PEAKS Studio Manual 2.0 - Bioinformatics Solutions Inc.
Contents
1. we will use Test in this example From here there are two options Next or Finish Select Finish for the AAs PTMs set to be created as Trypsin without PIMs AAs PTMs set Selecting Next will activate the Residue PTM Editing dialog ll Residue PTM Editing xj Select Enzyme Trypsin v Residues at N terminus wj A MR lr N ei D ei E eK E ei le G MH HA 17114 L le KE le Mm ei FLOP eS 41 T e 4 e Y e Y 0 SelectAll Residues in the Middle 4 A MR lr N ei D lel el E lr O le G HA 711 L le E lem el F e P eS 41 T e 4 e Y e Y 0 Select All Residues at C terminus JA HRITNDDOclENDa US H HUL Wi kid mF OP Us TOW OY OY Selecta Figure 11 Enzyme Selection Select a pre defined enzyme from the drop down list and customize residues for different positions Once residues are chosen select Next to continue defining PTMs for this AAs PTMs set or select Finish to save this set 19 PEAKS STUDIO QUICK START Pi ResiduePTM Editing EE xj PTiis that can be selected Select As Fized gt selected Fixed PTMs Builtln Hydroxylation Oxidation lt Builtln Lipoyl lt Builtin Methylation Builtln Myristoylation lt Builtin HN acyl dighceride cysteine tripalmitate BuiltIn O GichAc Select As Varied gt lt Unselect Edit PTM selected Varied PTMs New PTMI Remove PTM hall lt Builtln Palmitoylation Figure 12 PIMs choosing All PTMs2. 1 Trypsin without PTMs 728 30 613 20 542 20 NNSCCCCK lt 1 288 10 GGNSCCCCK lt 1 GNGSCCCCK lt 1 NGGSCCCCK lt 1 AJO O O O P 0O M jo 00 Aa M0 J 00 co ho ho a E gt F ik ho a Color Code gt 90 80 90 60 80 lt 60 387 9 316 29 No tasks Figure 6 Inspecting the spectrum Protein Identification PEAKS protein identification functionality uses peptide sequences from the auto de novo process to search fasta databases Select the file positive pkl located in the Peptide Data panel on the left To begin protein identification select the Automatic De Novo toolbar icon 2 The Auto de novo options window will appear the same as the option window for auto de novo sequencing If the FASTA database was not configured during installation it must be configured to enable protein identification functionality To do so either e Click Edit PEAKS Properties in the option window e Select Edit PEAKS Properties from menu or toolbar For details of how to configure FASTA database please check Chapter 4 Configuration Once the database setup is complete check the Search results in database box in the option window choose the appropriate database and then click OK 14 PEAKS STUDIO QUICK START PEAKS will perform auto de novo sequencing on all spectra in the file then use the sequence results to search
3. and overwrite local PEAKS Properties with the information from XML file PEAKS Environment Preference Environment Options for default Data Input Output directories can be changed to increase efficiency The Default Configuration File Directory is where the PEAKS configuration file located Environment Preference x Em ronment T Ca to Mo Ki ir cai eee ey L om i L Default Data Input Directory Dleiguo Previous directory 0 User directory Browse Default Data Output Director Drileiqua 0 Previous directory C User directory Browse Default Configuration File Directory Documents and Settingsileiguo BSll peaks Figure 23 PEAKS Environment parameter Color Spectrum viewing color options 26 PEAKS STUDIO QUICK START Environment Preference E i x Environment Color Auto De Nowo Manual De Novo Please choose color for Spectrum _Peak Freezed Peak a ie Red 255 se Current_Peak a st i 255 Y_Alignment B Alignment Green 0 Yon Md Blue Ml Preview k YE e Current color i waal Figure 24 Spectrum viewing color option Manual De Novo Parameters Change the default parameter for tag searching and random machine error Length the default maximum length of the sequence tags to be searched Number the default maximum number of sequence tags to be searched It is also possible to set the default random machine error for Manual De Novo Auto De Novo Param
4. area in the A enment View panel to let only the selected area be shown in the Spectrum View panel Add remove ions to from a peak select a peak then trigger a popup menu by right clicking the mouse Select Ion Edit from the popup window to view the Ion Editor dialog box From here it is possible to add or remove ion designations to from a 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 277 14 374 14 Set lon K2 350 23 A 100 203 14 A a Sal len Remove bon 54125 342 2 ae 29411 a41 2143NH3 pe 4775 tr 564 22 f ooa Ha Search lefi FE i ba H20 203 78 320 380 Search right th 560 605 08 a A Search C terminal by 15 pa A oman EA Search N terminal by Y FE A rida 10 o1 1 SearchNterminalbyB l eH f E Search C terminal by B 0 100 200 200 400 600 ri 800 Using sequence tags Search C N terminal by Y B tight click anywhere in the Spectrum View panel to trigger the popup dialog From the menu select the terminal search of interest The corresponding terminal tags will appear in the top right panel for testing the round radio button or insertion check box into the working peptide Two short cut keys can be used F5 for search C terminal by Y and F8 for search N terminal by Y S
5. be 0 ES Z ni mian ae t Sha i m 2 am aa a ii ii a merei e net Gra se L on a aE gt rie oe 27492 400 500 60 700 BOD 900 970 48 E Ao pa 100 fi i i i i i i i i I i i i it 1 j i I i I i I ii i i i I i l i i i i i Hr i i E I I I i i I I I de i ee ee AC E A A E a o 00 200 20D 1200 1500 130 06 Figure 32 Spectrum View and Alignment View Panel Tool Bar of Main Process Window Deconvolution button turn MS deconvolution on to filter noise from the data The default setting is off Undo Zoom button ES return to the previous magnification 1 1 button 1 11 to zoom back out to view the entire spectrum Ton editor button E edit add remove ions for the selected peak Export image button ce export the result spectrum image as image A l Print button E print the result image Report button e create a html format report for the current selected peptide candidate Redo button E Undo button E to redo or undo the edit to the spectrum ions Y ion alignment enable button E b ion alignment enable button m press to enable the y b ion alignment in the Alignment panel The defaults are on 37 Manual De Novo Sequencing To begin manual sequencing create a manual de novo peptide candidate using either copy an auto de novo peptide candidate for manual de novo Ot create a new manual de novo peptide candidate by choosi
6. but nothing happened First check the Tools menu and verify that Enable Tasks Running is checked If it is disabled the Task Queue will be shown in red and no tasks in the queue will be processed Another possibility is that PEAKS processes the auto de novo task in the background and usually this process takes several seconds You will find the job is still in the queue if you check the Task Queue panel After the process is done it will disappear from the queue Selecting the spectrum in the data file panel you can find the peptide candidates under Peaks item the spectrum image and ion alignment in the main process window 3 Can I edit the data file manually in PEAKS You can edit the precursor information m z and charge by right clicking on the precursor in the file panel but you cannot edit the spectrum itself 4 What enzymes should I use to digest the protein in order to use PEAKS to interpret the MS MS data The most popular enzyme for digesting proteins for MS MS analysis is Trypsin PEAKS comes with a Residue List predefined for unmodified tryptic digests to handle FAQ this common case Tryptic peptides typically show excellent MS MS spectra and produce good sequences If you wish to use a different enzyme or sequence small peptides in entirety you can use the Unknown Enzyme Residue List This residue list places no restrictions on the residues appearing at the C terminal end In addition you can
7. support staff tracing the bug For PEAKS Studio the logging file is located in the configuration file directory User can check the PEAKS Environment Preference to know the location of the configuration file directory The filename of the logging file is peaks log For PEAKS Batch the logging file will be saved in the output directory of the job PEAKS Studio Quick Start Getting started with PEAKS Studo EAKS is a software program for determining peptide sequences and identifying proteins from MS MS spectra It can be used in three principal ways e Auto de novo sequencing no user intervention no database requirement e Manual sequencing tools for a skilled user that assist the sequencing process e Protein identification using de novo and FASTA database searching Configure FASTA database The first time PEAKS Studio is started PEAKS will ask if you wish to configure the FASTA database required for PEAKS Protein Identification functionality A database configuration wizard will provide information indicating how to download and configure the database for PEAKS Protein Identification To configure a FASTA database 1 Choose a FASTA format protein database 2 Download the database and save the file to a directory accessible by PEAKS 3 Use the database configuration wizard or PEAKS Properties Editor to a Locate the database file b Specify a name for this database PEAKS STUDIO QUICK START Protein identification
8. 16 gt sp P02666 CASB_BOVIN BETA CASEIN PRECURSOR Bos taurus Bovine Mz Charge Mr calc Start End Score Peptide 437 48 854 47 113 120 94 61 VKEAMAPK 515 02 10095 64 72 418 IHPFAQTOS 646 48 6273 115 120 77 25 EAMAPK 780 57 761 48 185 191 28 69 VLPVPQK GP AKOO Mass 46296 598 Score 95 43 gt GP AK000922_1 Homo sapiens cDNA FLJ10060 fis clone HEMBA1001 407 unnamed protein product MASS 46345 Mz Charge Mr calc Start End Score Peptide 462 23 2 904 51 130 137 453 VIFLPFLS 509 92 2 999 58 148 157 2 28 FLGOGLSALL 515 02 2 1010 55 312 321 951 AYHLAVVLGS sp P027 Mass 19852 258 S Figure 8 Protein Identification Saving Results Select the data file positive pkl in the Peptide Data panel on the left Click the icon El or select Save from menu to save the processed file in ANN format Congratulations you just processed your first data file with PEAKS 16 Configuration Conjeenre PEAKS Properties and PEAKS Environment Preference Prior to processing data files it is necessary to configure PEAKS properties Also setting up proper PEAKS environment preference configuration may help increase efficiency PEAKS Properties Configuration PEAKS Properties dialog box allows you to configure Post translational modifications Amino Acid PTM sets and set FASTA databases for database searching For PEAKS Properties configuration there are three ways to invoke the configuration dialog e Click the i
9. BIOINFORMATICS SOLUTIONS INC Version 2 0 BIOINFORMATICS SOLUTIONS INC PEAKS Studio User s Manual Bioinformatics Solutions Inc 145 Columbia St West Suite 2B Waterloo Ontario Canada N2L 3L2 Phone 519 885 8288 Fax 519 885 9075 INTRODUCTION OF PEARS lina iia 2 PEAKS BASICOS ds 5 POST TRANSEATIONALMODIBICATION SARLINIS arre at AAA AAN A AA A ana 5 IA eR AE ASA A A 5 AT A EE SS T 5 AMINO ACI AA IMSS oli de 6 FASTA DATABASE o O e la ac lA a A A oada aai 6 TIRE FORMA S 3 diran cti td id e RE E E EAR vacia 6 HICE SAVE EXPORT et TA TU dE A TA E o 7 PEAKS PROPERTIES id A A A A SA AS AA A a 7 PEAKS ENVIRONMENT PREFERENCGE dd Ni St di a a ee 7 A ONG A OINA DA S E E e e o A e E E 8 LOGGING EILE ul A enka a ra a a E T E ARR 8 PEAKS STUDIO QUICK STAR Toons li A AAA A AA A AAA A AAA Aida 9 CONFIGURE PAS LA DATABASE naa A A A A A A A a sais 9 PENAS PE FROM PIE ip bce A A ESE A a AE EEEE la Pear thm O 10 MO DEN PON 5016 Sis CIDN CMe REE dl add at 11 INSPECTING THE SPECTRUM A SA SIA SNA ERES ASIA A AA SED 13 PROTEIN IDENTIFICATION is A Ai US ao Ec 14 VIEWING PROTEIN IDENTIFICATION RESULTS ia a AT AE E A E A EE E A bdo 15 DIVINO RESULTS id oa rt ria it a co tai tdo cal 16 CONFIGURATION ui A AA A A A A A aae eseas 17 PEAKS PROPERTIES CONFIGURATION ES EA A A AS AAA A A a ED tia 17 HAS TIVES EA e So le oe 18 PTM ld O A a as AE tg RA IA A EA A A ct 21 DADAS EMANAN E NS a E E RE LAA A A AAA e AAE E E Oa es 23 Import and Export PEA
10. KS Pr pertieS gt A A A A AA AAA AA AA EA EE E 26 PEAKS ENVIRONMENT PREFERENCE a ii caida 26 RA RR O E I A NN 26 COV OR LS ios 26 Manual De NOVO LIN A o oe Eno 27 Amo DEN AN A a eet a i E ca eae E E A N E N 27 PEAKS STUDIO USER INTERFACE viccsoostsessstesccssoossoossossdcsssossoessst ts eoesoo sree a asos sao o r eea ea aos ease ess eesosa sesso EE eesse aSo seses Eseese 29 ARATO EN BEN EA O EEA A E NN 29 ECGADDIRE O TOR SS herent cle thi id sane ta SS E a A A 30 PAT TRE DA NAE EEE EA E AESA EAE EASE EEIE EA EE E AA EAE EAE AAA IS 30 PROTEIN IDENTIFICATION RESULT WINDOW a e da a Nae e e eo 32 Protein Candidate erates ses A Cea tno ON EO S SEE EEEEPEPP ESS OSError 32 PROVARNAS ONE VIO AE VO AE IAE OSONA A REET ORE Eee TANNER E Corr ee earn eee 32 Index Paneli A NO 33 Information Panel anssi A A A AS E 33 MATARO CESSNA o e O 34 Ber EA A O A NI A O A O CARER 35 lon Table A A PEO AOE ER AEE T OPP ETE CCT ARIS eee OP OEE Ea ER CTR 35 Spectrum View and Alignment View PIDO Sind dt AAA DAA AA lt 36 LOO Bat ot Main Process Wind Wii il AAA A iaa 37 MANUAL DE NOVO SSEQUENCING 3d iia AA AR AAA A AA AA A A sansninadeawsecdeiasestaensencts 38 HOW TO START AUTO DE NOVO SEQUENCING AND PROTEIN IDENTIFICATION ccsssssssssssssssscscssssssscssssssssssscsscscscscsccecescscscscscscscsees 41 FREQUENTLY ASKED QUESTIONS iisissctsscccscccssseszcesesarcnaussenetcesscdsesusceusiwssveusesessacdsusbaconscsesndetessscesevndcesdevesvousesvestsited senusesess
11. ave all files Save data file If current data file or the sequencing result of the data file has been changed Peaks would save the data file as well as the result in ann format Save all files It is a batch mode of save data file If there are more than one data files loaded and changed using save all files would save these data files as well as the results in ann format respectively 9 We saw a green triangle on the m z axis of the spectrum What is that function 44 FAQ This green triangle is used to show the location of the singly charged precursor 10 Why I cannot delete a task from the Task Queue There are 2 steps to remove a task from the Task Queue disable the task running at Tools gt Enable task running unselect it right click the task in Task Queue from the popup menu choose remove 11 Where can I get the demo version of PEAKS The demo version of PEAKS can be found at BSI s web site You can either download the trial version or try the on line version from our web site 12 Where can I get the log file of PEAKS The log file can offer us some information for bugs tracking The location of log amp configuration file is set by PEAKS users can not change it You can find it at Configuration gt Environment gt Default Configuration File Directory 13 What is the differences between PEAKS and Pepseq PEAKS and Micromass Pepseq part of MassLynx ProteinLynx both do de novo sequencing Sa
12. con in the toolbar e Using the menu select Edit Configuration Peaks Properties e Click Edit PEAKS Properties button in Auto De Novo Options dialog Refer to PEAKS Quick Start for how to invoke Auto De Novo Options dialog Once invoked the Auto de novo dialog will appear PEAKS STUDIO QUICK START PEAKS Properties oOo Y xl AAs PTMs set Database List of Standard PT Med AA sets Euiltln Unknown Enzyme without PTMs Figure 9 PEAKS Properties Editor This PEAKS Properties Editor contains three tabs AAs PTMs Set PTM and Database AAs PTMs Set editor To edit AAs PTMs sets select the corresponding tab in the PEAKS Properties editor In this dialog you can create new AAs PTMs sets remove selected non built in AAs PTMs sets edit selected non built in AAs PTMs sets view the configuration of selected AAs PIMs sets The PEAKS Properties editor will show all existing AAs PTMs sets in the list PEAKS software contains two built in AAs PTMs set Trypsin without PTMs and Unknown Enzyme without PIM that cannot be removed or edited To select an AAs PTMs set move the mouse to it and click To create a new AAs PTMs set select the new button and the following dialog will appeat 18 PEAKS STUDIO QUICK START fill ResiduePTM Editing f xi Please Input a Name Test Next gt Finish Cancel Figure 10 Input AAs PTMs set name Specify the name for the customized AAs PTMs set
13. cting a spectrum in this window will present PEAKS sequencing results into the data viewing windows on the right side of the screen By default PEAKS displays five possible sequence candidates for each spectrum see the Configuration to change this number sorted by confidence levels Select a particular sequence the first one would be an obvious choice since it has the highest confidence to view the sequence alionment in the Alignment View panel at the bottom of the screen PEAKS also computes a Positional Confidence for each amino acid in the sequence and displays the amino acids in different colors according to their confidence score The Positional AUTO DE NOVO SEQUENCINGAND PROTEIN IDENTIFICATION Confidence Table will appear when you put the mouse on the sequence in the Peptide Candidates panel Selecting a data file in this window the protein identification result will be presented For parameters used in auto de novo sequencing please refer to Configuration 42 Frequently Asked Questions 1 What is the required system configuration for PEAKS PEAKS can be run on any computer that supports Sun s Java Runtime Environment JRE 1 4 On installation PEAKS will install a dedicated JRE for its use so it can co exist with another version of Java on your machine The following is the hardware requirements Minimum system requirement 512 MB RAM 512MB free space on hard drive 2 I started an auto de novo process
14. custuobscsaeadesascteesenesdecdeestuosseand 43 Introduction of PEAKS 2 0 Peaks makes the interpretation of MS MS data much easier and much faster EAKS is an innovative software system designed to derive amino acid P sequences and identify proteins from MS MS experimental peptide data Two versions of PEAKS are available PEAKS Studio and PEAKS Batch PEAKS Studio is the standalone module that provides peptide sequence and protein identification results via an intuitive interface allowing for rapid visual interpretation PEAKS Studio provides both auto and manual de novo tools for detailed examination of MS MS spectra providing the flexibility to manually modify auto de novo results when searching for additional sequence possibilities The y ion b ion searching algorithms can provide potential ion candidates previously not considered PEAKS Batch is a scriptable high throughput version designed for large quantities of MS MS data PEAKS Batch is a powerful part of the protein identification pipeline rapidly providing peptide sequences to facilitate data mining or statistical analysis Additionally the Batch version is a valuable tool when used in conjunction with database search methods Spectra that provide poor or negative database search results may yield valuable information when sequenced by PEAKS this information no longer needs to be ignored or discarded Both PEAKS Studio and PEAKS Batch share the following features e Supre
15. d 1024MB memory required PEAKS Studio Additional Features e Comprehensive manual sequencing tools including algorithms for y b ion search C N terminal search e Easy to use informative interface provides access to spectra sequence confidence and y b 10n information e Zooming labeling annotation and peak distance tools e HTML Report Editor provides easy access to spectra fragment and sequence information for fast document creation PEAKS Batch Additional Features e Scriptable command line driven interface e Rapid de novo sequencing less than 5 seconds per spectrum average on a single 1 GHz CPU with 1 GB RAM INTRODUCTION e Multi threaded for higher throughput on multi CPU systems e Both XML and FASTA output containing sequence confidence score results PEAKS Basics Terminolozies and concepts used in PEAKS chapter We will use these terminologies and concepts in the following T erminologies and concepts used in PEAKS 2 0 will be introduced in this chaptets Post translational Modifications PTMs A PIM is the modification of a newly formed protein This may involve amino acid deletion chemical modification of certain amino acids or the addition of small molecules e g phosphate groups or sugars to certain amino acids During the automated de novo and protein identification process PEAKS automatically detects user specified post translational modifications PIMs used in PEAKS can be grouped i
16. earch a sequence tag select a peak with a defined ion ie 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 These tags will appear in the top right panel for testing and or insertion into the working peptide Two short cut keys can be used F6 for searching the left side and F7 for searching the right side 39 4331 MANUAL DE NOVO SEQUENCING Select insert tags select a tag from the top right panel select the check box and this tag will be shown in the Adgnment panel All related ions will also be shown in the Spectrum View panel By using the check boxes on the right side of each tag multiple tags can be selected After clicking on the button these tags will be added to the working peptide to create one or more new peptide sequences Notes Testing vs editing mode if tags are shown in the Spectrum View panel it is in testing state no ion editing or tags searching is further allowed Editing can only be performed in Manual De Novo mode To edit an Auto De Novo result copy the peptide candidate to Manual De Novo first by right clicking on the sequence of interest and then selecting Copy For Manual De Novo After ions are added to two peaks a residue will be inserted automatically if its mass is equivalent to the mass difference between the two peaks Each editing can be undone
17. epsin without PThls lt Builtln Unknown Enzyme without PTMs I Edit View i Import Export Cancel Figure 13 After creation of a new AAs PTMs set Let s view the configuration of this AAs PTMs set by click View button Selected AA PTMs E xj AAIPTM set Test AA at W Terminus ARNOCEQGHLEMFSTYY AA at C Terminus RE AA at Other Positions ARNDCECGGHLKEMFRS TP Fixed PThis Oxidation Varied PTMs Figure 14 View AAs PTMs Set information To edit one customized AAs PTMs set select it from the list at left and click Edit button The edit procedure is same as creating new AAs PTMs set PTM editor To select PTM editor click the tab PTM in PEAKS Properties editor 21 PEAKS STUDIO QUICK START N PEAKS Properties E x List of PThS Buil In Acetylation Buil ln Amidation Buil lIn Beta methylthiolation Buil iIn Biotin lt Euiltin gt E Mannosylation Builtln Carbarmvylation Euiltin Citrullination Buil lIn Deamidation aoe Figure 15 PIM Editor Should a built in PIM be changed PEAKS will save the change and treat this PIM as a customized PTM it will overwrite the built in PTM the built in PIM will not visible until the customized one is removed Once this customized PTM is removed the built in PTM will appear again To create a new PTM click the new button and input information in the following dialog sii TM Editing EH xi Ma
18. eters m z error tolerance the allowed error in Daltons Residue and PTM Presents options for the type of enzyme used for digestion and any post translational modifications to amino acids The choices can be added removed edited in the Configuration dialog Report top the maximum number of peptide candidates displayed in the de novo results Source ionization source of the instrument 27 PEAKS STUDIO QUICK START Instrument PEAKS will do preprocessing with different parameter for data from different instrument In PEAKS 2 0 there are two set built in preprocessing parameters QTOF and ION Trap If the user s data is not from ION Trap it s suggested to use QTOF as preprocessing parameter Search result in Database When unchecked PEAKS will only provide de novo results for the spectra When checked PEAKS will use auto de novo sequence results for database search Use this configuration as default If this option checked PEAKS will not ask for input auto on auto de novo parameters PEAKS will use the parameters specified in this dialog as the default parameter 28 PEAKS Studio User Interface The user mtenace of PEAKS includes the Data Tree window lef the Main Processing window night and Protein Identification Result Windom night Tool Bar Open data file button Open a data file the file should be in PKL DTA MGF or ANN format Close data file button e Close the selected data file Save data f
19. functionality will remain disabled until one FASTA protein database has been configured for PEAKS Studio Open a Spectrum File To open a demo data file click the icon on the toolbar in the upper left corner of the PEAKS window or select Open from the File menu PEAKS demo data can be found in the DATA sub directory located in the PEAKS directory Each spectrum is represented by its precursor ion information m z value and the charge of the precursor ion that generated the spectrum In the following demo images we select the data file positive pk De gt fl aE 5 MOpe y aduwo pkl y aduwo111d pkl Ey aduwo111a ann Ey dirt ann E aduwo111a merge pkl y dir ttestann al E aduwo111a pki y dir2 pkl y aduwo111b pkl k Ey aduwo111c pkl positive pkl _open_ _corcal Figure 1 Select data file 10 PEAKS STUDIO QUICK START Figure 2 Open data file Auto De Novo Sequencing In the Peptide Data panel on the left select the file positive pkl To begin auto de novo a J sequence derivation click the Automatic De Novo toolbar icon 1 An Options window will appear that provides the opportunity to enter the relevant information about error tolerance enzyme digestion and modification information and the number of sequences to report The database used for protein ID is also specified in this window 11 PEAKS STUDIO QUICK START Figure 3 Auto De Novo Option After c
20. hoosing the appropriate instrument click OK button The demo file name positive pkl will appear in the Task Queue panel located at the bottom left corner of the screen The status panel will show which spectrum 1s being processed by PEAKS When PEAKS finishes the auto de novo process the file name will disappear from the Task Queue The round icon beside each spectrum will change from dark to light green when PEAKS completes de novo spectrum derivation User can choose from either one spectrum multiple spectra or multiple data files for auto de novo 1019 681 1050 98 2 sitive p Figure 4 Auto De Novo 12 PEAKS STUDIO QUICK START sale Figure 5 Finish of Auto De Novo Inspecting the Spectrum Select the spectrum 437 48 2 from the Peptide Data panel Then looking at the Main Process window on the right you will see the results of PEAKS auto de novo The upper left panel lists the Peptide Candidates and their possible peptide sequences with the confidence scores The upper right Ion Table panel provides information for the selected peptide sequence in the Peptide Candidate window The Spectrum View middle provides information about the spectrum and the Alignment View bottom provides alignment images of the peptide 13 PEAKS STUDIO QUICK START PEAKS Studio a 18 x File Edit Tools Windows Help aaa Reue HSG Ma ia lle alee Peptide Candidates PEAKS Auto De Novo 0
21. ication from FASTA format databases Before using the protein identification functionality in PEAKS the appropriate database s must be acquired and configured for use in PEAKS This process involves navigating to the location of the database file and specification of a database a name As this name will be saved in a result file it is recommended organizations standardize database names to ensure continuity File Formats PEAKS accepts four kinds of data formats for reading spectra Micromass PKL Sequest DTA Mascot Generic Format MGF and BSP s XML based ANN file The PKL format supports multiple MS MS spectrum datasets in a single file The first line of a PKL dataset contains the observed m z intensity and charge state of the precursor peptide as a triplet of space separated values Subsequent lines contain space separated pairs of fragment ion m z and intensity values Multiple MS MS spectrum datasets are delimited by at least one blank line The DTA format is very simple It only supports one MS MS spectrum dataset in a file The first line contains the singly protonated peptide mass MH and the peptide charge state as a pair of space separated values Subsequent lines contain space separated pairs of fragment ion m z and intensity values PEAKS BASICS Mascot Generic Format is described on Matrix Science s website It supports multiple spectra per file The ANN format is an annotated data file in XML format It supports bo
22. idate will appear in the Manual De Novo section of the Peptide Candidates window e El lale HE Peptide Candidates Fl PEAKS Auto De Movo 0 1 Trypsin without PIMs ASFPLE 1 ASENNE 1 ASENGGE 1 AS TAANE 1 5 Position confidence Table Tag Confidence Color Code 95 40 Figure 30 Peptide Candidate Panel lon Table Panel Select a peptide candidate from the Peptide Candidates window to view relevant fragment ion information in the Ion Table window If one cell in the Ion Table window is selected the corresponding Peak is highlighted in the Spectrum View panel 35 PEAKS STUDIO USER INTERFACE Peplide Candidates PEAKS Auto De Mowe 0 1 Tr e a limmon Seq yH20 NH H i Sin without PTs 13907 110 07 1 3 1950816709 G 11223 7611224 78 HTGWLTALGGLLK lt 1 32061426016 T M6876116777 HOTYWLTAGLGLLK 1 A 396 20 367 22 Be eal HOTVWWLTSPOGLLE lt 1 5 494 28 466 28 v 966 63 967 63 HGTVVLTALGAWLE lt 1 6 607 36 579 35 L__ 867 99 868 55 F T g 708 41 690 42 754 47 755 48 a 779 45 781 46 A__ 653 43 654 41 g 892 53 964 53 L 582 98 583 39 10 949 56 921 54 469 30 470 29 11 1006 56 97856 443 28 413 27 12 1119 68 1091 85 LE 395 21 356 20 1311232 774 204 754 C 124218 24318 14 129 10 130 08 oe A AA LP E 5 ea na E a a Fyi a X i Sn nn x ee af i TA 20 A E al
23. ile button El Save any changes made to the file a will appear next to any file that has been changed The file will be saved in the ANN format Save all files button 8 Save all files Any changes to files will be saved in the ANN format Automatic De novo button a perform auto de novo for a selected file spectrum or list of spectra If the Search results in database option is checked PEAKS will also perform a database search following derivation of de novo sequences Environment Preference Configuration button Ea configure the environment color de novo parameters and residue list for peaks Peaks Properties Configuration button configure the PEAKS properties parameter PEAKS STUDIO USER INTERFACE Import Database Wizard button E database download and configuration help Load Directory The protein identification process is file based which means PEAKS Studio will use all spectra from one data file to search a protein database As we know the DTA file and some other MS data files only support one spectrum in each data file To support protein identification for spectra from multiple data files PEAKS Studio 2 0 provides a tool to load all MS data files from one directory To start this process go to File Load Directory A PEAKS Studio File Edit Tools Windows Help aon o a ELS Load Directory Load data files from directory After choosing the appropriate directo
24. j x File Edit Tools Windows Help sicie miae le EL5 Ea aaa is Joe aja Peptide Candidates El PEAKS Auto De Novo 0 1 Trypsin without PTMs b 260 10 RCYHMFHEK 11 4 CYRHMFHEK 6 9 YCRHMFHEK 6 9 EHRHMFHEK 6 560 30 691 30 olo alo ma wn e n w elan 0 co l Color Code 95 90 95 80 90 lt 80 Y 4 430 129 1 PP y1 H20 R 391 3 271 79 Y 420 4 147 1 i sal 4 F ll L SI li akoa a d k Ilodi cia I i i 1 i 1 I I i i i I I I Eek La k Figure 29 Main Processing Window 34 PEAKS STUDIO USER INTERFACE Peptide Candidates panel There are 3 types of peptide candidates displayed in this window Manual De Novo created by manual sequencing Peaks Auto De Novo created by Auto De Novo User defined type for user defined peptide candidates For Auto De Novo all peptide candidates for a spectrum are listed with the sequence followed by the Global Confidence score To view Positional Confidence Scores for portions of the selected sequence place the cursor on a candidate string and the Positional Confidence Table will display detailed confidence information Auto De Novo results cannot be edited however an Auto De Novo sequence can be copied for editing purposes Right click on a candidate sequence to trigger a pop up menu then select Copy to Manual De Novo The editable sequence cand
25. me TestPTM Abbreviation TestPTM Mass Monoisotopicy 0 984 Neutral Loss Mass Monoisatopicy l0 0 Formula Residues that can be modified Mon location specific N terminus C terminus ARNDCEQGHILKM Rule os cancer Figure 16 Create new PIM User may specify the modification either by monoisotopic mass difference or by entering its empirical formula 22 PEAKS STUDIO QUICK START a PEARS Properties ListofPTMs Buil lIn Acetylation Buil ln Amidation Buil ln Beta methylthiolation lt EuiltIn Biotin Buil ln C Mannosylation lt Euiltin gt Carbamylation Euiltin Citrullination EE ean Figure 17 After creation of a new PIM Database Manager As there is no FASTA database shipped with PEAKS Software a protein database must be downloaded from the Internet This database must then be configured via the Database manager To configure a new database click New button ni PEAKS Properties Xx List of databases Add a new database Edit Sot ae defaut OK Cancel Figure 18 Create a new database PEAKS will ask for the location of the database file 23 PEAKS STUDIO QUICK START E xi Lookin Cluatapase O cal At cca BBY B Gor galt Select File Marne nr Files of Type All Files 4 eee Figure 19 Locate database file A database name is also required for example NR The database NR will then a
26. me Accuracy PEAKS correctly derives more peptide sequences from tandem MS MS data from any mass spectrometer than any other software currently available e Post Translational Modification Detection PEAKS automatically detects user defined post translational modifications INTRODUCTION e Full Automation PEAKS accepts a peak list in Micromass PKL Sequest DTA formats or Mascot Generic Format MGF and derives the peptide without the need of human interaction e Convenient Output Exports to both FASTA and XML formats e Superior Efficiency and Speed PEAKS typically derives one sequence in under five seconds on a standard PC e No Database Required for deriving the amino acids sequence PEAKS efficiently searches all possible combinations of amino acids using a global optimization algorithm No protem DNA database is required for deriving the amino acids sequence from MS MS experimental data e Great Error Tolerance PEAKS automatically estimates the noise level and the accuracy of the instrument that produced the data adjusts the calibration errors caused by temperature changes and accounts for most types of ions into the computation e Instrument Optimization PEAKS is optimized for TOF TOF and Q TOF instruments e Platform Independent PEAKS runs in any Java 1 3 1 environment with reasonable hardware and memory requirements Pentium 500 or better or any architecture where Java 1 3 1 runtime environment is supporte
27. me functionality The difference is PEAKS works and Pepseq does not even on Micromass own data A poster in ASMS this year by Terry Cyr has vividly described this he used 5 spectra from micromass machines Pepseq was able to identify just 1 sepctra the shortest 5 amino acids and badly failed on all other 4 PEAKS completely identified 3 peptides fully every single a a is correct and partially identified the other two Other facts Caprion Proteomics has partnered with Micromass and bought 10 20 of their high end machines But they are using PEAKS to do de novo not Pepseq It does not work Other facts PEAKS does PTM Pepseq hardly Other facts PEAKS works on other machines like MDS Sciex QStar too 45
28. ng from the pop up menu in peptide candidate panel All operations occur in the Spectrum View panel of Main processing window When the mouse is placed in the Spectrum View panel a blue by default bar follows the movement of the mouse This is the Position Bar and it is used as a cursor to indicate the current peak in the spectrum The information m z of the peak will be shown on the top of the Position Bar To select a peak click on it An orange by default bar called Freeze Bar indicating the selected peak in the spectrum 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 between the selected peak orange and the highlighted peak blue 374 13 277 44 bi 65 11 51 25 HE a WHS 3 ay 29411941 2154 19 47025 geas fa Ey 22 zo A ANHA bero are ea Basic operations MANUAL DE NOVO SEQUENCING Select a peak click on a peak to locate it with freeze bar an orange line by default Deselect a peak double click anywhere in the Spectrum View panel View the m z difference between two peaks select a peak orange line by default with the freeze bar and move the mouse to the left or right The number above the position bar is the difference between the two peaks Zoom in part of the spectrum in the Spectrum View panel drag between two peaks to be zoomed Or you can highlight the corresponding
29. nto two categories Built in PTM PEAKS integrates a number of typical PTMs as built in PIMs These built in PTMs cannot be deleted but they can be edited Once changed PEAKS no longer consider them as built in PIM However deleting this edited PIM will result in the restoration of the original built in PTM Customized PTM You can add a new custom post translational modification by specifying the modification either by monoisotopic mass difference or by entering its empirical formula Note that some PTMs such as phosphorylation can induce specific neutral losses e g beta elimination Accordingly it be necessary to simulate the possibility of these neutral losses through two separate PIMs e g one with beta elimination one without PEAKS BASICS Amino Acids AAs PTMs Set An AAs PTMs set contains enzyme and modification information When considering PTMs PEAKS requires this information for both auto de novo search and protein identification Both PEAKS Studio and PEAKS Batch provide tools with a graphical user interface to create custom AAs PTMs set PEAKS provides two options when setting up a PIM profile Fixed PTM PEAKS considers a Fixed PTM to be a modification present universally to every instance of the specified residue s or terminus Variable PTM PEAKS considers a Variable PTM to be a modification that may or may not be present on a specified residue FASTA Database PEAKS 2 0 supports protein identif
30. on panel 32 PEAKS STUDIO USER INTERFACE Index Panel The index panel is the upper part of the protein candidate panel Proteins are listed in descending order based on the score Detailed protein information is displayed in the information panel below Click on the Accession number in the index panel to view the information panel for the selected protein If there are more related proteins More will be labeled after the accession number Click on more another window will appear to show related proteins 2 sP 0 1 Trypsin without PTMs Result from database SP 0 1 Trypsin without PTMs Accession Mass Score P00921 284253 99 49 P009 22 24045 725 99 98 29209 91 63 73 29055 47 63 73 29077 5802 63 73 4rro9 945 63 73 Description CAH BOVIN Carbonic anhydrase ll Carbonate dehydratase Ih A I CAH SHEEP Carbonic anhydrase Il Carbonate dehydratase ID 4 11 eS CAH2_HUMAN Carbonic anhydrase Il Carbonate dehydratase Il CAIN Ca CAH2 MOUSE Carbonic anhydrase II Carbonate dehydratase In CAIN CAH RAT Carbonic anhydrase ll Carbonate dehydratase I 4 11 AP ARATH Floral homeotic protein APETALA POOS 1 Mass 28472 547 Score 99 99 CAH BOVINE Carbonic anhydrase ll Carbonate dehydratase Ih A I Mz Charge Mricalc Start End Score Peptide 490 52 2 360 4 0 507 03 2 995 52 146 509 92 2 399348 18 655 59 634 30 245 1012 58 99352 148 Information Panel Click on the accession number in Info
31. or redone An ion peak can be selected by clicking on the corresponding value in the lon Table panel 40 How to start Auto De Novo Sequencing and Protein Identification There are 3 ways to perform auto de novo sequencing and protein identification of selected spectra files Choose PEAKS De Novo from the Tools menu click on the Automatic De Novo toolbar icon or right click on the spectrum file then select Peaks Auto De Novo from the popup menu Protein identification is file based operation To perform auto de novo sequencing for more than one spectrum go to the Peptide Data window Either select the data file containing multiple spectra ie the pkl file or select multiple spectra by clicking on the precursor ion information while pressing the Ctrl or Shift key Then use one of the methods described above to begin the de novo sequencing process For a typical spectrum auto de novo sequencing requires about 5 8 seconds to complete As PEAKS derives auto de novo sequences in the background it is possible to continue working with PEAKS while auto de novo is running The Task Queue panel in the lower left corner shows any spectra that are still in the sequence queue Once each spectrum is sequenced the icon for that spectrum in the Peptide Data window changes from dark green Bi light green in the Peptide Data panel Protein identification operation will be executed after auto de novo sequencing all spectra Sele
32. ported into the PKL file format and FASTA file format PEAKS Properties PEAKS Properties contains the value of a PTM AAs PTMs set and FASTA database configuration This information is essential for PEAKS auto de novo and PEAKS protein identification PEAKS Environment Preference Options for default Data Input Output and Configuration file directories can be changed to increase efficiency In the current version of PEAKS the Default Configuration File Directory cannot be changed PEAKS BASICS User can also customize the options for color of spectra viewing auto de novo and manual de novo in PEAKS Environment Preference Configuration File PEAKS software requires a configuration file to store the value of PEAKS Properties and PEAKS Environment Preference for Studio The configuration file is in XML format For PEAKS Studio the configuration file will be saved in the configuration file directory User can check the PEAKS Environment Preference to know the location of the configuration file directory The filename of the configuration file is peaksconf xml For PEAKS Batch user can use the configuration file created by PEAKS Studio or create a customized configuration file with PEAKS Batch or write one with any text editor Logging File PEAKS software saves all runtime information in a logging file The logging file is text format When user reports a bug related to PEAKS the logging file will help BSI technical
33. ppear in the database list i PEAKS Properties List of databases per remo setas default ae ee a eee Figure 20 After creation of NR database in PEAKS If the database file is ever removed from the original directory the database information will be shown in red 24 PEAKS STUDIO QUICK START ni PEAKS Properties List of databases Remove Edit Set as default C aed Figure 21 Cannot find the database file As database contents change over time it is advisable to use the most current version of the database If the current database file is overwritten with a more recent file PEAKS will show the database information in light gray a PEARS Properties Listof databases Edit a aa Figure 22 Different size of database file Any database can be removed with the Database Manager Editing a database means re locate the database file The Set as default button will assign the selected database as the default database for auto de novo search and protein identification There is a in front of the name of default database 25 PEAKS STUDIO QUICK START Import and Export PEAKS Properties To export PEAKS Properties for use in either PEAKS Batch or PEAKS Studio licenses use the import export functionality in the PEAKS Properties Editor The export functionality will save PEAKS Properties information in a XML file and the import functionality can read the file
34. rmation Panel the following protein view dialog will appear to show the sequence of the protein The protein view dialog could be oo 81 4 DGPLTGTYR 157 99 VGDANPALOK 26 44 26 DEFPIANGER 250 63 73 PAQPLK 157 65 16 WGDANPALOK Figure 27 Related proteins viewed and printed in web browser Click on the peptide PEAKS will jump to the Main Process Window of related spectrum 33 PEAKS STUDIO USER INTERFACE Protein iew View with web browser Filename Ccitempidatatorpeaks Olpositive ann P00921 Mass 28472 5377 Score 99 99 CAH _BOVIN Carbonic anhydrase Il Carbonate dehydratase IM CA 1 Sequence Coverage 19 13 MCEI BLAST search of P00921 Links to retrieve entries containing this sequence from MEBI Entrez Accession Glf Database Database P00921 sp CAH BOVIN Carbonic anhydrase ll Carbonate dehydratase IN 4 11 Matched peptides shown in Fed 1 SHAWGYSEHR GPAHWHEDFP ITANGEROSPY NIDTES Y YOD PALEPLALYY 51 GEATSREMYN NGHSFNVEYD DSQODEAFLED GPLTSTYRLDY QCPFAFANSGSSB 101 EQGSERTYDE EEYAAELALDY HNNTEYGDFG TAAGOPIDGLA VW YELEYGD 151 ANPALOGERYVLD ALDDOSIETEGE STDFPNFDPS SLUPN YLDYW TYPGSLTIPP 201 LLESYIWIVL EEPISYSi00 MLEFRTLNFN AEGEPELLML ANWRPAGPLE 251 NROVYRGFPR Peptides List Mz Charge Mr calc Start End Score Peptide 490 52 2 960 47 80 88 81 4 DGPLTGTYR 507 03 2 993 52 148 157 99 VGDANPALGK 509 92 2 99948 18 26 44 26 DFPIANGER Figure 28 Protein View Main Process Window QPEAKS Studio io
35. ry a file chooser will help the user to select a directory to load Note Load directory will only load the first 500 spectra from the source directory not 500 data files And load directory will not load ANN file from the source directory Data Tree There are two parts to the Data Tree window the Peptide Data panel top and the Task Queue panel bottom After a data file a spectrum file in PKL DTA MGF or ANN file format is opened the file name is listed in the Peptide Data panel To expand it double click on the file name or click the round icon before the name Any of the MS MS spectra in the data file will be listed under the file name and have the precursor information m z charge as the name of the spectrum 30 PEAKS STUDIO USER INTERFACE Peaks can perform Auto De Novo on a single spectrum or on a file containing multiple spectra Once a job is submitted to Auto De Novo it is added to the Task Queue for processing Once completed the job is removed from the Task Queue list and the icon before the spectrum changes from Bio Click on the spectrum and its information and images are displayed in the main process window When a filename node is selected in the data tree and the selected file contains protein identification results related information will be displayed in the protein identification result window When a filename node is selected in the data tree right click to view a pop up menu that provides a short cu
36. t for some functions If this filename node is created by loading a directory user can choose sort the spectrum nodes by the source filename or by the precursor m z value of spectrum A PEAKS Studio File Edit Tools Windows Help elejeje ES ORES Fe Peptide Data Close Save Save As Export Peptide Sequence Export PKL File a Peaks Auto De Novo 490 6401 3 L 49064143 490 6423 3 490 6434 3 490 6493 3 492 7936 2 40279732 492 9025 2 495 9773 3 O 4992743 501 82722 501 5352 2 501 8356 2 505 9106 2 505 91182 505 8137 2 507 8429 2 Task Queue E 4 Ei VvorkiDataladuwea oil 461 9775 3 464 2791 2 464 2833 2 A suto De Novo 460 31523 De Movo 460 3152 3 Figure 25 Peptide Tree 31 PEAKS STUDIO USER INTERFACE Protein Identification Result Window The protein identification result window contains the protein candidate tree upper and the protein candidate panel bottom Protein Candidate Tree It is possible to perform protein identification for one data file in multiple databases with different parameters For each combination of database and parameter there 1s one protein candidate in the protein candidate tree Select a candidate by clicking on it in the protein candidate tree Information for this candidate will be displayed in the protein candidate panel In the following figure there are two protein candidates in the pro
37. tein candidate tree One of them is from database Human with error tolerance 0 1 and AAs PTMs set Trypsin without PT M the other is from database SP with same parameter y PEAKS Studio Ta ES File Edit Tools Windows Help sic e a alle e EL Protein Candidates SRE pde without PTMs Result from database SP 0 1 Trypsin without PTMs Accession Mass Score Description More 28472 537 99 99 CAH2_BOVIN Carbonic anhydrase Il Carbonate dehydratase II CAI More 19852258 99 79 LACB_BOVIN Beta lactoglobulin precursor Beta LG Allergen Bos d 5 More 25072256 99 78 CASB_BUBBU Beta casein precursor 25073 072 73 83 VMO2_VACCC Protein M2 3341 055 66 21 ARLY_BIFLO Argininosuccinate lyase Arginosuccinase ASAL P00921 More Mass 28472 537 Score 99 99 CAH2_BOVIN Carbonic anhydrase ll Carbonate dehydratase ll CA 11 Mz Charge Mr calc Start End Score Peptide 490 52 2 960 47 80 88 81 4 DGPLTGTYR 507 03 993 52 148 157 99 VGDANPALOK 509 92 99948 18 26 44 26 DFPIANGER 653 59 634 38 245 250 63 73 PAQPLK 1012 68 993 52 148 157 68 16 WGDANPALOK P02754 More Mass 19852 258 Score 99 79 LACB_BOVIN Beta lactoglobulin precursor Beta LG Allergen Bos d 5 Mz Charge Mr calc Start End Score Peptide 707 43 1 688 36 19 24 20 98 VIQTMK Figure 26 Protein Identification Result Windows Protein Candidate Panel The protein candidate panel is divided into two parts the index panel and informati
38. th a single MS MS spectrum and multiple spectra The ANN file format is consisted of ANN data file and ANN index file One ANN data file contains the MS MS information and peptide information of one spectrum If the user s data file contains only one spectrum the annotated data file will be saved as an ANN data file The ANN data file includes the peak list of the spectrum peptide results and protein id results The ANN index file provides a way to organize multiple spectra as a whole It contains ANN data file information and protein ID results of multiple spectra While the user s annotated data file contains multiple spectra each spectrum will be saved as an ANN data file that contains the peak list and peptide results of this spectrum These ANN data file will be saved in one directory One ANN index file will be created when the annotated data file contains multiple spectra This index file contains the following information the directory name where the corresponding ANN data files located the filename of ANN data files and the protein id results of these spectra Note The directory name saved in ANN index file is just the NAME of the directory not the absolute path of the directory So the ANN index file and the directory MUST be saved in same location This is designed to avoid problems during annotated data files sharing File Save Export Both PEAKS Studio and PEAKS Batch save result information in an ANN file Data can be ex
39. the database to identify the protein Auto De Novo xi miz error tolerance 0 1 T Residue and PTh Trypsin without PTMs T Report top 5 T Source ESI Instrument LIWadrupole TOF e Search results in database INR Use this configuration as default Edit PEARS Properties OK Cancel Figure 7 Protein identification Viewing Protein Identification Results To view the result from protein identification select the file positive pk in the Peptide Data panel on the left All identified proteins will be displayed in the Protein Window on the right The upper portion of the Protein Window lists protein candidates found User can choose one of them and the detailed protein information will be shown in the lower window 15 PEAKS STUDIO QUICK START Fie Eat Tous Windows He elefole axe 8 ES cae Protein Candidates GP AF21 More 28434691 29 84 gt GP AF218000_1 Homo sapiens clone PP222 unknown mRNA MASS 28471 SWeITAY More 115960 45 27 76 SVVITAV_HUMAN P06756 homo sapiens human vitronectin receptor alpha subunit precurso GP U351 9004 647 27 08 GP U35155_1 Human endogenous retrovirus polymerase gene clone HML 3 6 partial cds Si 78669 49 23 94 gt GP AK023921_1 Homo sapiens cDNA FLJ13859 fis clone THYRO1001033 weakly similar to 10812 008 21 59 gt SWWE7_HPV33 P06429 human papillomavirus type 33 e7 protein 6 1994 MASS 10837 sp P026 Mass 25073 242 Score 99
40. use the Residue and PTM configuration feature of PEAKS to define your own enzyme by specifying allowable residues at N and C terminal positions 5 Can I edit modify the result of Peaks Auto De novo You cannot modify the sequences returned by PEAKS Auto De Novo search in place but you can achieve the same goal by copying them to the Manual De Novo side of PEAKS Right click on the desired sequence and select Copy for manual de novo You can now edit the sequence and ion assignments in the Manual De Novo section of the Peptide Candidates tree 6 On occasion I cannot find the Freeze bar orange line by default to indicate the position of the peak in the ion edit window when I select one ion in the info window Why Make sure the peak of the ion is right in the display area of ion edit window 7 Howcan I save the results You can copy the predicted sequences by right clicking on the sequences and select copy For the images you can either print them by clicking the right most Print result image button at the top of the main process window or save them as files by clicking the second right most Export results as image button The printed or saved images look slightly different to those displayed on the screen The default output directory is the directory previously visited You can change it to another directory by configuring the environment 8 What is the difference between save data file and s
41. will be listed at left side If one PTM is a built in PTM there is a label lt built in gt in front of the name of PTM PTM as Fixed or Varied A fixed PTM indicates a modification that occurs in every instance of the specified residue A Varied PTM is a modification that occurs at only some of the specified residue locations To make the selection highlight the desired PTM from the list at left and then click Select As Fixed or Select as Varied Button If one PTM is already selected as a fixed PTM it cannot be selected as varied PTM and verse vise Selections can be removed from the list using the Remove button To select one PTM as Fixed Varied PTM click on the PTM from the list of Selected Fixed PTM or Selected Varied PTM To remove the modification from the list click Unselect button to remove it PEAKS software ships with some pre defined PIMs To create a new PIM other than the built in PTMs click New PIM to create a new one Once change are made to a built in PTM that PIM will be saved as a customized PTM and the label built in will be removed from the list For details of PIM editing please refer to next section Click on Finish button to save the new AAs PTMs set and there is one new AAs PTMs set will be listed in the AAs PTMs sets 20 PEAKS STUDIO QUICK START MII PEAKS Properties NE x AAS PTMs set Database Listofstandard PTMed AA sets Hew lt Euiltin T
42. y eae E cay Pag ul ia aea Laas D E 5 Te j 011 E EA 1 ia i al J I i l Color Code 95 90 95 BO J0 H0 oo 200 00 52000 780 00 1040 00 1200 00 Brera Ed 100 1184 73 yiz 447 44 Taa BBS SP pass 1294 6 4 1 1083 74 1007 y y va vid ie 1240 74 ai x11 Bar 4 GRENEN A ee T 0 200 200 aon ann 4000 1200 1170 70 Figure 31 Ion Table Panel An Error Plot image is also presented below the ion Table If necessary scroll down to view the Error Plot Select a column or cell y ions are selected here to view green dots in the error image These dots represent the location of y ion s in this example in the error image Spectrum View and Alignment View panels The upper panel is Spectrum View panel and the lower the Alignment View panel The Spectrum View panel is used for zooming in the spectrum to inspect it in detail it is also used in manual de novo to edit ions The Alignment panel displays the spectrum in its entirety along with a sequence alionment when a sequence peptide candidate is selected in the Peptide Candidates panel A blue bar along the horizontal axis of the alignment view indicates the range of the spectrum view in the Ion Edit panel Spectrum Image panel 36 PEAKS STUDIO USER INTERFACE ohs 2F b4 100 27718 des bz 4 20 Te 40521 wal ana T a ea ri a y a Hz0 i 940 6 353 7 a T 036 37 mae 7394 gisi ea doga a yeHeo v2 a ako BENKI vrao A
Download Pdf Manuals
Related Search