Home

PEAKS Studio Manual 4.2 - Bioinformatics Solutions Inc.

1. PEAKS DB Search NR 0 01 0 01 LIVTQTMK 42 4 769 43 TIVTQTK 42 4 Alz Hios ed E amp om E S SO ESE E E EON E r T 180 00 360 00 1 j 900 00 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 environment preferences and then choose either the Basic Ion Table Editor tab or the Advanced Ion Table Editor tab Both tabs look and work the same way lon Types lon Table Columns Immonium I Immonium a Add with charge e a a H20 I b a NH3 Remove b H20 b b H20 H20 b NH3 2 c c H20 c NH3 x 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 SCV SC SCV 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 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 i
2. Sl Reed etl eel ed ed aed dd ed ed RININSISISISISISISISIS Peptide Details Links to retrieve spectrum information 458 73856 2 De novo LDALNENK Database LDAINENK gt gi 229460 pri 732164A lactoglobulin beta Matched peptides shown in Red LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLQK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLVCQO CLVRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI NCBI BLAST search of de novo sequence LDALNENK NCBI BLAST search of database search sequence LDAINENK Protein View The protein view is most useful as a summary 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 Peptide Match Reports bottom section PEAKS presents each protein candidate with a peptide match list beneath it Each peptide that matched the protein sequen
3. Cancel Here we can form 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 33 SCV This dialogue allows us to create or edit a PTM SCV The Basic and Advanced lon tables differ only in the number of ions they can display The Basic table displays up to six ions PTM Editing Dialogue PN 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 term
4. OK button this will commence printing 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 SVC Toolbars SCV 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 c Close data file button Close the selected data file Press this after 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 and not yet saved The file will be saved in the ANZ format Press this after selecting a data file in the Peptide Data Frame SC SC SC a a e E Eal RT Save all files button Save all files Any changes to files will be saved in the ANZ format 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 single MS MS spectrum and remove the old ones Data Refinement
5. Cancel Make sure that Peaks database Search and X Tandem Search ate 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 already 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 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 ta
6. E X gil229460 18 3677 m m 99 50 00 12 a lactoglobulin beta X gil2194 18 309 8 i m 99 50 00 12 Chain B Bovine Beta Lactoglobulin Latti X gil4925 18 351 M E e 99 50 00 12 Chain X The Cys121ser Mutant Of Beta L X gil7207 18 267 M mem 99 50 00 12 beta lactoglobulin water buffalo X gil7576 18 367 M mmm La 99 60 00 12 Chain D The Structure Of Bovine B Lacto X gil2173 18 281 M mm E 99 50 00 12 O Chain A Bovine Beta Lactoglobulin Com R167 17 167 ME fea 99 53 64 12 beta lactoglobulin X gil7245 18 375 TE E 99 50 00 12 Chain A Structural Changes Accompanyi X gi1259 19 883 T E I 99 45 51 12 Beta lactoglobulin precursor Beta LG A X gil2780 19 921 T E E 99 45 51 12 lactoglobulin beta Bos taurus X gil3808 20 023 T Ee E 99 45 00 12 heta lactoglobulin Bubalus bubalis X gil2237 18 177 E EE ae 99 30 25 tg lactoglobulin beta gil4949 19 976 T EE EE 99 27 22 7 beta lactoglobulin Capra hircus X gil1658 19 947 T Ea E 99 27 22 7 m beta lactoglobulin X gij1279 17 678 E a 99 15 00 5 beta lacto globulin Ovis aries X gil5403 18 151 m a 99 14 81 5 Beta lactoglobulin Beta LG X gil1314 19 921 8 a 99 13 33 5 beta lactoglobulin Ovis aries Ngis308 651 Emmm 99 22 81 3 beta lactoglobulin X gi6579 400 E 96 25 71 2 beta lactoglobulin Bubalus bubalis R373 5 1900 M 81 53 19 2 beta lactoglobulin variant D Bos taurus X gil1151 17 8647 E 55 3 12 1 Chain A Structure Of Porcine Beta Lacto gil4067 19 7207 I 55 2 8
7. PEAKS 4 PEAKS Studio PEAKS Client PEAKS Viewer BIOINFORMATICS SOLUTIONS INC PEAKS 4 2 User s Manual PEAKS Studio S PEAKS Client C PEAKS Viewer V Bioinformatics Solutions Inc 470 Weber St N Suite 204 Waterloo Ontario Canada N2L 6 2 Phone 519 885 8288 Fax 519 885 9075 Written by Iain Rogers Please contact the author for questions or suggestions for improvement S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer INTRODUCTION soenen cd aa aee vas eunde tude snpascsewaesinsouinns sade ansesenewmwesmseiaweraaauaes 4 INTRODUCTION TO PEAKS 4 2 arisini iaee cdeverssncivebvesusevesasteviasadensdzsstaaelassdebeeussddeinus REA aeea ai 4 HOW TOUSE THIS MANUAL roer cess s8tuchvee rie aeae aer ote od aeea TE aa E Rao cupdovedle aee e ie 4 DEON A E EEA E T E TE T E ATE 5 Terminology and Abbreviations GloSsSafy sssssssssssssesessersrsrsrensereseseeneneenenennereesenrnnenrerenensenenresenrenrnrerenrenenees 5 Files and Format GLOSS ALY sijivunii aan i eiii itia ons eiss 7 GETTING STARTED WITH PEAKS 4 2 esesssseseesseeesessesesessooesessooesessooeressooesessooereeseoeeee 8 SCV WHATWE WILL NEED na e a a a e a a a a a A a 8 Package Contents eneit esas sos escheat Fuse Sea AE AEE SE AEEA ENEE AEE ETE REEERE EAE AEE TE esa eve TSt 8 System Tegur emen tE a ient rei ieia D AEE ET EE EPI AE R amas Moa OET 8 EA Sg ULI saiae eea a PESTA STD EAE EE BNR PEER
8. gi taxid taxdmp 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 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 be sure to select the FASTA database not the compressed file of the same name see step 5 Database header e If we chose one of the public standard databases in step 3 its format format is important style will be displayed in the advanced options box The selected database for protein ID result format is shown in the dropdown list Accession number information and reports If parsed the way PEAKS parses the database headers i e the parsing rules are correctly accession numbers and protein names will be shown in full shown in the textboxes below e If our database is an EST database containing DNA sequences check the EST database checkbox 14 If we chose 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
9. Apply Filters button Digging for a protein by name To illustrate this process let us find a protein that contains the word lactoglobulin in the database entry s description Add the Protein Filters called Desc Jn the Edit filter section were required to type in a regular expression regex This allows us to use wildcards A nice common wild card is that means anything of any length For example lactoglobulin Will find anything that contains the word lactoglobulin with anything before and anything after Press the Apply Filters button Setting a protein mass range If we know the approximate mass of the proteins we re interested in we should eliminate all proteins that are not close in mass Add two filters Protein Filters Mass gt 12000 and Protein Filters Mass lt 32000 Press the Apply Filters button Saving Loading Filter sets Were sure to use some common filters repeatedly To save a Filter Set press the Save As button on the filter pane This saves all filters that we ve added to the Selected Filters list To recall a saved Filter Set select it from the drop down list in the top right 103 SCV The bottom panel in protein view shows the columns selected for peptide view SCV SCV We can export only one view one part of the report at a time corner of the filter pane This will simply populate the Selected Filter
10. 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 e PTM library A listing of all possible built in and custom entered post translational modifications that PEAKS can use as a part of its analysis 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 a spectrum this is reflected by how close together can two peaks be and still be told apart 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 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 leas
11. Spectra explained by database Spectra explained by de novo Spectra explained by database Spectra explained by de novo Spectra explained by database Spectra explained by de novo 102 How Filters are linked Filtering out removing a de novo result does not affect what is displayed in the Protein View tab or Peptide View tab Filtering out removing a de novo result also removes that spectrum from consideration in the Peptide View tab and Protein View tab Again filtering out removing a de novo result also removes that spectrum from consider ation in the Peptide View tab and Protein View tab SCV SCV Filter examples Any column of information in any part of the Protein ID report can be filtered Filters cascade throughout the report i e removing a protein removes all its associated unique peptides and removing all peptides from a protein removes that protein too Each filter can be applied several times over So it can get a little complex To illustrate here are a few examples Publication Show proteins that have two high scoring hits Add the Protein Filter called Query and in the Edit Filter section choose greater than and type 1 in the box without the quotes Then add the Peptide Filter called Score and and in the Edit Filter section choose greater than and type 50 in the box without the quotes Press the
12. Peptide View 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 shown below 97 Protein Pane MSA Pane NCBI BLAST search of gil229460 Links to retrieve entries containing this sequence from NCBI Entrez AccessioniD Description gi 229460 lactoglobulin beta Peptides List Index m z z Mass Peptide Mr Calc Start End UniqueID Peaks 1 452 28497 2 902 5554 TKIPAVFK 902 5589 76 83 0 Y 2 458 73856 2 915 4626 LDAINENK 915 4661 84 91 i z 3 467 2729 2 932 5312 LIVTQTMK 932 5366 1 8 3 v 4 533 2925 2 1 064 5704 VLVLDTDYK 1 064 5752 92 100 5 Y 5 545 92804 3 1 634 7623 TPEVDDEALEKFDK 1 634 7673 125 138 6 v 7 597 3386 2 1 192 6627 VLYLDTDYKK 1 192 6702 92 101 7 v 8 623 2947 2 1 244 5748 TPEVDDEALEK 1 244 5771 125 135 8 Y 9 673 38513 1 672 3779 GLDIQK 672 3807 9 14 10 v 10 674 4233 1 673 4160 IPAVFK 673 4163 78 83 13 Y 12 697 7167 3 2 090 1282 LDAINENKVLVLDTDYKK 2 090 1260 84 101 15 v 13 771 75635 3 2 312 2473 VYVEELKPTPEGDLEILLOK 2 312 2517 41 60 17 y 14 818 3876 2 1 634 7606 TPEVDDEALEKFDK 1 634 7673 125 138 18 v 16 903 1304 3 2 706 3694 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 3687 15 40 19 v 7 903 5675 1 902 5602 TKIPAVFK 902 5589 76 83 20 v 18 933 5426 1 932 5353
13. fesidues at the end of a peptide start of a new peptide bracket Use Xto 3 z F denote any residue residues at the end of a peptide start of a new peptide Specificity Parameters example of what peptides can be returned I 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 FLMVVY K P semi 0 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 peptides 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 w
14. Default machine error 0 1 E random machine error 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 the Manual de novo section in the next chapter We can choose how many search results we d like to see and choose how the maximum number of amino acid residues length To choose how long tags will be we click on the Maximum tag length dropdown list box and making a selection To choose the number of search results displayed click on the Maximum return dropdown list and make 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 60 Loading and preparing data A t
15. Then register with PEAKS If problems still occur please contact BSI technical support 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 Configuration In addition to ae nove sequencing of peptides PEAKS 4 2 also has the ability to search through a database search to identify 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 ot download one Additionally we can associate taxonomy with certain databases This is database configuration 11 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 4 2 s main functionality using the NCBI nr database We can use PEAKS without the database search PEAKS can perform de novo sequencing WARNING Downloading a database can take a long time 8 hours depending on connection speed Most only take 20 30 minutes To config
16. displayed for reference under Accession No If the report was generated after inChorus protein ID each peptide that had a search program agree identify the peptide is given a checkmark under its column The columns themselves ate customizeable Right click anywhere in the report and choose Toggle Column from the pop up menu The sub menu that appears shows a checkmark in each of the columns that are currently showing Click any one of them to show or hide a column These settings will apply to all our reports The peptide view list is sort able Click on a column header to sort the list using that column s values For example sort by score Clicking again on the same column header will toggle ascending descending sort Use ctrl click to set a second sort one can sort by Accession then by score in this way When sorting the grouping by MS MS scan number will be retained except when sorting by Accession No Scores associated 21 Nov 06 11 22 04_NR inChorus protein ID results De Novo View Peptide View Protein View Search parameters Filter Pane with each peptide represent the 7 miz Peptide Mr Calc Score Start Accession quality of the 45228407 ITKPAWK 907 5680 eal 76 gil229460 match and the number of 458 73856 _2 IDALNENK 915 4661 gil4388846 prograna iii 467 2729 _2 LIVTOTMK 932 5366 gil229460 467 2729 _2 INTOTMK 932 5366 gil72079 agreement on the 533 2925 _2 VLVLDTDYK 1 064 5752 100 gi
17. the Peptide View In this mode the De Novo View only shows peptides for spectra that could not be satisfactorally explained by searching the 38 See the section Post analysis of results preparing for publication for help on using filters database As such removing a peptide spectrum from the database search results the Peptide View list will cause the de novo sequence for that spectrum to appear on the De Novo View The de novo peptides still have to pass the filters defined for the De Novo View Option 2 De Novo View shows all peptides that are not filtered In this mode the De Novo View shows de novo sequences for all spectra that have not been removed by filtering out database peptides or proteins As such removing a peptide spectrum from the database search results the Peptide View list will cause the de novo sequence for that spectrum to be removed from the De Novo View The de novo peptides still have to pass the filters defined for the De Novo View Checkbox remove de novo peptides with no database hits This option removes all peptides that have no corresponding database peptide hits Selecting Option 2 above and this checkbox shows essentially the reverse of Option 1 Save As Button Save and re use your filters between different sessions of PEAKS All active filters and selections from the Options Frame are saved 39 SCV Main Processing Window
18. 51 SV F PEAKS Properties Enzyme list PTM Library Database List of databases NR Dilirogersidatabasesinr_20051 120 fas SPROT Dilirogersidatabasesisprotfas gt If we choose to update the database perhaps by downloading the latest database file and overwriting the old database file PEAKS will show the database information in light gray A Light grey colour could also mean that the database does not have header information P PEAKS Properties Enzyme list l PTM Library Database List of databases NR Diirogersidatabasesinr_20051120 fas SPROT D irogersidatabasesisprot fas Importing and Exporting PEAKS Properties We may wish to use PEAKS 4 2 on another system However if we have a large number of user defined PTM enzymes and saved parameters 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 PEAKS Properties with the information from XML file If we wish to use our PEAKS properties on a colleague s system we must remember to export our colleague s properties to a separate file so that it will not be lost and can be imported later A note on sharing sequences with PTM Seq
19. 56 SCV CHAMOIS Ol OUTS oei eerie ti i AE S A AAE EEEE EEEE Ei ts 56 SCV Display Changing typefaces and peak width s sssessssssersesesessssrsensrsrsnenrereseneeneneesrnennenrerenrnnenenrenes 57 SCV Changing Manual de novo defaultS ssessesssssessesserrsrsserenresesesnenersrsrsnenrerenensenenersrnennrnrerenrnnenrereses 58 LOADING AND PREPARING DATA woesccdicsusg cdinsidseketueesstieuaspedaaudes chinveusedihvess chieusesediewasd 61 SCV LOADING DATA INTO PEAKS 472 oietatik etrean sath a ee Teee E Ee E E e esek 62 SCY Op nmgdata 1c NPR EERE ee a EE E AAA AE E AEAEE 62 SCV L ading directory Jull of DTA files isiisc ninini i a oE E EE OE E 63 SCV Loading Thermo RAW d t monsenssure nacii on S E ETAETA S ATAA E 64 SCV Importing Applied Biosystems WIFF data ssssssssesssssssrssrnseresseersrsesnenrereseneenenersrnennrnreresrnneneereses 64 SCV Importing Masslynx RAW data cea neniu nnie Aid anai wld ean Cantina A AE TEN Rs 64 SCV Importing Data from the ABI 4700 or ABI 4800 nssssssesssssesssseesrsesserrnresesesnenensrsennrnreresrneenereses 65 DYStEMLREGUITEMENtS are n E EN E E A EE A AE R OE 65 C nfigur ti N aerer sneme ntaa a i a e a e i e eaa aa aii i a 65 Dat extraction pr ced retnoni nnna le RE A A E E E E A a 66 SC REFINING DATA BEFORE ANALYSIS vdani ieie iien ieor ieiet i aiee aE onata ea irio pear taden i iE 66 SCV MANUALLY MANIPULATING DATA FILES sessessssceseeeceeceesecseeeccesecuaceaceeesaecaeseneesecnae
20. 673 4163 697 7167 3 LDAINENKVLVLDTDYKK 2 090 1 260 771 75635 3 VYVEELKPTPEGDLEILLOK 2 312 2517 818 3876 2 TPEVDDEALEKFDK 1 634 7673 903 1304 3 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 3687 903 5675 1 TKIPAVFK 902 5589 933 5426 1 LIVTQTMK 932 5366 1065 586 1 VLVLDTDYK 1 064 5752 1157 1289 2 VYVEELKPTPEGDLEILLQK 2 312 2517 1354 1898 2 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 3687 Matched peptides shown in Red LIVTOTMK OKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLQK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEOSLVCQ CLYRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI Color Code gt 80 60 40 20 Search parameters This tab displays the protein identification parameters that were used to guide the search that generated these results Filter pane The Filter Pane allows us to control what to display on the other tabs of the multi part protein ID report by setting up customizeable filters The various parts of this screen are described as follows Possible Filters list a list of the filters currently available that can be used to build a particular filter set Active Filters list a list of the filters currently selected that will be used to filter your results Add Filter Button clone the currently selected filter in the Possible Filters listing to the Active Filters 37 Apply Filter Button Apply your filters any all selected filters will not be used until this button is actually
21. Dialogue serasetreranrnr n E E a aar ia eano 32 SC PIM Selegtor Dialogue serrie ii EA E EEN E lant 33 SCV PTM Editing Dialogue ssneiisiciiieiniiinisianiien iinis esa tiii re ess 34 SCV Enoironment Preferences Windowsie torrir iiri RaT TEE A E a RETT NEART 34 SCV Protein Identification Result Window ssssssssssesesrsrsserssreseesneserersesnenrereneneenenreseennenrerenennenenreses 35 De Novo VEW E sate E A E E S E E 35 Peptide View smn an E R EE E EE E ee E E pe ia Honea ie ERN E 35 Protetty VIOW such sea ariii ti ieii Teseo iee Toe Eee eat eas eena SEEE a EE EEEE E oboe SEa Ee andetasiedn 36 Search Parameters an an a a E T E aaa Se A ae A ER 37 Filter pane sirrinin eoii hs sua aden anes EA a EE E E AES EE aE a due E e O E Ea E a eie 37 SCV M in Processing Window isiro iari i E A E E A E 40 SCV Jom Ettr re Tiuri EE EAO OEE ATERAT E AEO A OEE 42 SCV Export TMA SE Dialogues ciyin T E NEE EET ATT T AS 43 SCV Print Image Dialogue se sesriini oan Ao n aE REEN EO AE R E E any 44 SVC AKE D is AN ASAA NE TER NTT ET A E EEE E ERETTE T 44 SGV Miata wii oui to OLD AP z Syiir iien r EAE sepa KA E anges 44 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer SGV M in Processing Window Toolbar corse eect pha cre site lire tee cine siete ae ca cleat ra stilted ot 46 PEAKS STUDIO CONFIGURATION 3 siscscsapisvevaseuventarewadevanscveesassvnen tone seessnssvadesenbessasasenes 47 SCV PEAKS PROPERTIES CONFIGURAT
22. E eas 104 SCV WYSIWYG Tepot S ei ecen terenie en aa E ara Ee E aie ee e aE E tag EE Ea e r iseia ta 104 SCV Exportine peak listassa ts cates o Aa AE E EAE AE V O EET 105 SCV Exporting Sequences by SpectrUM s e sssssssersereseressesersrressereeersrssenenresenseneneeseennrrerenrnnenrereneneenene 105 SCV Exporting EXCLUSION Lists sssini tinerii iini en patyreason ge iiiiie 105 SCV Exporting high resolution spectral images ssssssssssssssesssssesesrsserenreserernenersrenneneerenrnnenrnreneneenene 105 SCV SAVING RESULTS ninani a a e a a E a a T A TT S 107 ABOUT BIOINFORMATICS SOLUTIONS INC ssesssssesccccecsesesessescecccceeseseeessssecoeseeeseeeee 108 PEAKS SOFTWARE LICENSE icsccsscdanineswienswesiddunseonacastcosadusies VA EEES TEn Enasi 109 REFERENCE PEAKS PAPER cicsttnrcccnksninece dans cvacdanunnesdshecscsaimensesannees ei ibana tane iraa 111 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer Introduction Introduction to PEAKS 4 2 PEAKS makes the interpretation of MS MS data easier and 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 4 2 provides peptide sequence and protein identification results via an intuitive inte
23. Fiveze 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 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer 641 26 342 2 ps 294 11341 2199 NHS 2 sits 2 HON Sy ago eeo vt aml bawo Measure the m z difference between two peaks Select a peak orange line by default with the Frveze 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 to from a peak Select a peak then right click the mouse anywhere in the Spectrum View Frame Select Set Y Ion from the pop up menu to designate the peak as a y ion Set B Ion from the pop up menu to de
24. LIVTQTMK 932 5366 1 8 21 Y 20 1065 586 1 1 064 5787 VLVLDTDYK 1 064 5752 92 100 24 v 21 1157 1289 2 2 312 2432 VYVEELKPTPEGDLEILLOK 2 312 2517 4i 60 25 w 22 1354 1898 2 2 706 3650 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 3687 15 40 26 v Matched peptides shown in Red 1 LIVTOTMK VAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLQK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLVCQ CLYRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI Color Code 80 60 40 20 Scrolling down the Matched Peptides panel reveals the complete sequence of the highlighted protein with the matched peptides highlighted in red The strength of the red colour is a visual indication of the confidence in that portion of the protein Where an EST database was used the translated sequence is shown with all six reading frames concatenated De Novo View usually shows de novo sequences for spectra that could not be explained by matching to the identified proteins as listed in Protein View but the exact behaviour is customizable See below for a discussion of filters and how they relate to the De Novo View The list of peptides in the De Novo View behaves quite like the list of peptides in Peptide View Differences are as follows Note that since PEAKS 4 2 uses de novo sequences to help out in the database search it is sometimes useful to see which de novo sequences were use to help in the search Each sequence that is used in this way has
25. Novo Parameters Dialogue De Novo Options De Novo Sequencing Parameters Saved parameters SampleData Instrument Quadrupole TOF Parent Mass Error Tolerance 0 1 Fragment Mass Error Tolerance 0 1 Enzyme Semi Trypsin Report up to peptides 5 Edit Enzyrnes PTM selected for search max variable PTM per peptide Add Remove PTM _ Deconvolute the data for analysis use for non deconvoluted data 2 Save As Ok Cancel Instrument choose the type of spectrometer that produced our data Choose from a dropdown list Parent mass error tolerance determines how much random and systematic experimental error on the patent precursor ion PEAKS will account for in its analysis Select a tolerance from the dropdown list Daltons are the units here 27 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 Daltons are the units here 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 up to 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 s
26. The main pakaka 1 processing Peptide Candidates PEAKS Auto De Novo 0 1 Trypsin with window is used to 472 473 perform manual de DYVEK lt 1 357 358 novo sequencing VLTEK lt 1 ga Z 258 259 and to examine VDPIE lt 13 129 130 the results of auto DISER lt 13 PE re denn aaa e A T a See de novo PEAKS DB Search sprot0 1Trypsinw T sequencing VDVEK 99 0 11 oor Color Code gt 90 80 90 60 80 lt 60 an 276 16 314 13 258 14 72 08 100 03 147 11 y b3 375 22 443 18 490 25 2 420 iB mE a Ce dese A ob t 0 500 paeme mn Main Processing Window Toolbar quick access to many processing functions See Toolbars in the next section 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 this shows the confidence in each of its parts Under the Manual de nove heading the masses of the residues are displayed That is to say the mass of the whole peptide minus water 40 The ions displayed in both
27. a checkmark in it s PID column The parameters used to generate a de novo sequence may be slightly different from the database search parameters As such PEAKS 4 2 records how each de novo sequence was generated this information is in the Source program column Positional confidence information is available in de novo sequencing results Colour codes show the strength of confidence in an individual amino acid The colour codes are the same as those used in the main processing window SCV red is the best then purple then blue then black Positional confidence can also be seen by holding the mouse over a sequence Result filters are accessed by clicking on the Filter Pane tab These allow us to manipulate the contents of the whole multi part report For instance one may choose to discard any protein hit that was not supported by at least two high scoring peptides The filters are very flexible allowing you to create filters on any of the fields score mass search engine etc and any number of fields The filters are also cascading such that removing a protein from the results removes all peptides associated with only that protein and vise verse But all this can be a bit complicated so please read on to the section Post analysis of results preparing for publication Comparing proteins multiple sequence alignment After having identified some proteins it may be useful to see how their sequences comp
28. and prompt us to save a separate copy 68 SCV SCV SCV Manually Manipulating Data Files Editing Precursor information MEETA File Edit Tools Windows Help SSe ee e e FT eonan ME eS 7409 22 F mass 678 5 7479 21 Pept p Intensitiy 1 _ Charge 2 662 31 Apply Cancel It is possible that the precursor information as listed in the Peptide Data Frame is incorrect If the charge listed is wrong or if the m z listed is even slightly incorrect more than 0 1 Daltons depending on the accuracy selected it could really affect the quality of the results In this case it is imperative that we 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 while holding it in position over the name A small menu will appear click on Edit Precursor In the dialogue that follows type the new precursor information into the appropriate textboxes Click the Apply button when finished to apply the changes Click the Cancel button to exit discarding changes 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 information pertaining to that spectrum Manually merging MS MS sc
29. 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 dialogue will allow us to choose and to set parameters for each of these refinement tools Auto 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 PEAKS Protein ID 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 inChorus Protein Identification button perform protein identification on a selected data file using multiple search engines Press this after selecting a data file 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 Import Database Wizard button help user download and configure database 45 SCV Main Processing Window Toolbar m y ion Alignment
30. 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 toggle show hide the location of PEAKS corresponding to b ions and the corresponding proposed peptides between them a Deconvolute button toggle on off deconvolution of the mass spectrum scale 1 4 1 1 zoom button return spectrum to original 1 1 zoom ka Undo Zoom button return to previous zoom ratio 4 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 g Next Peptide button redo changes to the peptide in manual de novo ke Previous Peptide button undo changes to the peptide in manual de novo Ey 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 o 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 46 PEAKS Studio Configuration How to set up PEAKS Studio just the way ne like it his chapter deals with configuration PEAKS 4 2 is a versatile and flexible tool But
31. 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 tight 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 the 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 sequencin
32. 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 4 2 allows us to set up many defaults and presets to help us be quick and precise We can use PEAKS 4 2 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 4 2 before processing data files S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer SCV SV PTM Post Translational Modifications affect the mass of modified proteins and residues PEAKS Properties Configuration One of PEAKS 4 2 preferences PEAKS Properties configuration sets the parameters that the algorithm will use while processing our data files PEAKS properties include enzynme PTM and database When using PEAKS Client all PEAKS properties are controlled by the server please contact your administrator PEAKS Studio 4 2 provides tools to edit PEAKS properties for convenient use in de novo sequencing and protein identification in PEAKS Studio To edit PEAKS Properties Click the 4 icon in the main window toolbar Or from the Edit menu select Configuration then PEAKS Properties The PEAKS Properties dialogue will then appear This dialogue box has three tabs Enzyme li
33. most SVC Windows Dialogues Frames and Reports SVC PEAKS 4 2 main window P5 PEAKS Studio File Edit View Tools Help aja e a El Peptide Data 21 Nov 06 11 22 04 _NR 452 28497 2 459 73856 2 467 27292 533 2925 2 545 92804 3 561 7268 2 597 3386 2 623 2947 2 Comprises Peptide data frame left This displays a listing of parent ions by m z and charge Clicking on one will bring up its MS MS spectrum The colored dot by each spectrum appears 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 ie 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 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer SC 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
34. nodes Make sure the new node is selected before running protein ID or any other function on it SCV Using the Mass Calculator car ia ual gs me tool to help us determine the molecular weight of a peptide To Hie Edit Windows Help access the Mass Calculator open z P M Enable Tasks Running the Tools menu and click auto De Novo Mass Calculator The Mass BY Protein Identification Calculator will appear 47971 access the mass calculator without Pe kd 1639 8 1 having to load PEAKS click on ed chrome Cnkk a the Mass Calculator s icon in the start menu It will appear in the same program group as PEAKS We can also load the mass calculator outside of PEAKS To When using the mass calculator remember to start W Mass Calculator Edit Help with water AVWEICLSK H20 gt lt H lt Acetyl gt Mono Mass 1104 5759 laverage Mass 1105 3322 Clear Water Proton PTM Num Back D 4 hydroxynonenal HNE pplied Biosystems original ICAT TM dO pplied Biosystems original ICAT TM d8 pplied Biosystems cleavable ICAT TM light pplied Biosystems cleavable ICAT TM heavy pplied Biosystems ITRAG TM multiplexed quantitation chemistry We can click any of these buttons multiple times to repeatedly add that mass Amino acids are represented by their single letter symbols Clicking
35. nr or Swiss Prot database is selected and the Apply button has been pressed or either database was selected on a previous screen Best practices configuring databases for use with X Tandem 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 PEAKS 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 documents 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 The database we downloaded may be in a compressed file perhaps a zip or a
36. on an amino acid s 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 lt H gt 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 the dialogue and the mass will be added to the sequence To remove a mass that we ve just added to the peptide press Undo 93 Working with Results Vening Post Anabsis Exporting and Saving of results present them simply effectively and allow you to get them out of PEAKS easily This chapter covers how to interpret the results how to get the most out of them with minimal effort how to present them to others and how to save data and results together for future reference R are what count So the PEAKS 4 2 developers have made sure to This chapter assumes that the user is familiar with all preceeding chapters 94 SCV The walkthrough in Chapter 3 gives a good summary of this topic Viewing Protein identification resul
37. s containing the data that we wish to tefine 2 Click the NI 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 Merge scans ofthe same peptide yes O no Retention time window for raw files only miz tolerance Correct precursor charges yes O no Remove low quality MS MS scans O yes no Preprocsss MS Ms scans no already done yes no 3 Choose the data refinement tools we wish to use by clicking the yes radio button next to each one 67 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 The units here are m z values in Daltons For retention time we use whatever units are recorded in the data file usual
38. that could not be Filter proteins based on the number explained by peptides from of peptide matches they contain the Peptide View 1 De novo view shows all peptides that are not filtered Options C Remove de novo peptides Equal to with no database hits ay eiroater than Lesserthan Not equal to Now both filters appear in the list Press the Apply Filters button to use them Going back to the Protein View tab and expanding the plus sign reveals that we are only left with variants of lactoglobulin inChorus Searching 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 Peaks database Search EX Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines Z XTandem Search FD Options _ Omssa Search C Options Spider Search Ea Options port Database Search Results C Mascot Result dat M Browse _ Sequest Result summary html gt Browse
39. the task queue list and the dark green icons beside the spectra in the Peptide Data Frame change to light green and or an asterisk appears 76 SCV 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 nove In this example the highlighted sequence is 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 PIM was defined created by another PEAKS user on another system the PTM would still be shown and it can be imported into the local PEAKS configuration as desired Right 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 r
40. them on our results For larger reports this can take a few seconds during which time the tabs of the multi part report will be greyed out After completion we can click on any of the tabs to see how the results have been affected 101 SCV How filters act on de novo sequences PEAKS 4 2 automatically relates de novo sequencing results to protein ID results This makes it easy to see which de novo sequences correspond to known contaminants and which are truly new peptides The relationship is always drawn back to the data not necessarily the sequence which helps highlight important sequence variations PEAKS 4 2 users can set what is displayed on the list of de novo sequences according to the following diagrams Option Selected Options De novo view shows peptides that could notbe explained by peptides fram the Peptide View De novo view shows all peptides that are not filtered LCI Remove de novo peptides with no database hits Options O De novo view shows peptides that could not be explained by peptides fram the Peptide View De novo view shows all peptides that are not filtered Remove de novo peptides with no database hits Options De novo view shows peptides that could not be explained by peptides from the Peptide View De novo view shows all peptides that are not filtered Remove de novo peptides with no database hits What De Novo View Displays
41. to PEAKS is 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 patameter set that refers to it The lists on the right show what PTM 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 72 Modification PTMs that can be selected lt BuiltIn 4 hydroxynonenal HNE Selected Fixed PTMs Carbamidomethylation lt BuiltIn Acrylamide adduct lt BuiltIn gt Amidation lt BuiltIn Applied Biosystems cleavable ICAT TM lt BuiltIn gt Applied Biosystems cleavable ICAT TM lt Built In Applied Biosystems iITRAQ TM raultiple lt BuiltIn Applied Biosystems original ICAT TM d lt BuiltIn Applied Biosystems original ICAT TM lt Built In Beta methylthiolation lt Built In gt Biotinyl iodoacetamidyl 3 6 dioxaoctang lt BuiltIn Biotinylation lt BuiltIn C Mannosylation lt B
42. 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 SERVER 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 running 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 65 SC 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 Op
43. to match the market 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 108 PEAKS Software License This is the same agreement presented on installation It is provided here for reference on 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 purposes 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 c
44. 1 1 beta lactoglobulin Sus scrofa X gil3455 19 7347 I 55 2 81 1 oO beta lactoglobulin Sus scrofa X gil6347 29 0917 8 55 1 92 1 PREDICTED similar to 605 ribosomal pr X gil5564 31 223 1 55 1 80 1 g PREDICTED similar to 60S ribosomal pr gil7400 33 1471 55 1 69 1 PREDICTED similar to 605 ribosomal pr Remember all proteins in this group contain the same set or a subset of the same peptides We ll see that there are still some hits down there that aren t very relevant Yes they contain a good peptide but only one Let s see if we can remove these one hit wonders Go back to the Filter Pane tab The Selected Filters list still shows our Peptide Score Filter gt 50 Let s add one to it Scroll down the list of Possible filters and select the protein filter called Query filter In Edit Filter select the greater than option and type 1 in the box then press the Add Filter button 21 Possible Filters Selected Filters Ly Delta Mass filter m G De Novo Filters C Mass filter eee Peptide Filters C Accession filter Remove filter C Score filter gt 50 0 Apply filters Bi Per spectra filter Protein Filters UniquelD filter Cy Query filter gt 1 0 J Protein Filters B Score filter C Query filter B Desc filter B Mass filter G Coverage filter B Acc filter Selected filter Options Edit Filter De novo view shows Query peptides
45. 60 v v la 673 3 _1 672 37 IGDLQK 672 38 0 0028 50 619 624 _gil28972720 v la 673 3 _1 672 37 LGDLOK 672 38 0 0028 50 136 141 gilS2782259 v a 673 3 1 672 37 LGDIQK 672 38 0 0028 50 736 741 gil63586345 v a 673 3 1 672 37 GLDIQK 672 38 0 0028 19 9 14 gil229460 v Mo 674 4 1 673 41 IPAVFK 673 41 0 0002 76 78 83 gil229460 v 10 674 4 1 673 41 LPAVFK 673 41 0 0002 76 24 29 gil62659199 lv m 12 _ 697 7 3 2 090 LDAINENKYLYLDTDYKK 2 090 0 0022 100 84 101 gil229460 liv lv 12 697 7 3 2 090 IDALNENKVLVLDTDYKK 2 090 0 0022 100 84 101 gil2194089 ral I 13 771 7 3 2 312 VYVEELKPTPEGDLEILLOK 2 312 0 0044 100 41 60 gil229460 v m 14 818 3 2 1 634 TPEVDDEALEKFDK 1 634 0 0067 100 125 138 gil229460 v m 16 _ 903 1 3 2 706 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 0 0007 99 15 40 gil229460 v m 17 _ 903 5 1 902 56 TKIPAVFK 902 55 0 0013 3 76 83 gil229460 v 18 _ 933 5 1932 53 LIVTQTMK 932 53 0 0012 34 1 8 gil229460 v Me 933 5 1 932 53 IIVTQTMK 932 53 0 0012 34 1 8 gil72079 l 20 __ 1065 1 1 064 VLVLDTDYK 1 064 0 0035 5 92 100 gil229460 Vv 21 Mr ANR VYVEELKPTPEGDLEILLQK 23125 0 0085 100 41 60 gil229460 v y 22 1354 2 2 706 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 0 0037 90 15 40 gil229460 v PEAKS displays the same Peptide View as before Now X Tande
46. 72 37787 LGDLAGK 9 26 673 38513 672 37787 LGDLOK 79 79 452 28497 902 5554 KTLAVPFK 7 82 they appear Ked 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 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 these good spectra that did not match This is essential so that we can organize our data well and because 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 shift click and ctrl dick the paste button Pressing the node The new node appears as Then press the cut button next to Datal will expand it Datal and reveal the pasted spectra PEAKS Studio PEAKS Studio File Edit Tools Windows slaiele elxie it l aiae xE 91 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
47. CENSEE 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 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 trans
48. Cancel The top section of this view shown above 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 Albumin node has been expanded to reveal several similar proteins Clicking in the same place now a sien 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 a 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 The columns themselves are customizeable Right click anywhere in the report and choose Toggle Column from the pop up menu The sub menu that appears shows a checkmark in each of the columns that are currently showing Click any one of them to show ot hide a column These settings will apply to all our reports 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 The columns in this list are the same as the ones we choose for
49. ION c cssccssseseeesceseeseeeceesecseeecesecuaeeseeseeeaecaeeeceesecsaeeaeeaeesaeeaeees 48 SV Creating and defining PIM eects eeec utacea se cad ide eves Nadie ees ples 8s ob eiiiai ii ba Poe 48 Creating aiNew PEM A AET E T E E E T 49 Edine a PIM a ter nea Me ae a ed eee Reeciatn 50 Removing a PIM eiee a aerea slice donned thal dada IPESE E Era SE aiora aSa dist Ean SEEE EENEI EESE ESAE 50 S MARIUS Mance noite onu aa o n ascot a a a Aa O AAE a S 50 Load Configure a new databases rinn n e ana N EE NE EERE ETEEN E EAER E 51 Remover Database ss cesta tsiettsaeieevinejedstencasuevee E E E A E EE E S 51 Edita Data ase arene an n e OR EA E RAR E NE RONN E e RE ETEN RE 51 Moving Updating a Database 00 ccs cscscscscsesesesesssesesescavseseseseseseseseececececscecsesesesssssesssaseanavaesesesesseeseses 51 SV IMPORTING AND EXPORTING PEAKS PROPERTIES ssssss ccc cetoncedeahaxtepestuatvsnss tntuperonutadtnsheneventnadoiasoien 52 SVC PEAKS ENVIRONMENT PREFERENCE CONFIGURATION cccccscssssensnssorssonsssesnsnsesecacncsenenenenenenenenes 53 S gt WRENCHES SOC Parameters asnih e iaa e EA aa AE AEE E EDS A AE SAE 54 SCY C nfiguringthetofi table raniecenadenanonienanannadannnor n i aia Matec Sead Pale 55 SCV Environment Changing default file handling settings sssssssssrsrsssrrereseresrererersesnrnrereseeenenreses 56 SC Environment client to PEAKS Online s sssessseersessseseeeresesersererersereeeereeseseenerresesernevsrersrerenerseseseereree
50. TS OP E A AE ERE EEE e A 8 SCV TINEST WN E EAN D EIN EEEE EEEE E EE A 9 SC CONNECTING TO A PEAKS ONLINE SERVER sssssessesesrssesseseseesessrstseestsssestestsesteesteseseestsessesteetseseee 10 OC REGISTERING PEAKS naie eE E E tenia Gas bia E E E E 10 SC DATABASE CONFIGURATION isoro aeni aita a E a e a i a oso a ei 11 FEATURES WALKTHROUGH siesssrscesviriccaviiccccosiricocsvcicensricdiors edicien ciie ciiin ea 17 SC BEGIN THE WALKTHROUGH noonoo ni e E E A E A E E A E aa a i 17 SVC VIEWING RESURS Toa fides Tre torte Bees n e tent kanes eN kde A Cote ae A ee ae E shh T S e a 19 SVC MANIPULATING RESULT Sirota nenie a a a aaa aaea araa a i e ea a en 21 S CHORUS SEARCHING miei ereen netoa ee aerar Ne CEER Era Ee eer aie idesosbosbavesasdouganvesoeedisvasebiavsdeaseesvineonayy 22 GRAPHICAL USER INTERFACE cccsssseresrorerccisorcrsroracinrtrrovdsstriesor tcner stenose seed staan 25 SVC WINDOWS DIALOGUES FRAMES AND REPORTS cssssssesseesecseeeeeeseceseeaeeeesaecseeeeeseceaeeaeesecnaeeatees 26 SVC SPE AIS 22M AERO OU ites aa aaa st Face pha se Sad va tae ats teen RO a Ran ew Geeta amen St 26 SC Auto de Novo Parameters Dialogue ce cipccccicticcest hesegsesstecnecanescesstotevenes sige ccsisipkea yiasenssees sean ebaabeeseoeeeehs 27 SC Protein Identification Parameters Dialogue ssssssssssssrsserssreseresseersrsesnenenreneneenenensrnennrnrerenrnnenrereses 29 SCY PEAKS Properties 1D lO SUC ar ales cok tii ces iiae ei n a e a aiai 31 SCY Enzyme Editor
51. 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 16 SC Features Walkthrough Lets familarize oursehes mith PEAKS his section of the manual will walk us through most of the basic functionality of PEAKS 4 2 After completing this section we will have seen how easy it is to load and view a data file perform de novo sequencing and database search protein identification Begin the walkthrough Run PEAKS 4 2 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 S 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 Studio data folder Load the file OrbiOrbi pkl by clicking on it then clicking Open The data file will appear in the left hand frame Make sure OrbiOrbi pkl Le the data file is s
52. Y L E 0 aS LLN e EPMLAYLK lt 1 LEDLAYLK lt 1 FPLLAYLK lt 1 s PEAKS DB Search SwissProt 0 1 0 1 Trypsin with Cam EDLIAYLK 99 f AS Dte F r pt o 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 83 Short forms for the modifications may also be used 482 7 ali a ROSIE Peptide Candidates Sequence Parameter Config e alap sump to protein A 945 38 PEAKS Auto De AA PTM Set Config EDLLAYLK DELLAYLK New Candidate for Manual De Novo EPMLAYLK S E PEAKS DB Seartt EDLIAYLK 99 n _ ma ll Pa ts f F aa ra WISSPTOUU T UT Trypsin 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 09 JELAYLK DE 226 168 AYLK EDLLA phosphorylation Y LK DE 226 168 A phosphorylationY LK Then press the OK button The sequence we just entered will appear under the Manual De Novo heading and when
53. abase to download Find one download it and unpack it If taxonomy is available for this database download those files too Return to PEAKS 4 2 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 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 the procedure for configuring a new database at any time Edit a Database This tool allows us to change the name that PEAKS 4 2 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
54. ale PEAKS multiplies the m z of ion s that were doubly charged by two Note that the deconvolved scale PEAKS shows is at 1 Fixed modification sclecting 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 Enzyme The residues PEAKS can find in different positions in the sequence This is based on information about the enzyme used to digest our protein sample ESI Electrospray lonization A method for ionizing a sample into the mass spectrometer m z mass to charge ratio 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 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 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
55. all data as opposed to using instrument vendor software if the data is to be used by PEAKS The 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 If we choose to do pre processing on the fly during protein ID as opposed to using the Data Refine tool beforehand PEAKS preserves the original data and does not save the results of its preprocessing 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 87 saved de novo parameter sets for the de novo portion Select one 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 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 Note the Advanced Options selections will not be saved 4 Press the OK button to comme
56. an add to the filter pane and add a filter that shows only database peptides with scores greater than 50 or any other value we choose Scroll down the Possible Filters list and select the Peptide Filter called Score Filter In the Edit Filter frame remember to select greater than and type 50 then press the Add Filter button The filter now appears in the list of Selected Filters The fier can be edited while in either listing and can be added multiple times in case for some reason you only wanted peptides with a score greater than 50 but less than 90 Hit the Apply Filters button and let s examine the results Click the Protein View tab De Novo View Peptide View Protein View Search parameters Filter Pane Accession PEAKS DB Search U X gil229460 18 367 E 99 50 00 12 lactoglobulin beta Display Score Coverage Query mat Marked Description Ok so we removed the other protein and we re left with Lactoglobulin beta The other protein only had one hit IGDLQK with score 50 That peptide was removed from the report Since the other protein no longer had any supporting peptides it was also removed from the report Now we re left with our lactoglobulin beta group Click the plus sign here to expand this group further
57. ans 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 peptide s 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 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 SCV Changes made to the original spectrum after duplication will not affect the duplicated spectrum SCV 2 Next right click in the Peptide Data panel and choose Merge Spectra from the popup window that appears OR click the Pad 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 OR 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 Copying spectrum data will duplicate
58. are This is particularly useful when trying to identify specific isoforms of proteins or antibodies To display a multiple sequence alignment between two proteins click on the time and date stamp underneath a filename in the peptide data tree left This brings up a multi part protein ID report Click on the Protein View tab to see a list of identified proteins Mark a few proteins by clicking on their checkboxes in the Marked column Then in the bottom panel of this view click the MSA tab It will be blank to start with but click one of the two buttons on this panel to C generate the MSA and display in the default web browser generate the MSA and display it on the MSA panel De Novo View Peptide View Protein View Search parameters Filter Pane Accession Mass Display Score Coverage Query matc Marked Description PEAKS DB See LI ES gil229460 18 367 E m 99 50 00 12 x lactoglobulin beta X gil2194 18 309 E 99 50 00 12 LJ Chain B Bovine Beta Lactoglobulin Latti X gil4925 18 351 M m 99 50 00 12 LJ Chain X The Cys121ser Mutant Of Beta L X gil7207 18 267 a 99 50 00 12 beta lactoglobulin water buffalo E Ry 18 367 M mamm 99 50 00 12 LJ Chain D The Structure Of Bovine B Lacto X gil2173 18 281 E m 99 50 00 12 fal Chain A Bovine Beta Lactoglobulin Com R71
59. ask based guide to getting our data into PEAKS 4 2 he following chapters deal with usage They are 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 4 2 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 61 SCV SCV PEAKS demo data can be found in the DATA sub directory located in the PEAKS directory Loading data into PEAKS 4 2 PEAKS 4 2 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 PKL The file format associated with Micromass instruments DTA The file format associated with SEQUEST software MGF The file format associated with Mascot software ANZ the zip compressed XML based file format associated with PEAKS XML format files using the mzXML schema XML format files using the mzData schema RAW files from Thermo Electron instruments WIFF files from ABI QSTAR instruments RAW files from Waters QTOF instruments BAF YEP and folders of FID files from Bruker instruments XML format files from Waters ProteinLynx software DAT files created by BSI s ABI converter software O
60. at the protein level 21 Nov 06 09 47 31_NR inChorus protein ID results De Novo View Peptide View Protein View Search parameters Filter Pane Accession Mass Display Score Coverage Query mate Marked Description PEAKS DB Seal HS gi229460 18 367 mm cece scad E X gil6264951 57 150 z gil5564 64 701 HiS options X gil7421E 75 548 X gil6673C 75 657 X gil3269E 76 121 X gil73967 76 784 X gil7420 77 885 gil7243C 78 127 gil28972 78 207 X gil52782 85 124 I gil6797C 85 095 1 X gil55631 247 X gil6358 261 1 HS gil2170390 93 652 ED similar to mKIAA1321 protei D similar to 82 kD FMRP Inter protein product Mus musculus Format xis v Filename C Documents and Settingstirogers BSNDesktop 21 Nov 06 lt a C Export protein sequence D similar to 82 kD FMRP Inter protein product Mus musculus protein Homo sapiens 1 protein Mus musculus ctokinase muscle Macaca fas protein product Macaca fascicul D similar to PHD finger protein D similar to mKIAA1030 protei rotein activator 1 Mus musculus Report Types Protein and peptide report for currently highlighted homolog group Complete protein list peptide details omitted Current marked proteins and corresponding peptides O Full protein and peptide report homologous proteins grouped Ok
61. at 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 4 2 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 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 yellow and red and b ion alignment kaj yellow and purple icons in the main processing window toolbar To view another peptid
62. bsi peaks PEAKS Studio PEAKS Online Host Name or IP Address User Email E Peaks Invocation v Close the main processing window before loading a new one for a different spectrum Load sample files at start up Show wiff file options Show bruker file options Show GNU Public License Ok Cancel Removing Saved Parameters After months 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 SCV Configuring the ion table The ion table displayed in the top right of 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 from the View menu j 467 2729 2 Main processing window al Peptide Candidates b H20 Immonium PEAKS Auto De Novo 0 01 0 01 209 16 199 18 LLVQTIMK 49 2 LLVASATMK lt 1 LLVSAATMK lt 1 409 28 LLVAASTMK lt 1 537 34 627 35
63. ce 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 peptides highlighted in red Protein View showing two proteins in the index and beginning the full report MSA Pane bottom section This panel displays a multiple sequence alignment between any protein that is marked in the 36 Index top section The MSA is only displayed when the button is pressed E 21 Nov 06 11 22 04_NR inChorus protein ID results De Novo View Peptide View i Protein View Search parameters Filter Pane Accession Display Score Coverag Query m Mark Description PEAKS DB E X gil22946 18 3 X gil43 18 2 X gil72 18 2 X gil21 18 3 X gil43 18 3 X gil49 18 3 X gie 17 1 X gil72 18 3 57 41 lactoglobulin beta 57 41 Chain Bovine Beta Lactoglobulin Com 57 41 beta lactoglobulin water buffalo 57 41 Chain A Bovine Beta Lactoglobulin Latt 57 41 Chain Structural Basis Of The Tanford 57 41 Chain X The Cys121ser Mutant Of Beta 53 64 beta lactoglobulin 53 09 Chain A Structural Changes Accompan x Protein Pane MSA Pane 697 3386 2 VLVLDTDYKK 1 192 6702 623 2947 2 TPEVDDEALEK 1 244 5771 673 38513 1 GLDIQK 672 3807 674 4233 1 IPAVFK
64. ching with Lactoglobulin Beta Most peptide matches 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 entry for spectrum 3 467 2729 This will bring up the main processing window for spectrum 467 2729 2 Look in the top left frame to see the de novo and database results Color coding shows positional confidence scores By the letters coded in red we can see that the PEAKS auto de novo analysis returned with gt 90 confidence the partial peptide sequence LLVXXTMK but was not as sure of the middle two residues The PEAKS DB Search was able to confirm this result returning the peptide LLVTQTMK Selecting another spectrum from the Peptide Data 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 SVC Manipulating Results We already know that the sample was a simple digest of Lactoglobulin Beta let s see if we can filter out the other spurious hits from the report Click on the Filter Pane tab Here we c
65. ctrum Alignment Frame O Peptide Candidate Tree Error Map Annotated Spectrum with Alignment Cancel File Format select an image file format from the drop down list Bitmap JPEG Portable Network Graphic and Graphics Interchange Format are supported Width and Height together these determine the size of the output image measured in pixels Filename type in the textbox or browse to a file E to enter the file name of the image that will be created Resolution in most cases the image resolution can be upscaled for printing large pictures More resolution i e more pixels means a higher quality picture when blown up or printed Image types Select one of these options to choose the image that will be output The annotated spectrum with alignment will suit most purposes 43 SCV Print Image Dialogue 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 m Paper A 7 appropriate dropdown list Size Letter 7 Source Automatically Select z Printer button pressing this will bring up another dialogue m Orientation Margins inches where we can select from a list of printers installed on our machine Portrait Left fi Right fi C Landscape Top fi Bottom fi i Cancel Printer
66. d our data Choose from a dropdown list Parent mass error tolerance determines how much random and systematic experimental error on the patent precursor ion PEAKS will account for in its analysis Select a tolerance from the dropdown list Daltons are the units here 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 Daltons are the units here 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 PTM selected for search this box displays the modifications currently selected for analysis these will be considered during database seatching To change this click the Add Remove PTM button Database to search This dropdown allows us to choose which FASTA format database to search Taxonomy selection if the Database to search has been configured for taxon based searching this list will allow us to select limit which taxa ate searched Paste fasta sequences Paste a few sequences in this box and PEAKS 4 2 will search through those sequences as opposed to the database selected Preprocess this data on the fly PEAKS Studio has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data C
67. d view and choose Export Report from the pop up window that appears P Protein Report Export File Options The report export dialogue will appear One must choose either HTML or XLS fomat and specify a file name PEAKS 4 2 will load the report after it has been created but it makes sense to always place the report in a place we can find it later on By default the filename of the report will include the time and date the report was created To change the location filename one can either type it in to the Fileame box ot click the folder icon to browse to a new location Format fylg Filename c report Export Peptide Report File Options Report Types Format html 7 Export peptide details Protein and pepi k Filename cireportstexample2 htmi Complete protei Current marked Report Scope D Full protein and Export selected peptides Export all peptides OK Cancel Ok J Cancel a The bottom part of the Report Export dialogue asks us what we would like to include in the exported report When exporting from Peptide View or De Novo View we have 104 SCV SCV SCV SCV the choice of exporting the whole list or the selected ones When exporting from Protein View we have some more options allowing us to include peptide details the complete protein sequence a listing of proteins containing the same set or a subset of the same peptides or to include only marked proteins Press t
68. dd 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 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 75 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 should 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 4 2 it is added to the Task Queue for processing After processing the job is removed from
69. e 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 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 Summary generation done Click the Protein View tab PEAKS 4 2 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 De Novo View Peptide View I Protein View Search parameters i Filter Pane Accession PEAKS DB Sea O R220 18 367 M mmm E 99 57 41 18 lactoglobulin beta E S gil1094913 64 669 I 29 0 83 1 O PREDICTED similar to 82 kD FMRP Interacting P Display Score Coverage Query matc Marked Description 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 4 2 groups them all together thus avoiding cluttering the report Click the plus sign next to gi 229460 for a listing of other possible lactoglobulin Th
70. e XCalibur software is installed on the same computer as is PEAKS 4 2 To load Thermo RAW data simply choose File Open and browse to the file Importing Applied Biosystems WIFF data PEAKS 4 2 can load WIFF data from our Applied Biosystems QSTAR mass spectrometer provided that the Analyst QS software and the MSX plug in are installed on the same computer as is PEAKS 4 2 The MSX tool is produced and sold by Infochromics Ltd and is available at cost from Bioinformatics Solutions Inc Please contact a BSI sales representative to obtain a license To load QSTAR WIFF data simply choose File Import wiff raw data and browse to the file Importing MassLynx RAW data PEAKS 4 2 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 PEAKS uses a utility called wolf exe originally created as part of the Sashimi Project to access MassLynx libraries and convert the data PEAKS ships with a version of wolf exe designed to work with MassLynx 4 0 If we need a different wolf we can check www bioinfor com products peaks support watersmicromass phi Additionally we must make sure that the following MassLynx libraries are installed on the same computer as PEAKS and wolf exe DACServer dll Genutil dll MetaGD32 dll raw dll securityAccess dll securitySettings dll
71. e 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 32 SC 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 Applied Biosystems cleavable ICAT TM lt BuiltIn gt Applied Biosystems ITRAQ TM raultiple lt BuiltIn Applied Biosystems original ICAT TM i lt Built In gt Applied Biosystems original ICATCTM lt BuiltIn gt Beta methylthiolation lt Built In 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 Citrullination mOxidation lt BuiltIn Deamidation New PTM lt BuiltIn Dimethylation lt Built In Farnesylation Remove PTM lt Built In Flavin adenine dinucleotide lt BuiltIn gt Formylation lt Built In Garmma carboxyglutamic acid lt Built In Geranyl geranyl lt BuiltIn Glucosylation Glycation lt Built In Guanidination lt Built In Homoserine Select As Fixed gt
72. e 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 pop up menu will appear 78 3 We can select the pop up 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 Spectrum Alignment Frame Now we are ready to edit the sequence using manual de nove techniques 79 SCV SCV SCV 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 scratc
73. e peptides matching these homologues will be the same set or a subset of Lactoglobulin beta 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 dealing with complex mixtures Clicking any protein s gi number will display the peptides matched to that protein in the bottom pane 19 NCBI BLAST search of gil229460 Links to retrieve entries containing this s AccessionID Description gil 229460 lactoglobulin beta vy 467 2729 2 Main processing window EE Peptide Candidates Peptides List Index mz zPeptide amp PEAKS Auto De Novo 0 01 0 01 TEMP 1 452 28497 LLVTQTMK 49 2 2 458 73856 LLVQTIMK 49 2 3 467 2729 LLVASATMK lt 1 4 533 2925 LLYSAATMK lt 1 5 545 92804 LLVAASTMK lt 1 oe E PEAKS DB Search NR 0 01 0 01 TEMP g 673 38513 1 GLDIQK LIVTQTMK 33 4 10 674 4233 1 IPAVFK TIVTQMK 33 4 12 697 7167 13 771 75635 14 818 3876 16 903 1304 17 903 5676 18 933 5426 20 1065 586 21 1157 1289 22 1354 1898 2 VAGTWYSLAMAASDISLLDAQSAPLR 2 706 3687 Matched peptides shown in Red 1 LIVTOTMK VAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLOK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLVCQ CLYRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQOEEQC HI Above 18 of the original 22 spectra indicated a peptide sequence mat
74. elect the typeface from the dropdown list and or change the font size then press ok The width of peaks on the spectrum display can be adjusted from here as well 57 Environment Preferences Parameters Basic lon Table Editor Advanced lon Table Editor Environment Colour Display Manual De Novo Font settings Main processing window Graphic Book Anti i labels Font type oo qua Font size De novo and Peptide view Font type Lucida Console Font size Protein view Peptide list Font type Lucida Console Font size Misc settings Peak width 1H Display Colour code r Curentcoly W Cancel SCV Changing Manual de novo defaults We may wish to sequence a peptide manually using spectrum data PEAKS 4 2 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 This can be done on an individual basis by right clicking on the sequence we re working on but we can set defaults To adjust Manual de novo options open the Environment Preferences dialogue and ensure that the Manual de novo tab is selected Environment Preferences Parameters Basic lon Table Editor Advanced lon Table Editor Environment Colour Display Manual De Novo i search parameter Maximum tag length 3 h A Maximum return
75. elected In the Tools menu select PEAKS Protein ID The protein identification options dialogue will appear 17 Enter the settings as shown Settings can be changed by clicking on the drop down list and selecting one of the options File Edit View EERE M Enable Tasks Running E Peptide Data D Work Flow 482 7 Merge Spectra 584 auto De Novo 539 634 7 678 2E inchorus protein ID 728 amp Database Search Options Protein Identification Parameters Saved parameters OrbiStandard Precursor mass search type Instrument i v p EAE E E Monoisctopic D Average Parent Mass Error Tolerance 0 01 v Database to search Fragment Mass Error Tolerance 0 01 v NR v New database Edit database Enzyme Semi Trypsin 4 Taxonomy selection max missed cleavages 2 Edit Enzymes Mammalia PTM selected for search Set Taxa Paste fasta sequences Add Remove PTM max variable PTM per peptide y Preprocess this data on the fly deconvolute filter noise centroid Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search Ihave already run de novo don t run it again QTOF S
76. elected for analysis these will be considered during auto de novo sequencing To change this click the Add Remove PTM button Max variable PTM per peptide this allows us to restrict the number of variable PTM that will appear on any given peptide SC Protein Identification Parameters Dialogue Database Search Options Protein Identification Parameters Instrument FT Orbitrap CID SORI Parent Mass Error Tolerance 0 01 Fragment Mass Error Tolerance 0 01 Enzyme Semi Trypsin Saved parameters OrbiStandard Precursor mass search type Monoisctopic Average Database to search NR v New database Edit database max missed cleavages 2 Edit Enzymes PTM selected for search max variable PTM per peptide Add Remove PTM Preprocess this data on the fly deconvolute filter noise centroid Advanced Options Taxonomy selection Mammalia Set Taxa Paste fasta sequences PEAKS uses a hybrid search technique that requires some sequence tags to help in the search Ihave already run de novo dont run it again Run de novo using different parameters than the above Run de novo using the same parameters as above detautt QTOF Standard Instrument choose the type of spectrometer that produce
77. en 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 Select a job run There is a radio button before each job run only the MS MS job tun can be selected for export because we need the precursor information Select a job run and click Convert to do the extraction Choose a filename to save After clicking the Convert button the user needs to input a file name and the peak lists of the selected job run will be exported 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 When PEAKS is connected to a PEAKS Online server we ll also save time by uploading smaller preprocessed data Data refinement is done locally before 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 the MS MS scans themselves can be centtoided filtered for noise and deconvoluted uploading to the server To begin the refinement of data from a whole MS MS tun 66 1 In the Peptide Data Frame select the data file
78. eneessenaeeaeees 69 SCV Editing Precursor info ATOM siai eanna ely tps situa Ea ecg EERE TAE T EAT AES 69 SCV Manually merging MS MS scans of the same peptide ocscersscecessssecrssensenoronsssonsecnsrrsnsnenesevnens 69 SCV Cutting and Copying Spectrum Data ssssssssssssssrsrersresesensesersresneeeresennenenresenennrnresrnrenenrereneeenenee 70 SGV Pasting Spectrum Data ririn ta ea a O A E NE ONE EN 70 ANALYSING DATA WITH PEAKS 4 2 pisi pornasi oeaan a N Ea EaR SEA S a Saalis 71 SCV USING PEAKS STUDIO WITH MODIFICATIONS PTM eseeseseseseeeseereseerersesesesesrsrssssrsrsrerererserersns 72 SC AUTO DENOVO SEQUENCING 3 issseiscssssscsessesscncvoscongesnesneosdasadntocscesaeesicnsonavanesecossaiagenneshaveesausteniesnaesds 73 SCV VIEWING AUTO DENOVO RESULTS ace a deci sieges os ooa a Teotia Ea RE o EEA Ser Eo eE a ES EE a e Eest 77 SCV EDITING SEQUENCING RESULTS PREPARATION sesesesesesessssssetersereresesisestsesesssstststststereteeeestere 78 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer SCV MANUAL DE NOVO SEQUENCING sccssssscesscsseesceeeeesecececeesecuaeeecesecsaeeaceeessecaeeeesecsaeeasessenaeeasees 80 SCV Creating a fresh spectrum for GEGUCTIONIO ccc fice rclalistieattnt ny teetesccideitaecet tian ratty Mekelet format eae 80 SCV _Manyal TIE NO COM IDEM ONS Siac eatecereasl n anera E EEE E aae 80 Selectie a POA K csees fs dosxesuscsesssss Sacytu doves odo desesesevacsssbedugve
79. epresents 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 wm Bl CE vii 6 Tea e wuto De Novo 0 a WNE i n anoal wt amp Wern 1 Trypsin aithi 0 2 uae no 3 Ml 589 3 1 maa Ai CRANE PEAKS DB Search sprot 0 1 Trypsin w VDVEK 99 Peptide Candidates A E PEAKS Auto De Novo 0 1 Trypsin with Cam DVVEK lt 1 Color Code 90 80 90 60 80 lt 60 EEE VLTEK lt 1 zA VDPMK lt 1 p LTVEK lt 1 PEAKS DB Search Swiss Prot 0 1 Trypsin with Cam VDVEK 99 Color Code 90 80 90 60 80 lt 60 a 2 2 west se 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 close to the centerline indicates a more confident 77 Zoom in far enough and we may resolve the isotopic ladder depending on our instrument data SCV 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 th
80. equence parameter E PEAKS DB Search Original View and click the import modification VDVSAY 53 8 button GAKGEAG 41 aus 2 Lab 2 custom PTM Y BA _showmoancanon innon Moaetion T s SVC PEAKS Environment Preference Configuration One of PEAKS 4 2 preferences PEAKS Environmental Preference allows us to customize PEAKS 4 2 to our needs PEAKS Environmental Preferences include Environment Color Manual de novo and Parameters To edit PEAKS 4 2 Environment Preferences Click the Ez icon in the toolbar Or from the Edit menu select Configuration then Environment Preference 53 The Environment Preferences dialogue will then appear This dialogue box has seven tabs Parameters Basic lon Table Editor Advanced lon Table Editor Environment Color Display Manual de novo and Parameters Clicking a tab will allow us to edit the Environmental Preferences corresponding to that tab Environment Preferences Parameters Basic lon Table Editor Advanced lon Table Editor Environment Colour Display Manual De Novo Default Data Input Directory CADocuments and SettingsibsiiDesktop Tutorial Data Previous directory User directory Browse Default Data Output Directory CADocuments and SettingsibsiiDesktop Tutorial Data Previous directory User directory Browse Default Configuration File Directory CADocuments and Settingsi
81. ering PEAKS The first time we run PEAKS we 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 Media Access Control address of the machine we are going to use PEAKS on If we are connected 10 SC to the internet registraton HIPEAKS Studio will be completed z automatically If all is well File Edit Tools Windows ajai el wlxle a dialogue will show Registration Successful Peptide Data and PEAKS will load The software uses your computer s MAC address a as a unique identifier for aP P rs the computer As such if you have more than one MAC address for your computer you may experience problems in registering PEAKS The display will be something like this PEAKS Studio 4 0 Registration Please input registration information Registration Key PEo4k3lak32KLD your key User Name Your Name Organization The Company Machine IP Address O0 27 3G FF FF 17 O0 12 3H 0G BB 99 11 12 GH G 7 BB 20 NOTE 3 MAC addresses First MAC Second MAC Third MAC Previous Register Cancel If this occurs try disabling your wireless network card restarting windows and plug into an Ethernet cable only
82. ferred 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 110 Reference PEAKS Paper Phase use the follaving reerences when publishing a study that involved the use of PEAKS Bin Ma Kaizhong Zhang Christopher Hendrie Chengzhi Liang Ming Li Amanda Doherty Kirby 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
83. g it is possible to undo the change With the Peptide candidate still selected in the Peptide Candidates Frame click ihe l previous peptide 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 el next peptide button We can click this button multiple times to proceed to successively later stages in our edit SCV Suggesting a sequence to see how it fits the data If the data is ambiguous PEAKS 4 2 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 4 2 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 choose New Candidate for Manual De Novo PEAKS Studio File Edit View Tools Windows Help aaae xe aa Ee T aan DR NTS es 4 9 e a1S19 amore E PEAKS Auto EDL LA
84. ge the Default PEAKS Invocation to PEAKS Online Then specify the IP address or URL of the PEAKS Online server the port number to use and a username and email address Press ok Now we can use PEAKS 4 2 in the same way we normally would except that processing will occur on the PEAKS Online server The email is used to notify us that a job has been completed in the event that we close PEAKS 4 2 before finishing Changing Colours For ease of viewing we can choose which colours we would like to represent which items on the spectrum view To change the colour of an object on the spectrum we SCV open the Environment Preferences dialogue and ensure that the Colour tab is selected Environment Preferences Parameters Basic lon Table Editor l Advanced lon Table Editor Environment Colour l Display Manual De Novo Please choose color for Swatches HSB RGB Spectrum_Peak Freezed_Peak Current_Peak Y_Alignment B_Alignment Y _lon Curentcolbr W Cancel Choose the object whose color we d like to change from the list at the left Then we select a swatch to choose the colour After we ve chosen colors we may click the OR button to exit and save changes Display Changing typefaces and peak width We can customize the various typefaces to use on certain parts of the PEAKS 4 2 interface To do so open the Disply tab on the Environment Preferences window S
85. h PEAKS 4 2 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 nove or any user defined type heading This will bring up a pop up menu 2 Select New candidate for manual de nove from the pop up 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 of the peptide less the mass of water 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 movement 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
86. he OK button to complete the export Exporting peak lists 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 not be preserved Exporting Sequences by spectrum For most users the WYSIWYG reports exporting will be enough but PEAKS 4 2 also provides other ways to export 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 table 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 The difference here is only that the results are organized by spectrum rather than by peptide as in the WYSIWYG exporting Exporting Exclusion Lists If we ate repeating an experiment it can be useful to exclude the peptides we have already scanned and anlalyzed In this way we can focus on some
87. he list of Selected Filters If we d like to add another filter we can repeat the process continuing to add as many filters as necessary In this way it is also possible to have two filters on the same property we can set a range of protein mass for instance by applying one filter on the upper bound of the mass and 100 adding another filter to be the lower bound of the mass We can also have more complex filters that involve multiple properties The example above shows filters that allow only proteins with more than one high scoring greater than 60 score peptide a standard requirement for publication Filters are grouped into three basic types to reflect what they act on De Novo filters act to remove proposed de novo sequences Peptide Filters act to remove peptides found in the database from the report and Protein filters act to remove proteins from the report The filters cascade through each view in the multi part report For instance filtering out a protein by mass perhaps will remove it from the Protein View and remove all peptides associated with that protein from the Peptide View list The manner in which the De Novo View is linked can be specified by the user illustrated on the next page Filter sets can be saved and re used between sessions but remember to hit Apply Filters to use them Once all the filters we need are set up and showing in the Selected Filters list we must press the Apply Filters button to use
88. heck 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 30 SCV PEAKS Properties Dialogue F PEAKS Properties Enzyme list PTM Library l Database List of Enzymes lt Built In Arg lt Built In Asp N lt BuiltIn gt Asp N N terminal Glu Built In gt Chymotrypsin lt Built In 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 Duilt Ins Dancin soli 4 3 vr 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 PEAKS We may make new databases available to PEAKS from here 31 SCV 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 a residue to denote any residues at the end of a peptide start of a new peptide amino acid except the ones enclosed in these
89. ical user might perform It assumes we can identify parts of the Graphical User Interface and that we are familiar with how PEAKS 4 2 can be configured Such detail has occasionally been omitted from this chapter in the interest of succinctness 71 SC For PEAKS Client the PTM library is stored on the server Ask the administrator to make changes Using PEAKS with modifications PTM PEAKS 4 2 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 run PEAKS the box will be blank meaning no PTM are selected PTM selected for search Fixed Carbamidomethylation Var Deamidation Var Oxidation Yar Phosphorylation Add Remove PTM To add modifications to this list click the Add Remove PIM button The Modification dialogue appears The entire PTM library e the list of all lt built in gt and user defined modifications that are available
90. iew 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 41 SCV The lon Editor is used when performing manual de novo sequencing lon Editor ion Editor es Please choose ion type Selected peak information C Term lons mz 136 0692 intensity 4 0323 t N Term lons current ion H20 NH3 Add Remove Comments Selected peak information displays information about the currently selected peak Under Please choose ion e the radio buttons set whether the gt ions in the ton choice list are C terminal ions or N terminal ions Ion 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 42 SCV Export Image Dialogue Image Export Image Options File format pmp v Width g4q Height 480 Filename esktop Tutorial Datal458 73856 bmp Resolution Upscale to 10H resolution Image Types lon Table and Error Map Current Spectrum View O Spe
91. ifference 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 ORK button we return to the PEAKS Properties dialogue to find that our new PTM is listed at the top of the PTM list 49 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 Builtln Applied Biosystems cleavable ICAT TM heavy lt Built In gt Applied Biosystems cleavable ICAT TM light lt BuiltIn gt Applied Biosystems ITRAQ TM multiplexed quantitation chemistry lt Built In Applied Biosystems original ICAT TM d0 Import Export 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 PTM button The PTM Editing dialogue will appear Now we follow the same procedure see above as we would if creating a new PTM Removing a PTM To remove a PTM we open
92. imental 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 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 up to 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 often returns better results if
93. inus GHI Middle Only L Rule This is not areal 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 Environment Preferences Window This window controls the basic user configurable environment Colours displays file handling and default settings can all be changed from here The window contains 7 tabs Parameters tab Our saved parameters for de novo sequencing and protein ID are shown and can be deleted from here Basic Ion Table Editor tab allows configuration of the basic ion table Advanced Ion Table Editor tab allows configuration of the advanced ion table Environment tab Allows
94. ion applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer 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 PIM 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 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 PIM button while selecting PTM The PTM Editing dialogue will appear VS 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 OK Cancel 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 d
95. ion of our start menu Click Next SC SC 9 Review the choices we have made We can click Previous if wed like to make any changes or click Next if those choices are correct 10 PEAKS 4 2 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 4 2 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 Connecting to a PEAKS Online Server PEAKS Client sends all its intensive computation jobs like PEAKS auto de novo and PEAKS Protein ID to a PEAKS Online server installed somewhere in the lab or computing facility PEAKS Studio can optionally do this too In the Environment tab of the Environment preferences dialogue change the Default PEAKS Invocation to PEAKS Online Then specify the IP address or URL of the PEAKS Online server the port number to use and a username and email address Press ok The email is used to notify us that a job has been completed in the event that we close PEAKS 4 2 before finishing It s important to monitor the task queue when running jobs on the PEAKS Online server Hold the mouse over a job that s in the queue to see it s status El Task Queue E C Program Files Peaks Stu Task IS_WAITING Peaks Online SampleData pkl Regist
96. 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 4 2 provides a tool for doing so Under the File menu click Load Directorry Now browse to the directory we wish to load PX PEAKS Studio Do not select a file within the directory rather select Ele Edt View Tools Help ___ the directory itself Press the Open button Open Ctri 0 iy ah Load Directory P Import MassLy1 gad data fies rom arecioy After loading the spectra we can choose to sort the Tae spectrum by the source filename or by the precursor Import bruker raw data R H Load 4700 Data m z value of spectrum To do so right click the Gi Close parent node on the Peptide Data and choose to sott Save Cts Save As ar Pee The data file we just opened appears in the Peptide Export gt Data Frame on the left It is represented by the Exit folder name Each spectrum contained in the data 2 K Si 41 ly wa file is represented by its precursor ion information i f m z value followed by the charge of the precursor 63 SCV SCV SCV Some versions of MassLynx may differ ion that generated the spectrum and file name Loading Thermo RAW data PEAKS 4 2 can load RAW data from our Thermo Electron mass spectrometer provided that th
97. l229460 peptide 545 92804 TPEVDDEALEKFDK 1 634 7673 138 gil229460 623 2947 TPEVDDEALEK 1 244 5771 gi 229460 673 38513 IGDLQK 672 3807 gi 1094913 673 38513 GLDIQK 672 3807 gil229460 673 38513 LGDL K 672 3807 gil62510779 674 4233 IPAVFK 673 4163 gil229460 674 4233 LPAVFK 673 4163 gil1 145653 Peptide Details Links to retrieve spectrum information 458 73856 2 De novo LDALNENK Database LDAINENK gt qi 229460 pri 732164A lactoglobulin beta Matched peptides shown in Red 1 LIVTOTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLOK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLYCOQ CLYRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI NCBI BLAST search of de novo sequence LDALNENK NCBI BLAST search of database search sequence LDAINENK 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 Vew 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
98. ly minutes or seconds 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 Ion 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 needs to 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 been designed for use specifically 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 a 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 4 2 will warn us when this may occur
99. m 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 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 4 2 s basic features 24 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
100. mem a 99 53 64 12 LJ beta lactoglobulin R725 13 E 99 50 00 12 fal Chain A Structural Changes Accompanyi _ amp 9 1259 1939339 E 99 45 51 12 LJ Beta lactoglobulin precursor Beta LG A X gil2780 19 921 T E i 99 45 51 12 m lactoglobulin beta Bos taurus amp 9 3808 20 023 T Em m 99 45 00 12 beta lactoglobulin Bubalus bubalis X gil2237 18 177 E EE an 99 30 25 7 Tl lactoglobulin beta EU gil4949 19 976 M ma an 99 27 22 7 m beta lactoglobulin Capra hircus X Protein Pane MSA Pane os gil229460 LIVTQTMKGLD IQKVAGTWYSLAMAASD I SLLDAQSAPLRVYVEELKP TPEGDLEILLQK gil223780 TIVTOTMKGLD IQKVAGTWYSLAMAASD ISLLDAQSAPLRVYVEELKPTPEGNLEILLOK gil229460 WENCECAQKKI IAEKTKIPAVE KLDAINENKVLVLD TDYKKYLLF CMENSAEPEQSLVCQ gil223780 WENgECAQKKI IAEKTEKIPAVEK IDALNENKVLVLD TDYKKYLLFCMENSAEPEQSLacQ gil229460 CLYRTPEVDDEALEKFDKALKALPMHIRLSFNPT1qEeQCHI gil223780 CLYRTPEVDNEALEKFDKALKALPMHIRLAFNPTqlEGQCHY Color Code 80 60 40 20 SCV Filtering results PEAKS 4 2 provides us with an exhaustive list of all proteins and peptides that can be found in the sample But since everyone has their own criteria of what information is required in the report and what is an acceptable result a good hit we must have a way of filtering out the less critical information to leave us with the essentials In order to manipulate results we must of course have done some protein identification using PEAKS Protein ID or inChorus sea
101. modes can be edited See the section in configuring PEAKS 4 2 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 H2O 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 area 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 horizontal axis of the alignment view indicates the range of the spectrum view in the Spectrum V
102. mzXML Step 2 Data Refine v Data Refine amp Options Step 3 Auto De Novo by PEAKS v Auto De Novo amp Options Step 4 InChorus Database Search Peaks Database Search Engine y Peaks database Search EX Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines Tandem Search Options Options Options Omssa Search C Spider Search port Database Search Results Mascot Result dat Mj Browse Sequest Result summary html gt Browse Step 5 Save Result phosB mzXML save as phoss anz a ecoli11 mzXML save as lecolit 1 anz T Il E v Close file after saving Step1 Load Files Click the browse button to open a file chooser From the chooser select several files by shiftt 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 Database Search Choose which protein identification programs to run the data PEAKS database search is required S
103. nce Auto de novo if we have so chosen and subsequent protein identification If we have chosen to perform auto 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 4 2 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 3 Or Select inChorus Protein ID from the tools menu The inChorus database search launch window will appear SC InChorus Database Search Peaks Database Search Engine Peaks database Search EX Options Peaks database search is mandatory the database and taxonomy will apply to other search engines Other Database Search Engines Tandem Search FL Options Omssa Search C Options Spider Search 4 Options port Database Search Re
104. ole eaue tiny 90 SC RUNNING PROTEIN IDENTIFICATION ON SELECT SPECTRA essseccesessreeseesecsceeceeseceaeeaeeseeeaecaeeeneenecues 91 SCV USING THE MASS CALCULATOR cve etase teert anser erea ernea Goat re e reS See ERa erena E eE ee eeta 92 WORKING WITH RESULTS ic conscsccenntssccanccnsddentecsccannosstcenseocdeanscestdensoesecanesesdcensocseean 94 SCV VIEWING PROTEIN IDENTIFICATION RESULTS svsxchentnentencheiconens cosdanevbnsbbsstunsh earthing toay vaca caywesumiyshonad 95 SCV COMPARING PROTEINS MULTIPLE SEQUENCE ALIGNMENT cscssseseseseseseseseneseseneseseseseneneneneneees 99 SCV FILTERING RESULTS 453 ne aee r E E EE E E E E E EE REE E a a 100 SCV How filters act on de novo SeguenceS ssssssessesssrssersrresserieresrssererreserernrnresenennenrerenrnnenrnreneneenene 102 SCV Filter examples 42 Grcs ceteris irin in EE EAS EEN O ARR A bass aE eiii 103 PUB ICATLON DEE EEEE ERT 103 Digging for a protein by NAME si ccssccssessseesesieenchsssssvevevsvanovbatssaebeviosocbsatesenssosaegusdabeanedovivonsbeveaderuastvagsvbobeigeesadonss 103 Setting a protein mass range ssib eio arasi eie rra a Seison araba EEES E E ASE ESES NEn ESES TESES SI ESES a DES Raas EES os 103 SCY Saving Loading Filter sets taco cans taitan cas nscehigetoanetieatenite onan ttiotuaahieitanasedctanletaneteateeatige onia 103 DEVE sTogoline CONTIG ei ces cous enak ae titan tate ate E a R E Te R Ea TEA 104 SCV EXPORTING RES LTS repeii ceinte aiser a ea E a i de leds e E
105. on 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 Environment Changing default file handling settings 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 PEAKS 4 2 can use the last folder we loaded from saved to as the current working folder or by togglig the appropriate radio button to User directory we can set it ourselves so that it will be the same each time The directory where PEAKS stores its preference information cannot be changed The option to load a new spectrum view window for each spectrum or open one at a time is available We can choose to show the sample data at startup Further adjustments can be made so that the GNU General Public license displays whenever GNU governed softwate libraries are called Click the appropriate checkbox at the bottom of the window Once we ve chosen from these options pressing the ORK button will exit saving changes The Cancel button will exit discarding changes Environment PEAKS Client to PEAKS Online In the Environment tab of the Environment preferences dialogue chan
106. ontained 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 whatsoever 109 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 LI
107. 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 4 2 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_staxid_prot dmp gz and the taxdmp file was called saxdmp zip Select these files A note on choosing the taxonomy files for Swiss Prot At the time of printing the gi _taxid file was called speclsttt and the taxdmp file was called saxdmp zip the same one as used by NCBI 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 15 Trouble shooting Some problems with a database may not appear until we run a search
108. oz file We must find the file and use a decompression utility such as WinZip 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 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 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 Next button NCBI nr bs UniProt Human Mouse and Rat IPI Human Mouse and Rat Other database 13 each field for more information Import database wizard Database Nickname Path Browse EST database Advanced Options Fasta header format set up ce Use built in database format UniProt gt Apply Rule to parse accessionid string from FASTA title gt is Rule to parse description string from FASTA title s URL to get detailed information with accession d http srs ebi ac ukisrsbinicgi bintwgetz e UNIPROT Accession ID Delimiter Taxonomy Options
109. pening data files In order to do any data processing we must first load our spectrum data into PEAKS 4 2 To open a PN PEAKS Studio File Edit View Tools Help data file click the S icon on the toolbar in the upper left corner of the PEAKS window or select Open from the File menu Load Directory Import MassLynx raw data Import wiff raw data Load 4700 Data Ef Close Open cto blew fF Save As Save Ctr S Save All Ctri Shift S Export Exit S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer SCV Lookin Data lalale multiple spectra phosphorylated IO single spectra OrbiOrbi pki 2 SampleData pkl Documents Computer FileName SampleData pkl Open My Network Places Files of Type ann anz dta mgf mzxml mzData pkl xml raw bt Spectrum Data File Cancel Select a file 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
110. potentially interesting but lower intensity peaks To tell the instrument to do this we must create an exclusion list PEAKS 4 2 has the ability to create an exclusion list that will be compatible at least with Waters Micromass instruments To create the exclusion list simply select a few spectra from the peptide data tree on the left then right click and choose Export to Exclusion list from the pop up menu that appears The selection of spectra can also be done using by clicking in the Peptide View or De Novo View of a Protein ID report Exporting high resolution spectral images It is often useful to provide visual aids in reports posters and papers As such PEAKS 4 2 provides the facility to export high quality images of a spectrum or part of a spectrum zoomed in In any case PEAKS allows us to export exactly what s shown on the screen So to export an image 1 Bring up the main processing window for an MS MS spectrum by double clicking on its precursor ion mz or by double clicking a 105 peptide in any of the reports The main processing window shows us the spectrum of interest along with any peptides matched to it Set up the colour typeface and size of labels and peaks on the spectrum To do so choose Edit menu gt Configuration gt Environment Preferences Within the Environment preferences dialogue choose the Colour or Display tab to get at the relevant settings optional If we want to export a pictu
111. pressed Edit Filter Frame Edit the settings for the currently selected filter Filters in the Possible Filters list and the Active Filters list can be edited Options frame The options listed here fundamentally change what is listed in the De Novo View regardless of the specific filters selected F 21 Nov 06 09 47 31_NR inChorus protein ID results De Novo View Peptide View Protein View Search parameters Filter Pane Saved Parameters Filter Parameters PEAKS example Possible Filters Selected Filters C Mr cale filter 4 De Novo Filters i Delta Mass filter Adafiter D Score filter 90 0 C Mass filter menpea J Peptide Filters Accession filter Supportfilter gt 1 CY UniquelD filter Apply fitters J Protein Filters C3 Protein Filters D Mass filter 10000 0 B Score filter C Query filter Bi Desc filter C Mass filter Coverage filter Bi Acc filter Selected filter Options Edit Filter De novo view shows Mass peptides that could not be Filter proteins based on their explained by peptides from mass the Peptide View z 10 000 De novo view shows all peptides that are not filtered Options C Remove de novo peptides Equal to with no database hits Grasterthan Lesser than Not equal to Save As Delete set Option 1 De Novo View shows peptides that could not be explained by peptides from
112. rching Click on the time and date stamp associated with this result under its file name in the peptide data panel on the left Once the report loads click on the Filter Pane tab to bring up the filtering options De Novo View Peptide View Protein View Search parameters Saved Parameters Filter Parameters N Possible Filters Selected Filters C Mr eale filter De Novo Filters 0 Delta Mass filter id Gites J Peptide Filters C Mass filter Remove filter C Score filter gt 60 0 C Accession filter J Protein Filters CS Uniqueld filter peoh iors C Query filter 1 0 C Protein Filters C Score filter D Query filter A Desc filter LD Mass filter LA Coverage filter D Acc filter B Selected filter Options De novo view shows peptides that could not be explained by peptides fram Edit Filter Score Filter peptides based on their PEAKS score the Peptide View A 60 J De novo view shows all peptides that are not filtered Options Remove de novo peptides Equal to with no database hits Greater than Lesserthan Not equal to Delete set Choose a filter from the Possible Filters list on the left by clicking on it Options for this filter will appear below in the Edit filter frame Once we ve set these options we press Add filter to bring it to t
113. re of the Ion Table we should configure what columns are shown in the ion table before the export To do so select one of the Ion Table Editor tabs from the Environment Preferences dialogue optional If we want to export a zoomed in region of the spectrum we must do so now by clicking and dragging horizontally over the area of interest Click the a export results button on the main processing window toolbar or right click on any frame within the main processing window and choose Export Image from the pop up menu The Image Export Options Dialogue appears On this window you can expott What the current spectrum Image Export view middle frame is displaying Image Options File format png w Width 7 q51 Height 295 Filename rs BS Desktap Genzyme 1 058 3 pno What the alignment bottom frame is displaying Resolution Upscale to 100 resolution All the proposed peptides as listed in the top left frame lon Table and Error Map Current Spectrum View Spectrum Alignment Frame z 5 Peptide Candidate Tree A combination of the alignment and spectrum annotations Error Map Annotated Spectrum with Alignment OK Cancel a Choose what you want to export b Select a file type Four popular formats are supported but either GIF or PNG formats are recommended c Choose a file name We can browse by clicking the folder icon or just type a file name in the bo
114. rface 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 manual This user s manual is intended to help us get started using PEAKS 4 2 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 term 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 Since PEAKS Studio PEAKS Viewer and PEAKS Client share the same uset s interface this manual covers all three software programs Where a section is S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer applicable to PEAKS Studio an S is noted C denotes PEAKS Client and V denotes PEAKS Viewer Some sections apply to all three so SCV is noted Scope PEAKS users are assumed to be familiar with computer usage and the operating sy
115. s list with our saved filters Don t forget to press the Apply Filters button to apply them Toggling columns Still the information in the report might be too intensive Some users might not care to see the start and end position of the peptide in the protein while some users may wish to see even more information PEAKS 4 2 allows us to customize which columns are shown Right click anywhere in De Novo View or the top section of Peptide View or Protein View In the pop up menu that appears hold the mouse over Toggle Column A sub menu will appear showing a checkmark in each of the columns that are currently showing Click any one of them to show hide a column PEAKS will remember which columns we select and present us with these columns each time we load a report Exporting Results WYSIWYG reports PEAKS 4 2 allows us to create interactive reports to share with collaborators colleagues and clients The reports are available in HTML or Microsoft Excell xls formats and follow a What you see is what you get WYSIWYG philosophy All the information you see on screen in PEAKS 4 2 will appear in the exported report For this reason it is important that we complete results filtering and toggling columns before exporting a report In order to export a report first choose the view to export by clicking on the De Novo View the Peptide View tab or the Protein View tab Next right click anywhere in the top section of the selecte
116. sdhdsoeescusueuts escesbssbo tases te secever gasusk n ET ENTE E TET 80 Measuring distance alorig the m z scalenie e N AE E E EE aE ESES 80 Measure the m z difference between two PEAKS cccecccsssssssseesceseeseesecseescessesecssseecsaecsesaecsesseenseeseeaeeaesaeeneeaes 81 Deselect 0 1 acc satst ie dseens actos tas cates ehst ate E tees ce oes ieaen ERE EE EE ENE E 81 ZOOM ii ON Part OF the SPECHCUDK eeir are peo raresa rr aa EE EaR a EER SEOSE E ES Ee E RES EINER ES ESSENER ENIE EPES ESES 81 Add rem vej ions t from a peak ws cocssssscciscesssseseeastdecn abbsasgekcbsvapedsessnnenents E ERE E EEE 81 Using s gueri e tags issis siisshivideiiihaelii ae dus Ea Aaa oa ESE ae EE EE EEEE E RSE E EENET ES Eei R EESE SEa 82 U doing an edit ropen a EE EEE oee EEEE EE EEEE EEEE EEEE EESE EREE E E n EE ENEE 82 Redone am editto e ae A E EE EE RE A e AE E 82 SCV SUGGESTING A SEQUENCE TO SEE HOW IT FITS THE DATA csssessessceseeseeeeeesecseeeceeaecsaeeaeesecnaeeaeees 83 SC PROTEIN IDENTIFICATION ee ta o eara E Ea EEE a aae Ea taeae 85 SCV PEAKS protein identification sssesesssssssessrnsrsesserresrsessenenreserennenersrernenenrenensenenersenennrrerenennenrnreses 85 S mCho tus protein tden ufan osere inni ei e ai A E Aea dicen A Cootidac 88 SC CREATING A HIGH THROUGHPUT WORKFLOW jyivcasssscieei tesiiens ecelitv evitvoutieaitivn nesters eli duerieneatea lenny 89 SCV SELECTING UNMATCHED DE NOVO RESUETS acuccio taenneteltauaio usenet H
117. securitySignature dll 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 SCV 2 We can then create a folder called C MassLynx on our computer 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
118. selected the ions that PEAKS has found to match the proposed sequence will appear on the spectrum spectrum alignment view and ion table 84 SC SCV Protein Identification The PEAKS 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 PEAKS also introduces an amalgamative approach to protein identification called inChorus With inChorus protein identification technology we can use PEAKS together with several other protein identification software programs This will deliver more protein coverage and more confidence in results than any one method on its own The following two sections deal with usage of PEAKS protein identification on its own and usage of inChorus protein identification PEAKS Protein Identification The PEAKS Protein ID search engine takes a hybrid approach that uses sequence tag information to filter the protein or EST database before fragment ion fingerprinting Thus 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 nove sequences to filter the database If we do not have existing sequence information or if we wish to refine our database search by providing brand new sequence information
119. signate the peak as a b ion Select Ion Edit from the pop up menu to view the Ion Editor dialog box and designate the peak as another ion 294 11 341 2112 NH3 c2 y3 H20N i Search N terminal by B E Sec Chote P RTE DESES Peet aan Vee NOE ir 81 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 on your keyboard 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 After setting two ions PEAKS 4 2 will estimate the residue found between them if a residue corresponds closely to the mass difference The peptide sequence
120. sk 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 23 F 05 Jun 06 14 47 33_NR inChorus protein ID results Protein View Search parameters Index miz_ z mass Peptide Mr Calc Delta Mass Score Start End Accession No Peaks Tandem 1 452 2 2 902 56 TKIPAVFK 902 55 0 0035 68 76 83 gi 229460 Y 2 458 7 2 915 46 LDAINENK 915 46 0 0035 71 84 91 gil229460 v 2 458 7 2 915 46 IDALNENK 915 46 0 0035 71 84 91 gil2194089 v 13 467 2 2 932 53 LIVTQTMK 932 53 0 0053 36 1 8 gil229460 v 13 467 2 21932 53 INMTQTMK 932 53 0 0053 35 1 8 gil72079 I 14 33 2 2 1 064 VLVLDTDYK 1 064 0 0048 85 92 100 gi 229460 Vv im 15 645 9 3 1 634 TPEVDDEALEKFDK 1 634 0 0050 100 125 138 gil229460 v lv E 697 3 2 1 192 VLVLDTDYKK 1 192 0 0074 100 92 101 gil229460 v Y 8 623 2 21 1244 TPEVDDEALEK 1 244 0 0023 100 125 135 gil2294
121. ss instrument s data in RAW format provided that PEAKS and MassLynx are installed on the same computer or pkl files All instrument s data as can be converted into mzXML mzData pkl dta or mef Installation To make sure we only use the latest information available to PEAKS 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 4 2 disc into the CD ROM drive 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 Select the top item Install 5 The installation utility will begin the install Wait while it does so Choose English as the language for installation instructions When the PEAKS 4 2 installation dialogue appears click the Next button 6 Read the 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 ditrectory in which we d like to install PEAKS 4 2 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 4 2 The default will put these icons in the programs sect
122. st PTM Library and Database Clicking a tab will allow us to edit the PEAKS Properties corresponding to that tab We can also import or export our preferences 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 open the PEAKS Properties dialogue and select the PTM Library tab PEAKS Client uses the PTM library defined on the server editing here will have no effect on the list available during analysis F PEAKS Properties Enzyme list PTM Library l Database List of PTMs lt Built In 4 hydroxynonenal HNE lt Built In Acrylamide adduct lt BuiltIn gt Applied Biosystems cleavable ICAT TM heavy lt BuiltIn Applied Biosystems cleavable ICAT TM light lt Built In Applied Biosystems iITRAGQ TM multiplexed quantitation chemistry lt BuiltIn gt Applied Biosystems original ICAT TM d0 lt BuiltIn Applied Biosystems original ICAT TM d8 Built In 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 S marks a sect
123. stem 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 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 group plus the intervening neutral amino acid residues BSI Bioinformatics Solutions Inc The makers of PEAKS and other fine bioinformatics software 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 Deconvolution rearrangement of the spectrum to show each monoisotopic peak as if it were singly charged Thus to reposition them on the sc
124. sults _ Mascot Result 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 Option screens for 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 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 4 2 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 SCV Work Flow Step 1 Load Files mport data phosB mzXML ecoli11 mzx lt ML phosB mzxmL nputfile list jecoli11
125. t an individual spectrum ot a few spectra within a data file auto de novo will proceed on only the spectra selected 2 Click the Automatic de novo toolbar icon Or Select Auto de nove from the Tools menu 73 We should begin by using the suggested error values then try some slightly higher or lower ones to find the best result Or Right click on the selected spectra or data files and select Auto de nove from the popup menu The Auto de novo Parameters dialogue window will appear De Novo Options De Novo Sequencing Parameters Saved parameters SampleData Instrument Quadrupole TOF Parent Mass Error Tolerance 0 1 Fragment Mass Error Tolerance 0 1 Enzyme Semi Trypsin Report up to peptides al Edit Enzymes PTM selected for search Fixed Carbamidomethylation Var Oxidation Var Phosphorylation max variable PTM per peptide Add Remove PTM Deconvolute the data for analysis use for non deconvoluted data Save As Ok Cancel 3 If we wish to change any of these parameters we do so now 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
126. t 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 Files and Format Glossary 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 ANZ file a PEAKS zip compressed XML based Annotated spectrum file format ANZ files preserve all the information from the PEAKS session DTA The file format associated with SEQUEST software FASTA Fast All A standard sequence database file format used for protein identification PEAKS can identify proteins from any FASTA format database of proteins MGF The file format associated with Mascot software PKL The file format associated with Micromass instruments SCV Getting started with PEAKS 4 2 Everything we need to know from the beginning and step by step his section of the manual
127. tandard x Run de novo using different parameters than the above Run de novo using the same parameters as above detault Save As Ok Cancel After entering the settings as shown click the Save As button to save these parameters for future use PEAKS Studio only 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 less than two minutes for this sample depending on the system s processing speed and 18 SVC 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 memory The PEAKS auto de novo algorithm derives sequence candidates for each of the 22 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 4 2 PEAKS uses a unique sequence tag plus peptide fragment fingerprinting approach to protein identification Viewing Results After the search is finished th
128. tep5 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 If it s a large run we should close each file after saving Selecting unmatched de novo results When working with unknown organisms abundant 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 4 2 s protein identification tool The proteins identified in the sample will be shown in the Protein ID Report Click on the Filter tab and make sure that Option 1 de novo view shows peptides that could not be explained is selected Then if desired add some filtering criteria then press the Apply Filters button Click on the De Novo View tab This now displays de novo sequences for spectra that could not be explained by the identified proteins as listed in Protein View Sort this list then click and drag to select some de novo sequences SC After pasting we must save Data1 to continue 21 Nov 06 11 22 04_NR inChorus protein ID results De Novo View Peptide View Search parameters Filter Pane M z Mass Peptide Score 1354 1898 2706 365 WLKWKDMYASDLSLLDAQSAPLR 673 38513 672 37787 LGDLGAK 9 26 673 38513 6
129. 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 Any built in PTM cannot be removed Database Manager PEAKS 4 2 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 database 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 4 2 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 an existing database PEAKS Client uses the databases configured on the PEAKS Online server We can t edit the database itself from within PEAKS 4 2 F PEAKS Properties Enzyme list PTM Library Database List of databases NR D irogersidatabasesinr_20051120 fas SPROT Dilirogersidatabasesisprotfas Set as default 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 dat
130. 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 function m z tolerance can also be adjusted separately for each phase to allow us to tweak the results 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 Paste FASTA sequences If we already know the sequence of the protein s we are studying we can paste it here must be FASTA format and run a search against it Alternatively if we search the same sequence regularly it is recommended to simply create a small text file and configure it as a database for PEAKS 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
131. 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 Us or Cut button S 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 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 in the main toolbar The pasted spectra will appear in the Peptide Data under the data file into which we pasted 70 Analysing Data with PEAKS 4 2 A task based guide to processing our data with PEAKS 4 2 his chapter will step us through the analytical techniques available to PEAKS 4 2 users It is broken up into tasks that a typ
132. trap instrument setting Fragmentation type can also be chosen from this drop down 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 74 PEAKS users should try setting this a little higher than past experience may suggest Fragment mass error tolerance Determine 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 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 its analysis Each is marked Fixed or Variable To edit this list click the A
133. ts 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 found 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 We can manipulate the contents of this multi part report by filtering out what we don t want to keep Finally we can export the report and sub reports in a number of ways Search parameters that we used when generating this report are preserved for future reference and are available by clicking on the search parameters tab 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 the MS MS scan When we first load the report it is sorted by MS MS index number This is analogous to the scan number or DTA file name unless spectra have been merged In the example above there was one match returned for MS MS spectrum 1 For spectrum 2 there were two LDAINENK and IDALNENK For spectrum 3 there were also two and so on Each peptide is given a score and the protein it matches is
134. uence data and protein identification results for a given spectrum ate stored in an ANZ file Any modifications that were found in the sequence ate 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 a 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 PIM of interest from the dropdown list in this example Lab 2 custom PTM lale aisle E PEAKS Auto De Novo 0 2 Unknown enzyme with L2custorm L2customa COPY Sequence to clipboard Ct i c L2customL Copy for manual de novo L2customL L2custonk View S
135. uiltIn Carbamidomethylation lt Built In Carbamylation lt Built In Citrullination lt BuiltIn Deamidation lt BuiltIn Dimethylation lt BuiltIn Farnesylation lt Built In Flavin adenine dinucleotide lt Built In Formylation lt BuiltIn Gamma carboxyglutamic acid lt BuiltIn Geranyl geranyl lt Built In gt Glucosylation Glycation lt Built In Guanidination 4 Il i Select As Fixed gt Select As Variable gt lt Unselect Selected Variable PTMs Deamidation Oxidation Phosphorylation Edit PTM New PTM 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 peptide will not be modified more than a few times and as such limit the number of variable modifications that can occur on each peptide Generally the more variable PTM we turn on the more ambiguous will be the results SC Auto de novo Sequencing To begin auto de novo sequence derivation 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 selec
136. umns in the table Selecting one or more click and drag or use shiftt click items in this list selects those spectra in the peptide data tree found on the left hand side The bottom panel of this view shows more details about the peptide identifiecation that is highlighted in the top section In this view we can see the protein that the peptide came from and a simple alignment between the original de novo sequence for this spectrum if available and the peptide found in the database 35 6 21 Nov 06 11 22 04_NR inChorus protein ID results De Novo View Peptide View Protein View Search parameters Filter Pane Peptide View 5 miz Mass Peptide Mr Calc Score Accession UniquelD showing peptides 452 28497 902 5554 TKIPAVEK 902 5589 gil229460 grouped by 458 73856 915 4626 IDALNENK 915 4661 gil4388846 spectrum 467 2729 932 531 2 LIVTQTMK 932 5366 gil229460 467 2729 33 2925 545 92804 597 3386 623 2947 673 38513 673 38513 673 38513 674 4233 674 4233 932 531 2 IIVTQTMK 932 5366 gil72079 1 064 5704 VLVLDTDYK 1 064 5752 gil229460 1 634 7623 TPEVDDEALEKFDK 1 634 7673 gi 229460 1 192 6627 VLVLDTDYKK 1 192 6702 gil229460 1 244 5748 TPEVDDEALEK 1 244 5771 gi 229460 672 3779 IGDLQK 672 3807 gi 1094913 672 3779 GLDIQK 672 3807 gi 229460 672 3779 LGDLQK 672 3807 gi 62510779 673 4160 IPAVFK 673 4163 gil229460 673 41 60 LPAVFK 673 4163 gil1 145653
137. ure a database 1 Load PEAKS 4 2 If we have not yet configured a database the wizard will appear automatically Otherwise In the edit menu select Configuration then Import Database Wizard The Import Database Wizard PEAKS Studio Edit View Tools Help Configuration gt PEAKS Properties BT Environment Preferences asl wt wen nstama a 2 Import Database Wizard P 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 Having selected a database the Import Database Wizard will provide us with some information about that database If this is in fact the database 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 whete 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 OSs Eep ebike uk 12 The taxonomy options are only available if the NCBI
138. us to set default file locations default file handling functions and to specify local processing or remote PEAKS Online processing 34 SCV Colour tab from here we can set the colours to use for various spectrum annotations Display tab from here we can set the type face to use across the PEAKS 4 2 user interface The width of spectrum peaks can also be set from here Manual De Novo tab default options for manual de novo sequencing can be set from here 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 De Novo view The De Novo view summarizes the de novo sequencing results for each spectrum in the data file The exact contents of the list are determined by filters set on the Filter Pane 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 brings those spectra in the peptide data tree found on the left hand side Peptide View The peptide view summarizes the results for each MS MS spectrum All peptides that match to each spectrum are displayed The exact contents of the list are determined by filters set on the Filter Pane By default the peptides are grouped by spectrum but the list is sort able by any of the col
139. utions 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 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 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
140. 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 Eal 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 S marks a section applicable to PEAKS Studio C for PEAKS client and V for PEAKS Viewer 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 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 exper
141. will guide us through the process of installation and configuration of PEAKS 4 2 If problems persist contact technical support What we will need Package contents The PEAKS 4 2 package should contain This manual PEAKS 4 2 System requirements PEAKS 4 2 will run on most platforms with the following requirements Equivalent or superior processing power to a Pentium at 500 MHz At least 1 GB of memory RAM 1 5GB is recommended For PEAKS Studio PEAKS Client PEAKS Viewer requires only 500MB JAVA Virtual Machine 1 5 or better provided on installation Instrumentation PEAKS 4 2 will work with data from any type of tandem mass spectrometer designed for proteomics work PEAKS will accept data in the following formats mzXML is a standard data format from the Sashimi Project It is an XML based format mzData is a standard data format from the Human Proteome Organization SCV 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 Bruker instrument s data in yep baf and fid formats Thermo Electron instrument s data in RAW format provided that 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 Microma
142. x 106 SCV d optional Specify width and height dimensions in pixels In some cases the size is predetermined by the size of the frame on our screen Otherwise we can increase these numbers to make the picture bigger e To export high resolution images increase the resolution by a few hundred percent f Press ok and you re done 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 save icon in the main window 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 pop up menu We may then change the file name To save all currently opened data files select Save all from the File menu 107 About Bioinformatics Solutions Inc BSI provides advanced sofovare tools for anabssis of biological data Bioinformatics Sol

PEAKS Studio Manual 4.2 - Bioinformatics Solutions Inc.

Contents

Download Pdf Manuals

Related Search

Related Contents