolerance determine how much random and systematic experimental error on the fragment daughter ion PEAKS will allow for in its analysis Select a tolerance from the dropdown or type in a value Again new PEAKS users should try setting this a little higher than past experience would suggest Instrument Type choose the type of spectrometer that produced the data to be analyzed If we are
78. of MassLynx may differ file is represented by its precursor ion information m z value followed by the charge of the precursor ion that generated the spectrum and file name Loading Thermo RAW data PEAKS Studio 4 0 can load RAW data from our Thermo Electron mass spectrometer provided that the masslynx software is installed on the same computer as is PEAKS Studio 4 0 To load Thermo RAW data simply choose File Open and browse to the file Importing Masslynx RAW data PEAKS Studio 4 0 can import RAW data from our Waters MicroMass QTOF instrument To do so we choose Import RAW data from the File menu As above the file browser appears Choose the RAW data and click the Open button Again we have the option to merge spectra or not For this to work PEAKS Studio 4 0 must have access to the following libraries which are part of MassLynx DACServer dll Genutil dll MetaGD322 dll raw dll securityAccess dll securitySettings dll securitySignature dil They should be stored in the folder C MassLynx as part of the MassLynx software If they are not stored here or MassLynx is installed on another computer the automatic loading will not work If the automatic loading is not working for either reason try this 1 We should be able to find the listed files on our computer or another computer in our lab If you can copy them do so 2 We can then create a folder called C MassLynx on our computer
79. ose an other database in step 3 we must enter parsing parameters ourselves by typing in the textboxes Alternatively if our database format is the same as one of the public databases we can choose to apply that database s format when PEAKS reads our database Select the database that is similar to ours from the dropdown list and press the apply button to fill the textboxes with the appropriate parsing rules The delimiter is the character used to separate multiple headers If we are configuring the NCBI nr database or the Swiss Prot database we may choose to point PEAKS Studio 4 0 to the location of the taxonomy files associated with that database Under Taxonomy Options we must type the location of the taxonomy files or click browse to find the file on our system If we do not specify these taxonomy files we will not be able to limit our database search to a specific taxon We can use the compressed zip or gz files no decompression is required for the taxon files A note on choosing the taxonomy files for NCBI nr At the time of printing the gi taxid file was called g Zaxid protdmp gz and the taxdmp file was called saxdmp zip Select these files 7 Press the Finish button to complete the database configuration We can repeat this process to configure a number of other databases Once configured a database need not be configured again unless we update the database itself 13 Trouble shooting Some
80. ose to the centerline indicates a more confident Zoom in far enough and we may resolve the isotopic ladder depending on our instrument data result We can also notice that the peak corresponding to the Ion we selected is highlighted on the Spectrum View Select a whole column to highlight all the points for that type of ion The types of ions displayed in the ion table can be configured choose Configuration gt Edit Ion Table from the Edit menu FTMS users might find this particularly useful when sequencing data acquired using ECD By looking at the Spectrum View Frame we can see the strength of the MS MS peaks that PEAKS Studio 4 0 has set as ions The view also displays the mass of the ions at that peak and the type of ion Click on a peak to mark it and display its information at the top left corner of the Spectrum View Frame Zoom in by clicking and dragging horizontally on an area of the Spectrum view The area over which we dragged will now take up the whole spectrum view To un zoom press the E undo zoom icon or press the 11 1 icon to return to the full spectrum view We may also zoom in on the spectrum using the Spectrum Alignment Frame Again click and drag horizontally on an area of the Spectrum view The area over which we dragged will now take up the whole spectrum view The blue bar beneath the Spectrum Alignment view shows where we ate zoomed in The white portion of the bar represents the area that we are zoomed in on
81. oses only 2 Ownership The Software is a proprietary product of BSI and is protected by copyright laws and international copyright treaties as well as other intellectual property laws and treaties BSI shall at all times own all right title and interest in and to the Software including all intellectual property rights therein You shall not remove any copyright notice or other proprietary or restrictive notice or legend contained or included in the Software and you shall reproduce and copy all such information on all copies made hereunder including such copies as may be necessary for archival or backup purposes 3 Restrictions Licensee may not use reproduce transmit modify adapt or translate the Software in whole or in part to others except as otherwise permitted by this Agreement Licensee may not reverse engineer decompile disassemble or create derivative works based on the Software Licensee may not use the Software in any manner whatsoever with the result that access to the Software may be obtained through the Internet including without limitation any web page Licensee may not rent lease license transfer assign sell or otherwise provide access to the Software in whole or in part on a temporary or permanent basis except as otherwise permitted by this Agreement Licensee may not alter remove or cover proprietary notices in or on the Licensed Software or storage media or use the Licensed Software in any unlawful manner
82. ot by each spectrum shows dark green for unprocessed or light green for sequenced or partially sequenced An asterisk next to a spectrum shows that it contains unsaved information Spectra ate grouped by data files or by nodes which act like data files Select a data file or node by clicking on its name i e click on CytC ESLanz in the above example or a spectrum within a data file by clicking on it Use the and boxes to expand and collapse the view Task Queue frame bottom left Shows running tasks sorted by priority Working area right This is where the Protein Identification Result Window and the Main Processing windows appear Menu bar access file edit view tools windows and help commands Main window toolbar quick access to many commands See Toolbars section below Auto de Novo Parameters Dialogue De Novo Options De Novo Sequencing Parameters Saved parameters Parent Mass Error Tolerance 0 2 Fragment Mass Error Tolerance 0 2 Instrument Quadrupole TOF Enzyme Semi Trypsin Report up to peptides 5 Edit Enzymes PTM selected for search Fixed Carbamidomethylation Var m xidation Add Remove PTM Deconvolute the data for analysis use for non deconvoluted data Save As Ok Cancel Parent mass error tolerance determines how much random and systematic experimen
83. present the quality of the match and the number of programs in agreement on the peptide Peptide view is available by clicking on the peptide view tab In tabular format it displays relevant information about each peptide found Since two peptides may match to one MS MS spectrum they are visually grouped together using colour by MS MS scan Peptide Mr Calc Delta M Score Start End Accession Peaks XTandem _Omssa Y TGPNLHGLFGR 1167 6150 015 99 44 27 CYC_BOVIN EAPPGNAKAGEK 1167 5 0 0116 60 0 5 CYC2_ARA TGPNLHGIFGR 11676150015 71 67 28 CYC_APTPA Y Y ISIS RIR PIRIPIRI IRIS LFVQK 633 385 0 015 71 11 CYC_BOVIN KEMVK 633 352 0 048 63 89 CTN2_MO TGQAPGFSYTDANK 1455 6 0 0631 99 72 CYC_BOVIN DAYAGGTKCSADLK 1455 6 0 0665 60 56 ACFD ECO mM KTGQAPGFSYTDANK 1583 758 0 042 100 0 CYC_BOVIN SEDTLYEYLLNPK 1583 7720 0281 61 11 CYC_SPIOL nN URREA N CREWFWLRLYYLYLK 2208 1 10 6658 70 0 GRT1_SCH A When we first load the report it is sorted by MS MS index This is analogous to scan number or DTA file name unless spectra have been merged In the example above there were two matches returned for MS MS spectrum 1 EDLLAYLK and
84. real people will use our software tools As such we hold in principle that it is not enough to develop solely on theory we must develop with customer needs in mind We believe the only solution is one that incorporates quality and timely results a satisfying product experience customer support and two way communication So then we value market research development flexibility and company wide collaboration evolving our offerings to match the matket user s needs Efficient and concentrated research development customer focus and market analysis have produced PEAKS software for protein and peptide identification from tandem mass spectrometry data RAPTOR and PROSPECT Pro software for threading based 3D protein structure prediction and PatternHunter software for all types of homology search sequence comparison PEAKS Software License This is the same agreement presented on installation It is provided here for reference onb If we are evaluating a time limited trial version of PEAKS and we wish to update the software to the full version we must purchase PEAKS and obtain a full version registration key 1 License Subject to the terms and conditions of this Agreement Bioinformatics Solutions BSI grants to you Licensee a non exclusive perpetual non transferable personal license to install execute and use one copy of PEAKS Software on one single CPU at any one time Licensee may use the Software for its internal business purp
85. roup plus the intervening neutral amino acid residues c ions an N terminal fragment holding at least one charge similar to a ions and b ions This is a prefix fragment of the peptide The c ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues plus the mass of ammonia x ions a C terminal fragment holding at least one charge similar to y ions and z ions This is a suffix fragment of the peptide The x ion s mass will be the sum of the masses of the C terminal group plus the intervening neutral amino acid residues plus the mass of Carbon Monoxide y ions a C terminal fragment holding at least one charge similar to x ions and z ions This is a suffix fragment of the peptide The x ion s mass will be the sum of the masses of the C terminal group plus the intervening neutral amino acid residues plus the mass of H z ions a C terminal fragment holding at least one charge similar to x ions and y ions This is a suffix fragment of the peptide The z ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues subtract the mass of ammonia Residue as used in this manual a residue refers to what remains of an amino acid once it has become part of a peptide or peptide fragment In this manual residues are referred to by their original amino acid names Resolution refers to the precision of an instrument On
86. s cleavable ICAT TM lt BuiltIn Applied Biosystems cleavable ICAT TM lt Built In gt Applied Biosystems TRAQ TM multiple lt Built In Applied Biosystems original ICAT TM 7 lt BuiltIn Applied Biosystems original ICAT TM lt Built In Beta methylthiolation lt Built In gt Biotinyl iodoacetamidyl 3 6 dioxaoctang lt BuiltIn gt Biotinylation lt BuiltIn C Mannosylation lt Unselect Selected Fixed PTMs Carbamidomethylation Select As Fixed gt Select As Variable gt lt Built In gt Carbamidomethylation Edit PTM lt BuiltIn Carbamylation lt Built In Citrullination New PTM Oxidation Selected Variable PTMs Deamidation lt BuiltIn Deamidation lt Built In gt Dimethylation Built In Farnesylation lt Built In Flavin adenine dinucleotide lt Built In Formylation lt BuiltIn Gamma carboxyglutamic acid lt BuiltIn gt Geranyl geranyl lt Built In gt Glucosylation Glycation Built In Guanidination 4 Il Phosphorylation Remove PTM Remember when doing auto de novo sequencing or PEAKS Protein ID on a complex mixture we will get best results if we choose the correct fixed PTM and a few variable PIM When using PEAKS Protein ID to characterize a protein it is best to search against a small database that contains only a few proteins and turn on all modifications Furthermore to limit spurious hits we can assume that it is less likely that a tryptic length
87. s we wish to use by clicking the yes radio button next to each one Instrument Type choose the type of spectrometer that produced the data to be analyzed Merge Scans of the same peptide in DDA mode a mass spectrometer will often produce several tandem ms i e ms ms scans of the same peptide To increase the intensity of real signal peaks within these scans and to reduce the size of the whole data set it makes sense to merge ms ms scans of the same peptide together To avoid improper merging of ms ms scans of different peptides we make sure that the measured parent ion masses of these peptides are very close and that they have similar retention times in the LC column Correct precursor charges Since a mass spectrometer measures mass to charge ratios we must know the charge on a peptide before we can determine its mass The standard method of finding the charge is to look at the spacing of the isotope ladder in the survey scan However many lon Trap instruments don t have enough resolution for this So PEAKS will look at the MS MS data to determine if it s charge 1 2 or 3 This tool need only be used on ion trap data Remove Low Quality MS MS scans Scans of contaminants and electrical noise should not be included in analysis Removing them from the data set will save time and reduce the risk of random matches to the database PEAKS presents an effective tool for removing these low quality ms ms scans This tool has bee
88. se the main processing window before loading a new one for a different spectrum Load sample files at start up _ Show GNU Public License Cancel Environment To change the working environment we open the Environment Preferences dialogue and ensure that the Environment tab is selected We can change the environment settings so that when we are browsing our system to find or save data files PEAKS always starts looking in the folder we specify The current working folders for data input and data output are shown We can choose to have PEAKS Studio 4 0 use the last folder we loaded from saved to as the current working folder or toggle the appropriate radio button to User directory to set it ourselves so that it will be the same each time The directory where PEAKS stores its preferences information cannot be changed We can choose to load a new spectrum view window for each spectrum or open one at a time We can choose to show the sample data at startup We can display the GNU license whenever GNU governed software libraries are called Click the appropriate checkbox at the bottom of the window Once we ve chosen from these options pressing the Ok button will exit saving changes The Cancel button will exit discarding changes 49 Colors For ease of viewing we can choose which colors we would like to represent which items on the spectrum view To change the color of an object
89. ssessessssessssasscsesensessessessssessees 90 REFERENCE PEAKS PAPER ccno E Ase EESE EEL OEE E ETE EE ASEC EEEO ECSS S OOCR ESE I ST E 92 Introduction Introduction to PEAKS Studio 4 0 PEAKS makes the interpretation of MS MS data much easier and muh faster EAKS is an innovative software system designed to derive amino acid sequences and identify proteins from tandem mass spectrometry data After running MS MS on a protein sample PEAKS performs de novo sequencing and database search identification of the protein s and peptides using raw experimental data PEAKS Studio 4 0 provides peptide sequence and protein identification results via an intuitive interface allowing for rapid visual interpretation PEAKS provides both auto and manual de novo sequencing tools for detailed examination of MS MS spectra providing the flexibility to manually modify auto de novo results when searching for additional sequence possibilities How to use this user s manual This user s manual is intended to help us get started using PEAKS Studio 4 0 acquaint us with its functionality show us how to customize PEAKS to our application allow us to work efficiently with the interface provide a task based reference and help us with troubleshooting As such this manual is organized into chapters based on these categories Use the table of contents at the front of this manual to access the relevant section If searching for the definition of a particular ter
90. t F PEAKS Properties Enzyme list PTM Library Database List of PTMs Valinol lt Built In gt 4 hydroxynonenal HNE lt Built In gt Acrylamide adduct lt BuiltIn gt Applied Biosystems cleavable ICAT TM heavy lt BuiltIn gt Applied Biosystems cleavable ICAT TM light lt BuiltIn gt Applied Biosystems TRAQ TM multiplexed quantitation chemistry lt Built In gt Applied Biosystems original ICAT TM dO Import Export Cancel Editing a PTM To edit a PTM we open the PEAKS Properties dialogue ensure that the PTM tab is selected and select a PTM from the list by clicking on it and click the Edit button To edit a PIM on the fly while setting up PEAKS auto de novo or PEAKS Protein ID dick the add remove PTM button to bring up the Modification window for that search then click the new PIM button The PTM Editing dialogue will appear Now we follow the same procedure see above as we would if creating a new PTM 43 Removing a PTM To remove a PTM we open the PEAKS Properties dialogue ensure that the PTM tab is selected select a PIM from the list by clicking on it and click the Remove button Built in PTM cannot be removed Database Manager PEAKS Studio 4 0 needs a protein or EST database in FASTA format to identify protein candidates Since databases are being constantly updated PEAKS does not ship with a protein or EST databas
91. tal axis of the alignment view indicates the range of the spectrum view in the Spectrum View Frame The Spectrum Alignment Frame can also show the positions of major ions that delimit the proposed sequence By default the Spectrum Alignment Frame displays b ion and y ion peaks and the derived peptide sequence between them The Spectrum Alignment Frame can also show the position of c ion and z ion peaks 35 The lon Editor is used when performing manual de novo sequencing lon Editor ion es Please choose ion type Selected peak information E intensity 4 0323 2 N Term lons current ion Add H20 Remove NH3 Comments Selected peak information displays information about the currently selected peak Under Please choose ion type the radio buttons set whether the ions in the ion choice list are C terminal ions or N terminal ions Ton choice list left lists the ions we can apply to the selected peak Selected ion list right lists the ions we have selected add or remove them using the Add and Remove buttons Apply button applies the ions in the selected ion list to the selected peak Export Image Dialogue Width 640 Height 480 Format gif Filename caProgram FilesiPEAKS Studio Dataimultiple spectra 927 4 gif a 7 Export selected area Peptide candidate tree n table and error map urrent spectrum window Co Cea
92. tal error on the parent precursor ion PEAKS will account for in its analysis Select a tolerance from the dropdown list Fragment mass error tolerance determines how much random and systematic experimental error on the fragment daughter ion PEAKS 24 will account for in its analysis Select a tolerance from the dropdown list Instrument choose the type of spectrometer that produced our data Choose from a dropdown list Enzyme choose from a dropdown list of enzymes that we used to digest our protein sample Click the Edit Enzymes button to edit the enzymes defined in this list or to add to it Report top set how many de novo sequence candidates PEAKS will report Choose from a dropdown list PTM selected for search this box displays the modifications currently selected for analysis these will be considered during auto de novo sequencing To change this click the Add Remove PTM button Preprocess before auto de novo PEAKS Studio has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data Check this box to turn preprocessing on Protein Identification Parameters Dialogue Database Search Options Protein Identification Parameters Saved parameters Parent Mass Error Tolerance 0 1 Fragment Mass Error Tolerance 0 1 Instrument Enzyme gtof standard Quadrupole TOF Database to search Semi Trypsin NR v
93. te EEE A A EE EEES 23 PEAKS Studio main Wii ow sais suas cuss A NN 23 Auto de Novo Parameters Dial essa sb 24 Protein Identification Parameters Dialogue PEAKS Properties Dialogue Enzyme Editor Dialogue s PIM Selector DiGlo gue niaren nre ETALE ERA aE EAE OEA IAEA AP DENON LA EAEAN EEEE arandos PIM Editing Dialogue osere oa RER EEE SEE ER EE E EAEE EIEEE E EERE CUL EAE iguseatcagavaxeeasayeaveces voxecagusensevahsuseccgeseuscctasvadecgusese Ion Table Settings Dialogue a Protein Identification Result WindOWiszz versar cestasa Knai alas ees areias esa a papos ade DER asses amaro aca ua CORONA EA SA LESNAR centra encon SESH UE eae 31 Peptid Viewers RRE NRO 31 Protein View Search parameters Main Processing Window ne Ton EUA SERE NAAA SIR ROA AIR EXPO AMA DIA lO UE tos EAA Print Image Dialogue TOBA bbuvanddulsvaie KEE EEEE P UEA EE EEEE EEE PTEE EEE NEE EFESE IRIARTE OELSE ET S OEELA SE ETLE ONEI ENAR OEE AESSR E E Main Processing Window Toolbar A A EE S 40 PEAKS STUDIO CONFIGURATION cccscsssssssssssssessssssssssssssssssssassssesscssssesssssscseseassssesesssssessesssscssseassssessessssessssssscsoseassesessessssessesesscseseasessessasessessees 41 PEAKS PROPERTIES CONFIGURATION eiii A At 41 Creating and defining PTM E Creating a New PM A career saida Lee sa ach ces ROUND E ENO AU ELSA saad ee ale acd ENTE ia adiar resales da Editing a PIM A NN Removing a PTM Database Manager Load Configure
94. ter setting two ions PEAKS Studio 4 0 will estimate the residue found between them if a residue corresponds closely to the mass difference The peptide sequence candidate name as displayed in the peptide candidate frame will change to show the residue and the mass remaining to be sequenced on either side of the residue Using sequence tags Searching the C N terminal by Y B right click anywhere in the Spectrum View Frame to trigger the popup menu From the menu select the terminal search of interest PEAKS will select the appropriate terminal tags and show them in the Ion Table Frame We may test the suitability of a tag by clicking on its radio button the tag will be shown in position on the Spectrum View We may insert one or more tags by clicking on their checkboxes then clicking the Apply button Press the Cancel button at any time to exit the search discarding changes Search a sequence tag select a peak with a defined ion i e an ion that has been labeled with a peptide Right click to trigger the popup menu then select Search Right or Search Left to search peptide tags either to the right or left of the selected peak PEAKS will select the appropriate terminal tags and show them in the Ion Table Frame We may test the suitability of a tag by clicking on its radio button the tag will be shown in position on the Spectrum View We may insert one or more tags by clicking on their checkboxes then clicking th
95. the main processing window displays all the ions that were found as evidence for the selected sequence There are two presets the Basic Table and the Advanced Table Select which one to display by choosing Show Ion Table gt from the View menu PEAKS Studio HE KKRELQCQDPCCCCR lt 1 KNLWLQCQDCPCCCR lt 1 QKRELQCQDPCCCCR lt 1 The Basic Table will display a maximum of 6 ions The Advanced Table can be configured to display as many as are available To configure the ion table we choose Edit menu gt configuration gt edit ion table and then select which table we d like to edit PEAKS DB Search NCBI nr 0 7 0 7 Tryg 157 88 EJES 1456 53 85 93 1271 24 1158 12 1030 03 927 46 798 96 683 92 483 87 mojojojo viD jojolol r The Ion Table Settings dialogue box will appear Please choose ion types charge combination and add selections to ion table columns list lon Table Columns Immonium 47 The ions types that will be displayed in the ion table are shown on the right The complete list of ion types available is shown on the left To add an ion type to the ion table i e add a column to the ion table 1 Select one or more ions from the list on the left Use Shift click and Ctrl click to select multiple list items 2 Select a charge
96. these parameters we do so now Parent mass error tolerance determine how much random and systematic experimental error on the parent precursor ion PEAKS will allow for in its analysis Select a tolerance from the dropdown list or type in a value New PEAKS users should try setting this a little higher than past experience may suggest Fragment mass error tolerance detetmine how much tandom and systematic experimental error on the fragment daughter ion PEAKS will allow for in its analysis Select a tolerance from the dropdown or type in a value Again new PEAKS users should try setting this a little higher than past experience may suggest Instrument Type choose the type of spectrometer that produced the data to be analyzed If we are using a hybrid instrument choose a setting that matches our fragment ion mass analyser For example if we measure the parent ion in an FT and the fragment ions in an ion trap choose the ion trap instrument setting Fragmentation type can also be chosen from this drop down 63 Enzyme Tell PEAKS what kind of enzyme was used to digest the sample Choose from a dropdown list of enzymes or if our enzyme or combination of enzymes is not in the list click the Edit Enzymes button Report top set how many peptide sequences PEAKS will report from its de novo sequencing analysis Max missed cleavages determine the most missed cleavages to allow internal to the peptide in a de novo seq
97. to from a peak Select a peak then right click the mouse anywhere in the Spectrum View Frame Select Set Y Ion from the popup menu to designate the peak as a y ion Set B Ion from the popup menu to designate the peak as a b ion Select Ton Edit from the popup menu to view the Ion Editor dialog box and designate the peak as another ion 342 2 294 11 341 2112 NH3 E 42221 e2 HON b3 H20 Search left 70 See PEAKS Environment Preference Configuration to find out how to change the sensitivity of the residue estimate Two short cut keys can be used F6 for searching the left side and F7 for searching the right side The Ion Editor dialogue allows us to add or remove ion designations to from a peak Select an ion from the ion choice 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 We can type any comments we wish to make about the ion peak then press the Apply button to apply the changes to the selected peak Two short cut keys may also be used to label a peak Select a peak then hit the y key to add a y ion and or the b to add a b ion to the peak After setting an ion both the alignment view and the peptide sequence candidate name as displayed in the peptide candidate frame will change to reflect the mass remaining to be sequenced on either side of the ion Af
98. to de novo prior to our database search the Auto De novo process will appear first in the task queue Once this is finished the database search will begin If PEAKS finds protein candidates after searching the database a Protein Identification results window will appear inChorus protein identification inChorus protein identification will call upon several search engines for protein identification Once we load our data into PEAKS we can invoke start searches running on several search engines at once When all the results are returned PEAKS Studio 4 0 will compare the answers and summarize everything in one simple report 1 In the Peptide Data Frame we select the data file s that we wish PEAKS to use to identify our protein s This can be done by clicking on a data file s name in the peptide data frame 2 Click the Protein identification toolbar icon del Or Select inChorus Protein ID from the tools menu The inChorus database search launch window will appear 77 Help is sometimes available by holding the mouse over or clicking on a part of the screen InChorus Database Search Peaks Database Search Engine Peaks database Search FS Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines C XTandem Search Options Omssa Search RA Options Spider Search Options port Database Search Results C Mascot Res
99. to from a file Creating and defining PTM If we know that our sample protein may have been modified since translation we need to apply this information to our analysis To edit the list of PTM available to PEAKS Studio we use the open the PEAKS Properties dialogue and select the PTM Library tab F PEAKS Properties Enzyme list PTM Library Database List of PTMs lt Built In gt 4 hydroxynonenal HNE lt Built In gt Acrylamide adduct lt BuiltIn gt Applied Biosystems cleavable ICAT TM heavy lt Built In gt Applied Biosystems cleavable ICAT TM light lt BuiltIn gt Applied Biosystems TRAQ TM multiplexed quantitation chemistry lt BuiltIn gt Applied Biosystems original ICAT TM dO lt Built In gt Applied Biosystems original ICAT TM d8 lt Built In gt Beta methylthiolation Import Export All PTM are listed here including lt built in gt PTM and user defined PTM From here we can create a new PTM edit an existing PTM or remove a PTM from the list See the sections below for help with these operations Bwi t in PTM cannot be removed from the list but can be edited Editing a Built in PTM It is possible to modify a built in PTM PEAKS will save the modification and treat this PIM as a customized PTM It will temporarily overwrite the built in PTM we will not be able to see the original built in PTM until we remove the customized one We can remove this
100. toggle show hide the location of PEAKS corresponding to b ions and the corresponding proposed peptides between them 8 Deconvolve button toggle on off deconvolution of the mass spectrum scale 11 4 1 zoom button return spectrum to original 1 1 zoom A Undo Zoom button return to previous zoom ratio 21 Edit Ion button set or edit the type of ion associated with a peak in manual de novo Press this button after having selected a peak in the spectrum view frame e Next Peptide button redo changes to the peptide in manual de novo 8 Previous Peptide button undo changes to the peptide in manual de novo EJ Export Results button export the spectrum view ion table or to a picture bmp gif or jpg format with ions masses PEAKS and peptides marked E Print Results button print the spectrum view with ions masses PEAKS and peptides marked gt View Results button show in HTML format the spectrum view with ions masses PEAKS and peptides marked peptides and confidence scores the ion table and the error plot 40 PEAKS Studio Configuration How to set up PEAKS Studio just the way we lke ut his chapter deals with configuration PEAKS Studio 4 0 is a versatile and flexible tool But in order to use the software to its full extent we must learn how to configure it to make it do what we want it to Additionally PEAKS Studio 4 0 allows us to set
101. tra ate removed merged corrected and the data results relationship may be broken PEAKS Studio 4 0 will warn us when this may occur and prompt us to save a separate copy Editing Precursor information It is possible that the precursor information as listed in the Peptide Data Frame is incorrect If the charge File Edit Tools Windows Help listed is wrong or if the m z listed is even slightly slafala njx o a E incorrect more than 0 1 Daltons depending on the Peptide Data E Apo myoglobin ann accuracy selected it could really affect the quality of 409 22 the results In this case it is imperative that we sm change the precursor information The change will only affect the ANZ file we are working on To edit precursor information select a spectrum by clicking on its name then right click the mouse i while holding it in position oe the name A small xi menu will appear Click on Edit Precursor a 678 5 In the dialogue that follows type the new Mtensitiy 1 precursor information into the appropriate Charge 2 textboxes Click the Apply button when finished to apply the changes Click the Cancel button to exit discarding changes Apply Cancel The precursor information will be updated reflected by a change in the name of the spectrum in the Peptide Data Frame A will also appear in front of that name indicating that there is unsaved inform
102. uence For instance if we set this to 2 and Trypsin is the enzyme then PEAKS will return de novo sequences with up to 2 R s or K s internally PTM selected for search this list tells PEAKS what kind of post translational modifications to include in it s analysis Each is marked Fixed or Variable To edit this list click the Add Remove PTM button Max variable PTM per peptide To reduce uncertainty we can limit PEAKS de novo sequencing vocabulary by restricting the number of variable PTM we can find on a peptide Specify a number by typing it into the box To lift such restrictions type a very large number longer than the length of the peptide Saving Loading Parameters After setting up parameters we can save them for future use Click the Save Parameters button and choose a name for future reference when prompted Don t worry we can t accidently overwrite the defaults Any parameters we save will be available in the drop down list at the top of the window To see what s inside just select one and the parameters boxes will be populated Preprocess before auto de novo PEAKS Studio has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data Check this box to turn preprocessing on Notes on pre processing BSI highly recommends using PEAKS to preprocess all data as opposed to using instrument vendor software if the data is to be used by PEAKS PEAKS preprocessor sho
103. uence candidates PEAKS will report Choose from a dropdown list PTM selected for search this box displays the modifications currently selected for analysis these will be considered during database searching To change this click the Add Remove PTM button Preprocess before auto de novo PEAKS Studio has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data Check this box to turn preprocessing on Advanced options de novo sequencing The PEAKS approach to protein identification uses de novo sequences to help out in the seatch This section allows you to decide how to obtain the de novo sequences required for the search PEAKS Properties Dialogue Y PEAKS Properties Enzyme list PTM Library Database List of Enzymes lt Built In gt Arg C lt Built In gt Asp N lt BuiltIn gt Asp N N terminal Glu lt Built In gt Chymotrypsin lt Built In gt CNBr lt Built In gt Glu C bicarbonate lt Built In gt Glu C phosphate lt Built In gt Lys C lt Built In gt None eDuilt Ins Dancin mil 4 9 v Cancel Enzyme list tab Displays a list of built in and user defined enzymes We may edit and create Enzymes from here PTM library tab Displays a list of built in and user defined PTMs We may edit and create PTM from here Database tab Displays a list of databases available to
104. uld not be used on data that has already been pre processed as this will have adverse effects on the results unless it is ion trap data 4 Press the Ok button to commence Auto de novo sequencing Once a job is submitted to PEAKS Studio 4 0 it is added to the Task Queue for processing After processing the job is removed from the task queue list and the icon beside the spectrum in the Peptide Data Frame changes to light green and or an asterisk appears 65 Confidence scores are probability based on a scale of 0 to 100 Viewing Auto de novo Results After performing auto de novo on a spectrum we may wish to see what the algorithm determined the peptide sequence to be and review the results for ourselves To do so we click on the spectrum of interest in the Peptide Data Frame This brings up the Main Processing Window for that spectrum The most likely peptide sequence candidate as determined by auto de novo will be automatically selected This is found in the Peptide Candidates Frame as the top listed candidate under PEAKS Auto De novo In the example above this is the highlighted sequence VDVEK Any modifications that have been found will be shown abbreviated and in sequence before the amino acid residue they are associated with If the PTM was defined created by another PEAKS user on another system the PTM will still be shown and it can be imported into the local PEARS configuration as desired Right
105. ult dat Mi Browse Sequest Result summary html gt Browse Cancel 3 First select each of the protein identification tools we would like to use by putting a checkmark in their respective checkboxes Search parameters for each program can be set by clicking the corresponding Options icon PEAKS database search engine is mandatory 4 Then set search parameters for each search engine Options screens each of the programs available to inChorus are designed to work in the same way as options screens for the original programs For help in setting search parameters for each program please refer to that program s user manual In the case of PEAKS database search please refer to the above section Viewing Protein identification results To view Protein identification results for a data file we must have performed PEAKS protein identification or inChorus protein identification on that data file The result from each protein identification search is represented by the time stamp and database searched just under a data file s Protein ID Result Click on one to display the results report We can view results by peptide or by protein and check on the search parameters we used to generate these results Search parameters that we used when generating this report are preserved for future reference and are available by clicking on the search parameters tab 78 Scores associated with each peptide re
106. um Alignment Frame Now we are ready to edit the sequence using manual de nove techniques 68 Manual De Novo Sequencing We can use manual de novo sequencing to fine tune the results of an auto de novo analysis or to perform our own sequencing analysis from scratch PEAKS Studio 4 0 provides a set of tools to help us sequence a peptide using graphic cues from the spectrum Creating a fresh spectrum for sequencing We cannot change the results provided by PEAKS auto do novo or PEAKS database search Thus to begin manual de novo sequencing we must either copy a sequenced peptide see above section Preparing to edit sequence results or create a new peptide candidate for sequencing To create a new peptide candidate for sequencing 1 Right click on the Peptide Candidates heading the Manual De novo or any user defined type heading This will bring up a popup menu 2 Select New candidate for manual de novo from the popup menu A new candidate will be created under the Manual De nove heading or under the user defined type heading if we selected a user defined type The new candidate will not have been sequenced so it will be represented by the mass difference across the spectrum e g 945 15 Manual De novo Operations All operations occur in the Spectrum View Frame of the Main Processing Window When the mouse is placed in the Spectrum View Frame a blue by default bar follows the movem
107. up many defaults and presets to help us be quick and precise We can use PEAKS Studio 4 0 without the need to configure default settings will be used However to increase efficiency we should set environmental preferences and PEAKS properties This will enable us to customize the tool to our requirements It is recommended that we configure PEAKS Studio 4 0 before processing data files PEAKS Properties Configuration One of PEAKS Studio 4 0 preferences PEAKS Properties configuration sets the parameters that the algorithm will use in processing our data files PEAKS properties include Enzynme PTM and database PEAKS Studio 4 0 provides tools to edit PEAKS properties for convenient use in de novo sequencing and protein identification To edit PEAKS Properties Click the P icon in the main window toolbar Oy from the Edit menu select Configuration then PEAKS Properties Or Click the Edit PEAKS Properties button in the Protein Identification or Auto De novo Options dialogue that appears before each Protein Identification or auto de novo operation The PEAKS Properties dialogue will then appear This dialogue box has three tabs Enzyme list PTM Library and Database Clicking a tab will allow us to edit PTM Post Translational Modifications affect the mass of modified proteins and residues the PEAKS Properties corresponding to that tab We can also import or export our preferences
108. using a hybrid instrument choose a setting that matches our fragment ion mass analyser For example if we measure the parent ion in an FT and the fragment ions in an ion trap choose the ion trap instrument setting Fragmentation type can also be chosen from this drop down Enzyme Tell PEAKS what kind of enzyme was used to digest the sample Choose from a dropdown list of enzymes or if our enzyme or combination of enzymes is not in the list click the Edit Enzymes button Report top set how many peptide sequences PEAKS will report from its de novo sequencing analysis Max missed cleavages determine the most missed cleavages to allow internal to the peptide in a de novo sequence For instance if we set this to 2 and Trypsin is the enzyme then PEAKS will return de novo sequences with up to 2 R s or K s internally PTM selected for search this list tells PEAKS what kind of post translational modifications to include in it s analysis Each is marked Fixed or Variable To edit this list click the Add Remove PTM button Max variable PTM per peptide To reduce uncertainty we can limit PEAKS de novo sequencing vocabulary by restricting the number of variable PTM we can find on a peptide Specify a number by typing it into the box To lift such restrictions type a very large number longer than the length of the peptide Best practices for setting modifications PTM The developers have discovered that database searching o
109. y The settings that we used before should still be there If not select OrbiStandard from the drop down list in the top right corner Before pressing the OR button we can make one change Since we already have de novo sequencing results we don t need to do de novo sequencing again Click the option I have already run de novo don t do it again then press the OK button 19 Click the X Tandem search options button top This window allows us to set options for the X Tandem search tool This window is set up to behave almost exactly the same as the X Tandem interface so it may look familiar Leave the fragment error at 0 1 and make sure there are no modifications turned on Under 7 Predefined methods choose FTICR To learn more about X Tandem settings double click any of the question marks Press the OK button Now that we ve set everything up for the inChorus search press the OK button on the inChorus Database search dialogue inChorus will call on each search engine wait until they are finished then compile their results together ensuring the integrity of the data results relationship Watch the task queue bottom left of PEAKS Studio After everything is finished new search results will appear in the Peptide Data frame left stamped with the date and time The task queue will be empty and the results will display There s also a nice little report to tell us if there were any errors 5 05 Jun 0
110. y and Gilles Lajoie PEAKS Powerful Software for Peptide De Novo Sequencing by Tandem Mass Spectrometry Rapid Communication in Mass Spectrometry 17 20 2337 2342 2003
111. y PEAKS PEAKS preprocessor should not be used on data that has already been de convolved by instrument software as this will have adverse effects on the results unless it is ion trap data PEAKS preserves the original data and does not save the results of its preprocessing As such the decision to preprocess or not should be independent of what we ve already done with PEAKS Advanced Options De novo We must have some de novo sequences before database searching since PEAKS sequence tags to help in database searching As such the option of doing de novo prior to protein ID is presented here In most cases the same values for instrument error enzyme and PTM can be used in de novo and in protein ID but we have the option of using one of our saved de novo parameter sets for the de novo portion Select from the drop down list Saving Loading Parameters After setting up parameters we can save them for future use Click the Save Parameters button and choose a name for 76 future reference when prompted Don t worty we can t accidently overwrite the defaults Any parameters we save will be available in the drop down list at the top of the window To see what s inside just select one and the parameters boxes will be populated Note the Advanced Options selections will not be saved 4 Press the Ok button to commence Auto de novo if we have so chosen and subsequent protein identification If we have chosen to perform au

PEAKS Studio Manual 4.0 - Bioinformatics Solutions Inc.

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