Home

Untitled - GeneBio

Contents

1. eere 3 Navigation Dar eee Roe DUE ethos 3 JOD report z si o cu EE ERRARE RENE RES 3 Job menu Submission page eese B Database s Search engine Acceptance parameters Peak lists sss Server log Management Console page 22 My COPS ic ires EH DRE E ERIS PO ERE SES Define a new enzyme eise nennen nennen nnne Edit a user defined enzyme ssssssee Delete a user defined enzyme Define a new modification cecce Edit a user defined modification sess Delete a user defined modification Group defs sse RERUM AINE Account Settings Databanks 2 iiie nouit nin epa AE Jobs management neh 32 Browse files or export job results usnnnseeeesennnerneene nennen 32 Import Phenyx jobs or jobs from other search engines 34 Utilities cn SAME AR eae RR bout eteedi 35 Phenyx Web Interface Manual iii contents Status page ness eene DO Parameters page eene nnns DO Proteins Overview page ern OD Best scoring protein table Protein details 228er Peptide Match table reset Protein Match Details page s 45 Protein information table Peptide match table erede ec Sequence V EWS ccce Compounds Overview page 50 Functions tre ee espe te E HRS Dee APER SERIE po DOS 53 Vali
2. lt modRes gt Defines the modifications that change standard amino acids lt site gt Defines the position of the amino acids to be affected by the enzyme or modification rule lt sprotFT gt Defines what annotations to look for in the Swiss Prot feature table Phenyx Web Interface Manual 31 management console 32 Group defs The first Group Definitions menu will refer to the default enzymes and amino acid modifications that are automatically displayed to all users You may see additional Group Definitions menus that refer to the enzymes and amino acid modifications that are only displayed to users in a local group These views are available to all registered users Please contact GeneBio at support phenyx ms com with questions or comments about any predefined settings Account Settings Change Password This tool allows you to change your Phenyx password Enter the new password in the first box Confirm the new password by retyping it in the second box Click the Change button Databanks Private Databank This tool enables the user to add and refine new sequence entries that are FASTA formatted Copy and paste the sequence s in FASTA format into the text box Click on the Load button In order for the user s databank called privatedb_username to appear in the list of Database s on the Submission page log out and re log in to the Phenyx Web Interface Only licensed users of Phenyx are able to create thei
3. Description of the ion fragment with potential neutral losses is given with the corresponding delta mass Number in brakets refers to the position of bond cleavage in the ordered peptide sequence The color code for intensity rank is applied Description of the precursor is given with potential neutral losses regarding its charges Color code for intensity rank is applied to report the delta mass Immonium ion is labelled with the one letter code for the corresponding amino acid Color code for intensity rank is applied to report the delta mass Phenyx Web Interface Manual results comparison Results Comparison page Updated May 10 2008 This page allows the user to view one or more job results in tabular format The table is organized according to identified proteins It is easy to do a side by side comparison when multiple jobs are selected Output from other protein identification software such as SEQUEST Bioworks Mascot or X Tandem can also be opened in the table Insert jobs in comparison table There are two ways to create a comparison The most straightforward possibility is to use the Compare option on the Phenyx Desktop Select the desired job s by checking their boxes in the Job Report Choose Compare from the Actions on Selected drop down menu The selected job s are inserted in a table on the Results Comparison page In addition their ID numbers and title appear in the Jobs List Add Following Jobs Alternatively
4. Excel Word and Internet Explorer are trademarks of Microsoft Corporation Mozilla Firefox is a trademark of the Mozilla Foundation TRAQ is a trademark of Applera Corporation Rather than put a trademark symbol in every occurrence of trademarked names we state that we are using the names only in an editorial fashion and to the benefit of the trademark owner with no intention of infringement of the trademark Terms and conditions All goods and services are sold subject to the terms and conditions of sale of the company that supplies them A copy of these terms and conditions is available on request Limitation of liability The information in this User Manual is subject to change without notice and should not be construed as a commitment by GeneBio GeneBio is not responsible for the misinterpretation of results obtained by following the instructions in this manual Technical support GeneBio provides technical support for Phenyx in accordance with the terms of the License Agreement Please contact us at support phenyx ms com Databases Phenyx provides access to several databases on the Internet It is the responsibility of the user to acquire the database licenses if needed Phenyx Web Interface Manual contents Contents Introd cti n ar vessu cur aa iaa i a L Requirements ds teda ee ne 1 General workflow ie ei 2 Helpful tips sore A ti E aedes 2 Phenyx Desktop page
5. Phenyx Web Interface Manual submission Search engine Instrument Type Specify the type of mass spectrometer that was used to collect the data The algorithm that generates the score between experimental and theoretical masses is based on several instrumental properties such as the types and charge states of the fragment ions produced If the exact instrument that was used to collect the MS MS data is not listed then opt for a similar method Scoring Model Determine the detailed scoring scheme to be applied to resolve your data This parameter is dependent upon the instrument type Note that the mass tolerance for the fragment ions is included in the algorithm and cannot be set separately Note information into the Scoring Model top down menu refers to the fragmentation process Where the parent mass is detected is not considered For instance the scoring model CID_LTQ_scan_Orbitrap_6ppm refers to data that have been fragmented in LTQ and detected in Orbitrap with a fragment error tolerance as up as 6ppm Default Parent Charge The charge of the protonated M nH or deprotonated M nH parent ion as detected in the formatted peak list file by Phenyx The peak picking software that comes with your instrument assigns charges to the parent and fragment ions based on the observed mass to charge ratios These values are generally included in the peak lists If two or more integers are separated by commas then all charg
6. If a job is currently running then a progress bar at the bottom of the page indicates the two main cpu time consuming activities i e the DatabaseN_spectra_rnd and the DatabaseN_DB_blocks processes If ajob completes normally then you are automatically redirected to the Proteins Overview page The comments explaining the status of a given job number are described below e Reading source data The job is submitted e Writing idj The submission parameters and peak lists are transformed into a Phenyx XML file e Compressing input file Data is encoded to take up less storage space and less bandwidth for transmission e Submit to Database N The specified database is queried e DatabaseN spectra rnd Status of the random search of the MS MS spectra expressed as a percentage e DatabaseN_DB_blocks Status of the peptide matching procedure expressed as a percentage e Apply post processing command The results are transformed into a Phenyx XML file e Completed The job finished successfully e Error The job failed to process e Pending The job is in the queue awaiting processing Phenyx Web Interface Manual 36 status If an error occurs you can find out why a job failed and perhaps attempt to fix the problem yourself by clicking on the Comment link to go to the Troubleshooting page GeneBio s technical support is of course at your disposal should you have question or comment support phenyx ms com Once a jo
7. e If working with saved Results data then click on the Browse button to locate the Local File or archive on your hard disk Otherwise copy and paste the Remote URL of in the Add URL box If the data are only accessible using a login and password then type http login passwordOresult_url instead of a basic http result_url e Click on the Import button to convert the file into a job with a new identification ID number e Go to the Phenyx Desktop to start working with the job In the Title column of the Job Report an information about the origin of the imported job is given for example the original ID of an imported Phenyx job is referenced in brackets In case of an imported Mascot job the Title column displays imported from mascot and file title In the Comment column of the Job Report information about the imported file or URL is given Phenyx Web Interface Manual management console Utilities Convert MS MS Peak Lists This tool allows to transform one type of MS MS peak list into a different file format You can also filter the original peak list Click on the question mark links on the on screen form for further explanations Please contact GeneBio at support phenyx ms com with questions or comments about any specific needs Phenyx Web Interface Manual 35 status Status page Updated May 10 2008 The Status page or user s Desktop allows you to monitor the state of running pending or failed jobs
8. www genome jp kegg Enzyme Nomenclature Recommendations from the International Union of Biochemistry and Molecular Biology IUBMB found at http www chem qmul ac uk iubmb enzyme BioCarta Charting Pathways of Life found at http www biocarta com Define a new enzyme 1 24 Starting from the Phenyx Desktop click on the Management Console link The Management Console page opens in a new window Click on the Cleavages Enzymes link under the My Defs menu A form appears in the right hand pane Click on the New button Phenyx Web Interface Manual management console 4 Fill in the Name field e g type Chymotrypsin Management console testing user User environment Cleavage enzymes setup for user testing My defs testing my trypsin Name Cleavages enzymes Residue modifications XML user s def Cleavage site XML total def Group default defs Site O cleaw at adjacent Cleavages enzymes Residue modifications terminus XMLuser s def e 3 N Regular expression O Jobs Management Import archived job s Gain formula Utilities N term H Convert ms ms peak lists alle OH Job summary y C term mtu MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEF AKTCVADES AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEP ERNECFLOHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF
9. how far and in what direction the score deviates from the distribution s mean p Value The probability of a peptide match in a database occurring by chance with this score or better The lower the p Value the more significant the match Position The numerical start and end of the peptide in the main protein sequence MC The number of missed cleavages the allowed number of sites targeted amino acids per peptide that were not cut This is an error allowance for enzyme inefficiency partial cleavage e 0 Means that all sites were cleaved as theoretically expected Lessens the likelihood of random matches e 1 All combinations are computed for one uncleaved site including the case when zero missed cleavages occurred e 2 All combinations are computed for two uncleaved sites including the cases when zero and one missed cleavages occurred e 3 All combinations are computed for three uncleaved sites including the cases when zero one and two missed cleavages occurred e 4 All combinations are computed for four uncleaved sites including the cases when zero one two and three missed cleavages occurred e Half Enzyme digestion occurred at just one end specifically and the other end nonspecifically either the C terminus or the N terminus Modif Modification s any amino acid modifications undergone are cataloged The colon is a counter that surrounds each amino acid thus differentiaing the N and C termi
10. m User Manual Phenyx Web Interface PHENYX www phenyx ms com All intellectual property rights on this User Manual as well as on the Phenyx software belong to Geneva Bioinformatics GeneBio S A No part of this User Manual may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Geneva Bioinformatics GeneBio S A 2005 Geneva Bioinformatics GeneBio S A Avenue de Champel 25 CH 1206 Geneva Switzerland All rights reserved Phenyx uses Perl software 1989 1991 Free Software Foundation Inc 59 Temple Place Suite 330 Boston MA 02111 U S A Phenyx uses OLAV algorithms 2003 Geneva Bioinformatics GeneBio S A Avenue de Champel 25 CH 1206 Geneva Switzerland Phenyx uses Mascot result files 2003 Matrix Science Ltd 8 Wyndham Place London W1H 1PP United Kingdom Phenyx uses SEQUEST result files 2005 Thermo Electron Corporation 81 Wyman Street Waltham MA 02454 9046 U S A Phenyx uses X Tandem results files 2004 The Global Proteome Machine Organization Phenyx uses X Hunter results files 2005 The Global Proteome Machine Organization Phenyx Web Interface Manual Trademarks SEQUEST is a registered trademark of the University of Washington Phenyx and GeneBio are trademarks of Geneva Bioinformatics S A
11. Clears the Jobs List and table in order to start again Phenyx Web Interface Manual results comparison Results comparison table Identified proteins for the different jobs are compared in the table By default the results comparison table is sorted according to Group smallest to largest number This sort order can be reversed largest to smallest number by clicking on the header of the Group column Otherwise sort the rows in ascending order A to Z O to 9 or descending order Z to A 9 to 0 by clicking or re clicking on the header of any column Protein AC Database accession number By clicking on the AC link the Detailed Results Comparison page opens The suffix _WOSIGO denotes the protein sequence without the Signal sequence The suffix _WOPPO denotes the Peptide 1 in SwissProt The suffixes _CHAINO or _VAR1 denote Chain 1 from the original protein entry or Variant 2 from the original protein entry respectively To change the information displayed in the Protein AC column go to the Selected Proteins menu on the left side of the Results Comparison page Select one of the following choices and click the Update button Deselect an item and click the Update button to remove it from the table e All Every protein identified in each job is listed in the table e Main Proteins Only the proteins with the best scores for each job are displayed Main protein of the Proteins Overview page e Proteins in all Jobs Only the pr
12. a C terminal cleavage to occur A caret is used to indicate an absence It effects all the amino acids that follow with no commas between listed amino acids PE means proline or glutamic acid must not follow the targeted amino acid in order for a C terminal cleavage to occur Terminus Determines if the cleavage occurs on the C or N terminal side of the targeted amino acid Regular Expression Uses a a set of symbols to describe the cleavage rule For example lt RK P means to cleave on the C terminal side of R and K except if P occurs C terminal to arginine or lysine Phenyx Web Interface Manual 23 management console Gain Formula Gives the chemical formula to be added to the N terminus and C terminus of the peptide fragments Note that cleavage site is defined as cutting the C N bond of the peptide bond between two amino acids in a protein sequence Any registered user can define their own enzyme rules belonging exclusively to the individual Several online resources can assist you in creating these rules ENZYME Available through the Swiss Institute of Bioinformatics SIB at http www expasy org enzyme Biochemical Pathways Boehringer Mannheim s wall charts found interactively at http www expasy org tools pathways BRENDA Available through the Cologne University Bioinformatics Center at http www brenda enzymes info KEGG Kyoto Encyclopedia of Genes and Genomes found at http
13. siii ee teorema nennen 84 Display AA mods rn ann Hann man ta P e RES 66 Display following ACH ee le ren ero ran een eee 53 Display OPTIONS 2 aus as dc 65 Documentation uressesnersennnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nun een nn 3 DoNotCleave enzyme svete re cri 16 DOW I ais A A OBI een 4 E Empty lISE 42 9 ii lance entamhnusteeecalsaegtacnnededathbaatnuapacton De gran 60 66 ENZYME a EE 14 Experiment l ch rge Nasen sata sauna een anne anne 55 Experimental m Z ese ns nen ne nes 55 EXPO an Een IA CUI ERE EAR REM TEE EAM 63 66 EXDOFU EXG6Gl 2222 ea 1 dat IRURE ann ra RIA MUERE 6 2d E 33 Export last saved validated peptides cococcnccconnnnnnnnonononananraronnnnnns 53 EXPOMmtileXt HPLC sa nadas 6 Export dul a lA A REDE aa 6 F False positive Tea Da 33 FASTA TO Materna srta DEA dad a PLE dae ne 8 32 File O Mata E de 19 Fixed MoOdifiCatiONS iia nicolas dedicas 13 82 Fragment ion table ici nn nn an D Ir an an nennen 56 I DD a a e TS Immoniumr IoFiS vs ia an a Tran XR RECEN anat A 58 Importe PI Insert jobs in a results comparison table denia Instr ment Pei A Sia Renier lees Ion Series Selec econ n e dei Pi eese oes a a ii Phenyx Web Interface Manual 87 index 88 J JOD I Br as as 65 Job investigator un er ra en See cd dak cena a RER 35 JOD MEN Wis ee ee ee ee ee ee bed 6 JOD a010 p Eee CELERE MM LAM ERES 3 Jobs liSE ins nn ates en AR AA
14. which in turn has favorable repercussions on the protein coverage and score if the peptide matches are valid However by going from normal to half cleaved the number of theoretical peptides generated can increase by a factor of ten The increased search space size causes the p Values to deteriorate i e become larger The combined use of cleavage mode and taxonomy can counterbalance these effects For example a restricted taxonomy selection will limit the search space size and ameliorate the p Values while searching in half cleaved mode In Figure 2 the p Value decreases from Job 1 to Job 3 as the number of searched peptides decreases The z Score remains relatively constant when searching many peptides Jobs 1 and 2 but deviates slightly when only searching one protein entry Job 3 This deviation is caused by undersampling during the random process used to calculate the z Phenyx Web Interface Manual 77 understanding Phenyx Scores When searching only one accession number it is recommended to concentrate on the interpretation of the p Values Change the number of missed cleavages 10 uniprot_sprot taxonomy Homo sapiens Es no missed cleavage uniprot_sprot taxonomy Homo sapiens 22 1 missed cleavage uniprot sprot taxonomy Homo sapiens e 3 missed cleavages EU peptide VFDEFKPLVEEPQNLIK La Ad identification through the 3 jobs identified in job identified in job compound 041126AR_203 56
15. whole database s whereas the second round only looks at the proteins with satisfactory scores to have passed the first round By default only the Round 1 box is checked and the related search parameters are visible i e the red arrow is pointing down If you choose to do two rounds of calculations then check the box corresponding to Round 2 The same parameters specified in Round 1 will appear under Round 2 Click on the red arrow to collapse the search parameters i e the red arrow is pointing right Find an example of possible settings for a two round search below To learn more about the reasoning behind these selections go to Understanding Phenyx the effect of submission parameters on result accuracy at the end of this manual Parameter Round 1 Round 2 Fixed modification s Yes Yes Variable modification s None Yes Missed Cleavage s 1 3 Cleavage Mode Normal Half cleaved 12 Phenyx Web Interface Manual submission Parameter Round 1 Round 2 Conflict Resolution None Yes Peptide z Score 6 0 5 0 Peptide p Value 1 0e 6 1 0e 5 Turbo Yes None AA Modif Amino acid modifications select any chemical modifications post translational modifications PTMs cross linking etc from the drop down list The description of every listed amino acid modification is available in the Management Console Click on the Def Definitions link to go to the Management Conso
16. Change the number of missed cleavages Change the number of variable modifications 79 Search in one round or two ssssssse Hee Onesround usage tte Two round Usage Two round search parameters Activate conflict resolution esssses HH How Phenyx resolves conflicts Some points to consider sss v Phenyx Web Interface Manual introduction Introduction Updated May 10 2008 Welcome to Phenyx a software platform for the identification and characterization of proteins from mass spectrometry data This User Manual is dedicated to Phenyx users Depending on the Phenyx solution you have chosen e Phenyx Public server for initial and restricted submissions e PhenyxOnline for a secured environment without hardware IT issues e PhenyxServer for a highly customized platform also in large cluster environments the access to some features may differ It is then noticed in the present Manual Requirements To get started there are the following requirements e Web browser Mozilla Firefox or Internet Explorer 6 0 e Peak list files e Phenyx username and password Phenyx Public server users have to create their login credentials from the Login page at phenyx vital it ch pwi PhenyxOnline customers please contact support phenyx ms com for any login issue PhenyxServer customers please contact your admin to create your login credentials Phenyx Web Inter
17. FAKRYKAAFTECCOAADKAACLLPKLDELRDEGKASSAKORLKCASLOKFGERAFKAUAV ARLSORFPKAEF AEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR y Exe 5 Fill in the Cleav At field e g type FYLW 6 Fill in the Adjacent field if necessary 7 Select the Terminus e g C 8 Fill in the Gain Formula fields e g type H for hydrogen in the N term box and type OH for a hydroxyl group in the C term box Phenyx Web Interface Manual 25 management console 9 Click on the Save button The new enzyme is added to the scrollable list of user defined enzymes on the left hand side of the pane Management console testing user User environment Cleavage enzymes setup for user testing My defs testing mytrypsin Name Chymotrypsin Cleavages enzymes Residue modifications XMLuser s def XML total def Cleavage site Site O clea at Pruw adjacent Cleavages enzymes Residue modifications terminus XMLuser s def ec N Group default defs Jobs Management Regular expression O Import archived job s Gain formula Utilities N term H Convert ms ms peak lists C term OH Job summary Job investigator new Lies TEE delete save MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLOQCPF EDHVKLVNEVTEF AKTCVADESAENCDKSLHTLFGDKLCT
18. _ i to 1525 rt 91 7826 f 4 i 310 O PRESS Sum of sonne in range 1510 5 90 0434 4 5 205 nen to 1526 rt 91 8387 f 5 i 30 A BABA LNDLEDALQQAK blast 2 0 021 10 7 IP100220 t 2 0 039 6 34 IPIOOZ290 E 2 1P100009 NEK st 2 0 0070 713 progre FIGURE 5 On this Compounds Overview page there is one resolved conflict for Compounds 74 The z Scores and p Values for both peptides were good enough to apply the conflict resolution algorithm Phenyx then evaluated the quality of each conflicting interpretation in order to find out which match could be considered as correct In both examples the z Score difference is large enough to resolve the conflict The winner sequence is highlighted in green and has a valid status The loser sequence is highlighted in red and has an invalid status 3 When the scores of conflicting peptides with a priori valid status and scores above the Phenyx thresholds are very similar but the score difference or p Value ratio is below the discrimination threshold Phenyx decides that it cannot unambiguously resolve the conflict The peptides are considered as multiple winners They are labeled as conflict winners and can be subjected to manual validation for a final decision Sum of 10 scans in range 2012 rt 118 362 f 4 by TAATIENAQPILQIDNAR blast d E 87 Be a eran gee eaten ey IIAATIENAQPILQIDNAR 2 0013 7 75 Sum of 10 scans in range 2014 rt 118 474 f 2 i ATIENAOFILOI
19. can be opted for in a given submission file btdx File format defined by Bruker Daltonics dta File format defined by SEQUEST Thermo Electron mgf File format defined by Matrix Science e mzData File format defined by HUPO PSI e mzXML File format defined by the Institute for Systems Biology e pkl File format defined by Waters Corporation Peak List s Browse your computer s hard disk to find the file containing the peak lists to be determined A peak list is a delimited file format that includes the mass to charge and intensity for each peak in a mass spectrum Add one file at a time Phenyx automatically merges the selected files into a single continuous file You can also add several files that were previously compressed in ZIP GZIP or TAR GZ format i e one file with a zip gz tgz or tar gz extension Resubmitted Peak List s If you open the Submission page by using the Resubmit function for a selected job on the Phenyx Desktop the original data appears here and is automatically uploaded unless the corresponding check box is deselected You can also add new data with a different format Submit Click on the Submit button to send the submission file to the Phenyx Calculation Unit for processing A progress bar appears to display the process The user can effectively resubmit the search by successively clicking the Submit button the number of times desired Server log A message in green characters will appea
20. description is used for identification purposes It can contain a Cmpd compound or LC peak number MSn abbreviation to indicate that the spectrum was obtained in MS MS mode the mass to charge ratio assigned to the LC peak in parentheses and the LC retention time in minutes The various peak list formats generate different compound descriptions For example the spectrum title is inserted into this field for a mgf file For both dta and pkl files numbers are assigned to consecutive MS MS spectra and these values appear in the Compound description Sequence The amino acid sequence of the matched peptide is given in the parentheses The one letter amino acid codes are used Modifications Any amino acid modifications undergone are also given in the parentheses The colon is a counter that surrounds each amino acid thus differentiaing the N and C terminuses from the side chains of the starting and ending amino acids For example a sequence of 6 amino acids will be denoted by 7 colons For a peptide MSGECACK the code Oxidation M represents the oxidation of the side chain of methionine Theoretical Mass Calculated mass of the peptide Experimental m z Experimental mass to charge the observed mass to charge ratio of the parent ion Experimental Charge z observed charge state of the parent ion Experimental 4 Peaks Number of peaks the peak count in the mass spectrum Phenyx Web Interface Manual 55 peptide
21. page can be directly accessed by clicking on the ID links in the Jobs List In the job columns of the table the validity plus sign or invalidity minus sign of the match es is given By clicking on the or link the Peptide Match Details page opens Alternatively the table can be arranged by job Go to the Group by menu on the left side of the Detailed Results Comparison page Select Job ID and click the Update button The validity plus sign or invalidity minus sign of the match es is now given in the Protein AC columns Select Protein AC under the Group by menu and click the Update button in order to revert back to the default view To change the information displayed in the table go to the Display Options menu on the left side of the Detailed Results Comparison page Phenyx Web Interface Manual 65 detailed results comparison Select one of the following choices and click the Update button Deselect an item and click the Update button to change the view of the table e Display AA Mods Bold modified amino acids in the Sequence column e z Score The z Score is reported in the job columns By clicking on the z Score link the Peptide Match Details page opens e Best Peptide Match Show only the best match for each peptide All of the AC numbers selected to appear in the table are shown in the Protein AC List found at the left side of the Detailed Results Comparison page To delete a particular protein fro
22. process in the report Pending Click on the Pending link to display only jobs that are in the queue awaiting processing in the report All Click on the All link to display every job submitted to the Phenyx Calculation Unit Actions on Selected Perform operations on certain jobs Select a job by clicking on the box at the beginning of its row If a job is selected then the box is checked the row is highlighted in blue and the Job Menu is opened e Delete Remove the selected jobs from the report e Kill Cancel the processing of the selected jobs e Compare Insert the selected job s in a table on the Results Comparison page e Parameters See a comparison of the submission parameters for the selected jobs e Deselect all Dismiss every selection Refresh Click on the Refresh button to update the report Top Click on the Top button to move to the first i e most recent thirty jobs in the report Up Click on the Up button to move backwards through the report Thirty jobs are displayed at a time Down Click on the Down button to move forwards through the report Thirty jobs are displayed at a time 4 Phenyx Web Interface Manual phenyx desktop ID Job identification number Click on the ID link in order to open the Job Menu User The username of the person currently logged in and any other usernames whose jobs you have been granted permission to look at Status Current status of the job Click on the St
23. the Add Following Jobs feature on the left side of the Results Comparison page allows you to construct a new table or add jobs to an existing table Define a new comparison 1 Starting from the Phenyx Desktop click on the Result Comparison link The Results Comparison page opens in a new window 2 If necessary reset the page by clicking on the Empty list button 3 Enter the known job identification number s in the Add Following Jobs box More than one job number can be entered by using a comma and no space to separate them 4 Click on the Update button to open the results in the table The ID numbers appear checked in the Jobs List In order to insert additional jobs into a table repeat Steps 2 and 3 above Phenyx Web Interface Manual 59 results comparison 60 Jobs List A list of ID numbers and titles for the most recently compared jobs is given on the left side of the Results Comparison page If its box is checked then the job is currently shown in the comparison table A job that was previously deactivated can be reinserted into the table by selecting it box checked and clicking the Update button A green V next to an ID denotes a job that contains manually validated peptide matches By clicking on an ID link the Proteins Overview page opens Note that Mascot SEQUEST Bioworks or X Tandem output can be compared to your Phenyx results The data must first be converted into a Phenyx job using the Import tool on
24. 2 C terminal side of F orLorWorYorA orEorQ Pepsin_ pH_1 3 C terminal side of F or L Proteinase_K C terminal side of A orCorGorMorF or S or Y or W Trypsin KR C terminal side of K or R Phenyx Web Interface Manual 15 submission 16 Name Cleaves where Exceptions Trypsin_ KR_noP C terminal side of K if P is C term to K orR orR Remark Note that the selection of the DoNotCleave enzyme means that there will be no theoretical digestion of the proteins except the Methionine processing Thus this enzyme should be selected if the proteins you are looking for are already present in the database as individual entries Missed Cleavage s Allowed number of sites targeted amino acids per peptide that were not cut This is an error allowance for enzyme inefficiency partial cleavage A complete digest has zero 0 missed cleavages Any selection other than zero will lengthen the computation time As the number of potential peptide matches increases a search is rendered more ambiguous e 0 Means that all sites were cleaved as theoretically expected Lessens the likelihood of random matches e 1 All combinations are computed for one uncleaved site including the case when zero missed cleavages occurred e 2 All combinations are computed for two uncleaved sites including the cases when zero and one missed cleavages occurred e 3 All combinations are comp
25. 51 compounds overview 52 the box to select or deselect the validity A checked box means that the peptide match is considered valid by the user and should be computed in the final scoring of the protein An empty box means that the peptide match is considered invalid by the user and therefore should not be retained in the final scoring Click on the Save button to store your validations Note that when a search is done in several databases Phenyx displays the automatic validity status found in the first database as the User validity status for a given peptide match on the Compounds Overview page Ifthe match is Auto valid in the first database but not Auto valid in the next database due to the influence of the different database sizes on the results the Auto invalid status is reported on the corresponding Proteins Overview and Protein Match Details pages with the User status set as valid Sequence Amino acid sequence of the matched peptide Bolded amino acids were modified Click on the Sequence link to go to the Peptide Match Details page Search By clicking on the BLAST link the SIB Swiss Institute of Bioinformatics BLAST Network Service opens in a new browser window This tool enables you to find similar entries to the selected sequence in ExPASy s protein and nucleotide databases z Charge state of the theoretical peptide match d m z Delta mass to charge the value is the difference between the theoretical m z of a mat
26. 56 2 with 1 missed with no missed cleavage nn identified in job 2 with 3 missed 3 I f pes Lg 0 0001 0 000001 0 00000001 1E 10 1E 12 1E 14 1E 16 1E 18 p Value FIGURE 3 Going from O missed cleavages to 1 missed cleavage multiplies the probability of false positives by about two as the search space grows by a factor of about two In other words as the number of missed cleavages increases the worse the p Value for a given z Score Note that the peptide VFDEFKPLVEEPQNLIK is identified when zero missed cleavages are allowed since the rule for trypsin states that no cleavage occurs if proline P is C terminal to lysine K or arginine R this was fixed by the regular expression P on the Management Console page The higher the number of missed cleavages selected the larger the sample set of theoretical peptides This introduces a more important random component into your search process As seen in Figure 3 the p Value becomes worse for a given z Score The best practice is to use up to one missed cleavage allowing the search to truly reflect enzyme reactivity while working within the theoretical confines of Phenyx The effect of the number of missed cleavages on result accuracy is however less than the effects of the selection of taxonomy or cleavage mode Phenyx Web Interface Manual understanding Phenyx Change the number of variable modifications Three searches were performed in order to produce th
27. 6 244 R ONUTLWTSOI 2 1090 62 0 354 108 1718 23 226 244 K EAAENSLVAYK A blast 2 597413 0391 0 6 010 15 143 153 E blest 2 628498 os 0 4 5613 131 141 INTUS Bst 2 762378 0476 665 4326 08 132 ENS blast 2 498 837 0415 6 82 5 99e 08 87 94 ENS blast 2 488 943 031 678 8 02e 08 87 94 Bast 2 762474 038 674 936 08 A 3 EE 3 3 K GDYHRYLAEFATGNDR K Features a Sequence information Length 255AA Molecular weight 29174Da CRC64 07817CCBD1F75B26 This is a checksum on the sequence 19 20 20 ao so E MDDREDLVYQ AKLAZQALRY DEMUESHEXU AGMDUELTUE ERNLLSUAVE NUICARRASU 20 so E BIISSILUN ENKGGEDKLK MIREVRQHVE TELXLICCDI LDVLDENLIP AANTGESEU am E 150 160 au 100 FIGURE 2 Although not specified on the Submission page the N terminal modification of methionine M is retrieved by Phenyx The Swiss Prot protein entry is inset in order to show the modified residue information found in the database s Feature Table FT The acetylated peptides enclosed in red are found in the Modif column at the bottom of the Proteins Overview page Reproducibility of z Scores and p Values The random peptide population generated during scoring to determine the probability of a random match against a random database is unique for each calculation Consequently a small variation will occur in the results when resubmitting a job using the same parameters and peak lists This variability does not usually exceed ten percent of the z Sc
28. DNAR E 88 eda 66 2084 E120 50 fads 12227 El HAATIENAQPILQIDNAR blast 3 0 016 9 59 Sum of 12 scans in range 2131 rt 124 86 f 3 i 607 Te 5 B9 M 2174 rt2127 294 foe i452 LALDLETATYR blast 2 0 011 9 21 m Sum of 12 scans in range 2131 rt 124 86 f 3 i 607 LALDIEIATYR Moe ot ak Sen to 2174 rt 127 234 f 2 i 452 FIGURE 6 On this Compounds Overview page there are two conflicting sequences for Compound 89 both highlighted as winner sequences The z Scores and p Values for both sequences were good enough to apply the conflict resolution algorithm However the z Score difference is not large enough to resolve the conflict Both sequences are considered as winners and highlighted in green Note that the only difference in these particular sequences is an isobaric leucine isoleucine substitution Some points to consider There are several generalities that you should bear in mind Phenyx Web Interface Manual understanding Phenyx e A protein must have at least one valid match and satisfy the threshold AC Score protein score specified by the user on the Submission page in order to be listed as a result with all of its potential peptide matches e Only valid matches and conflict winners are used to calculate the protein score e The Phenyx threshold values for conflict resolution might be set higher than z Score and p Value thresholds specified by the user on the Submission page Therefore it is possible that a couple
29. E 60 Jobs management zr40ssuennnennonnnnenennnnnannnn nun nun nanann nnn nenne 32 60 K MIS 4 L Hofe roll Mer MEER aa a a Aa raa ae D 3 LOQGCd AS E E E T 3 M MIZ A AAA AAA PREND eaa A Pan TES 42 Maim Protein S eisern anane aeaa Raten anne nee 61 Management Console vaccine 3 Mascot eene 60 Mass spectrum 56 Match Score 56 Max ItenSitY iii ee e las 56 Maximum peptide p Value cere 18 82 Minimum peptide length 2 erret nice 18 Minimum peptide z Score errem arena 18 82 Missed cle vages eroe eise eran teh reme eh nhe anna nennen 16 74 82 Modification definitions cssssssssssssssssseen mmm 13 Modification NAME suere en nn ungen 13 Modifications eredt eor eua ineen a e extr rre sah trn reete e e RE para 43 47 55 MS MS scoring model eere nennen n enn nex nenne nenn 67 Multi ee FEAR lin reve EY Vera A ERES hie ee 51 My COS A FOTO PEE 23 N EIE 23 N vigation Bar ua Me rer ee nee 3 NCBInr 10 Neutral losses 58 Normalized SCOFe o cr ed deg ee 68 Number of missed cleavages omccccncncncnnnonnnnnronononanancncnnnnnnannnns 42 47 Numberiof peaks 2 ato ta Ram SRI a ae 55 Number of peptides obama Se es 40 62 Number of rounds iier roe een ne naa sera nr nt 12 80 Number of valid peptides uursusunnsananenanennnnnennanennanan anne nnn 62 O ONG TOUNG na nn ne iaaa 80 aoi ET EEE A da 62 P Pa
30. LC MS MS peak lists The description is used for identification purposes It can contain a Cmpd compound or LC peak number MSn abbreviation to indicate that the spectrum was obtained in MS MS mode the mass to charge ratio assigned to the LC peak in parentheses and the LC retention time in minutes The various peak list formats generate different compound descriptions For example the spectrum title is inserted into this field for a mgf file For both dta and pkl files numbers are assigned to consecutive MS MS spectra and these values appear in the Compound column In order to read the full description in the Status Bar at the bottom of the Web page move your mouse cursor over the truncated compound If this feature is not activated in your Internet browser then select Status Bar in the View menu Auto A plus sign means that the peptide match is considered valid by Phenyx and computed in the final scoring of the protein Note that valid peptides can also be not associated to a protein if the protein AC Score theshold is not reached A minus sign means that the peptide match did not fulfill the p Value and or the minimal peptide length thresholds or has been rejected after conflict resolution if activated and therefore is not retained in the final scoring User The validity of the peptide match as determined by the user The assignments made automatically by Phenyx appear by default Click on Phenyx Web Interface Manual
31. LC peak number MSn abbreviation to indicate that the spectrum was obtained in MS MS mode the mass to charge ratio assigned to the LC peak in parentheses and the LC retention time in minutes The various peak list formats generate different compound descriptions For example the spectrum title is inserted into this field for a mgf file For both dta and pkl files numbers are assigned to consecutive MS MS spectra and these values appear in the Compound column In order to read the full description in the Status Bar at the bottom of the Web page move your mouse cursor over the truncated compound If this feature is not activated in your Internet browser then select Status Bar in the View menu Click on the Compound link to go to the Compounds Overview page Phenyx Web Interface Manual protein match details Protein Match Details page Updated May 10 2008 ID Database protein identification code Swiss Prot refers to this as the entry name whereas other databases use the accession number AC Database accession number in parentheses The suffix _WOSIGO denotes the protein sequence without the Signal sequence The suffix _WOPPO denotes the Peptide 1 in SwissProt The suffixes _CHAINO or _VAR1 denote Chain 1 from the original protein entry or Variant 2 from the original protein entry respectively Click on the AC link to go to the actual database entry For example a protein identified in UniProt will open a page from the ExPASy
32. Proteomics Server developed by the Swiss Institute of Bioinformatics SIB DB Database description Description The description line from the sequence database that includes the official protein name for example Synonyms Other common names for the protein in parentheses Protein information table This table displays information on the current protein and is updated according to the manual validation Score Effective score the protein score that is recalculated based on user validated peptides It is the sum of the best scores per valid peptide sequences Peptides Peptide numbers the number of valid peptide matches followed by the total number of peptide matches found for the given protein Cov Coverage the percent ratio of all amino acids from valid peptide matches to the total number of amino acids in the protein Phenyx Web Interface Manual 45 protein match details 46 Peptide match table A peptide match is the pairing of an experimental fragmentation spectrum to a theoretical segment of a protein Only peptides that respect the z Score threshold are reported A summary of all matched peptides for the selected protein is given Auto A plus sign means that the peptide match is considered valid by Phenyx and computed in the final scoring of the protein A minus sign means that the peptide match did not fulfill the p Value and or the minimal peptide length thresholds or has been rejected after conflic
33. Search in one round or two Phenyx performs searches in one or two serial steps called rounds A classical one round search is performed on a defined set of proteins i e when a full database or taxonomy or accession number AC is selected Identified peptides that fulfill the z Score and p Value thresholds are accepted and the corresponding protein is validated The basic principle of two rounds is that the first round processes all the proteins in the designated search space and the second round only processes the proteins that passed the first round The first round parameters need to be stringent enough to sufficiently validate protein identification i e a few good peptides The second round parameters enable you to open the search criteria in order to increase the sequence coverage by searching for combinatorial modifications or other special features A two round search therefore identifies proteins according to a first set of parameters and then performs a more exhaustive search on the proteins while saving computation time and reducing the random match rate One round usage A single round is adapted to the following cases 1 You submit a large set of data and your aim is simply to retrieve a list of identified proteins The best strategy is to use a set of search parameters that will minimally validate your data Your goal is not to optimize the sequence coverage but to identify a protein with peptides having at least a certain
34. The start middle and end positions are labeled across the top of the diagram The solid bar indicates the total sequence The peptide matches are listed and highlighted in colored boxes green for valid peptides red for invalid peptides and orange for valid half cleaved peptides By moving the mouse cursor over one of the peptides in the second view the sequence is highlighted in yellow in the first view and details about the peptide match are shown at the bottom of the page Note that sequences up to 2000 characters are entirely displayed in the Protein Match Details view When a longer sequence is matched Phenyx displays and stores the portion of the sequence covered by the valid Phenyx Web Interface Manual protein match details peptides For example if a protein is 5000 amino acids long and the first valid peptide match is at position 1000 and the last one at position 4000 the view will display positions 1000 to 4000 plus some additional amino acids If a peptide is matched with an invalid status at position 500 the displayed sequence will still be the same Phenyx Web Interface Manual 49 compounds overview Compounds Overview page Updated May 10 2008 Identified peptide matches are tabulated according to their corresponding compounds i e spectra The matches are visually labeled in several ways in the Compounds Overview Multiple matches for a given spectrum appear with the letter M in the Multi column The descript
35. VATLRETYGEMADCCAKQEP E ERNECFLOHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLEKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAUAV ARLSORFPKAEF AEVSKLVTDLTEVHTECCHGDLLECADDRADLARYICENODS ISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR y 10 Try out the rule on a model protein by clicking on the Test button The predicted fragments cut by the enzyme are shown at the bottom of the pane 11 In order for the enzyme to appear on the Submission page log out and re log in to the Phenyx Web Interface Edit a user defined enzyme 1 Select an enzyme name in the scrollable list to view its cleavage rule e g Chymotrypsin 2 Make the desired changes e g modify the Cleav At field from FYLW to FYL 3 Click on the Save button 26 Phenyx Web Interface Manual management console 4 Try out the rule on a model protein by clicking on the Test button The predicted fragments cut by the enzyme are shown at the bottom of the pane 5 In order to implement the updated enzyme log out and re log in to the Phenyx Web Interface Delete a user defined enzyme 1 Select an enzyme name in the scrollable list e g Chymotrypsin 2 Click on the Delete button Chymotrypsin is removed from the list of user defined enzymes Management console testing user Jser environment Cleavage enzymes setup for user testing y defs testing y Name cleavages
36. alid 4 When you are finished validating and invalidating peptide matches click on the Save button to store the current selections The effects of user validations are immediate on the Compounds Overview Proteins Overview Protein Match Details and Phenyx Desktop pages If these pages were already open then you will need to click on the Internet browser s Refresh button to notice the changes e Compounds Overview The color coding in the Sequence column will change according to your selections e Proteins Overview The values for the Score Peptides number of valid peptides and Coverage in the Best Scoring Protein Table will be modified In other words selecting or deselecting a peptide match modifies the related protein s The new validity sign plus or minus will appear in the User column of the Peptide Match Table e Protein Match Details The new validity sign plus or minus will appear in the User column of the Peptide Match Table The color coding in the first sequence view will change according to your selections e Phenyx Desktop The Comment column in the Job Report will update with the number of valid peptide matches in parentheses 54 Phenyx Web Interface Manual peptide match details Peptide Match Details page A peptide match is the pairing of an experimental fragmentation spectrum to a theoretical segment of a protein Compound Description This is a label commonly attached to LC MS MS peak lists The
37. ame field to add new people Then specify the level of permissions Read access allows the job to be viewed only Write access allows the job to be viewed and edited Delete access allows the job to be viewed edited and erased by the specified user or network of users Click on the Save button The job is now listed on the Phenyx Desktop of the user s who received access rights to it The account name of the person who originally created the file is shown under the User column in the Job Report Phenyx Web Interface Manual 7 submission Submission page Updated May 10 2008 The Submission page is divided into several modules Profiles This function enables the user to manage different representative sets of data for submission The name appearing in the box is the current profile used in the Submission page To load a different profile select the corresponding name in the drop down list Click on the red arrow to expand the menu options Save As Profile Click on the Save as Profile button to store the current page settings as a new profile Enter the name of the profile in the popup window Click the OK button Set as Default Click on the Set as Default button to store the current page settings as the default profile Delete Profile Click on the Delete Profile button to remove the selected profile Title Descriptive name for the data identification query i e job Database s Parameters used to define the search spa
38. atus flag to go to the Status page If the job has already finished successfully then you link directly to the Proteins Overview of the results e Completed Jobs that finished successfully are green e Running Jobs that are currently processing are blue e Error Jobs that failed to process are red e Pending Jobs that are in the queue awaiting processing are yellow Date Year Month Day of job processing Title The search description specified on the Submission page Comment Encoded description about the processing of a job When a job completes successfully then the message tells you the e Number of proteins identified i e matches not including the proteins that are found in the subset e Number of identified peptides in parentheses e AC numbers followed by the Phenyx score for each protein in parentheses These values are updated upon manual validation The message user selected then appears followed by the number of user validated peptides If a job is Running a comment about the progress is given If a job did not proceed successfully a short error message appears To find out why a job failed and perhaps to attempt to fix the problem yourself click on the Error Text link to go to the Troubleshooting page Phenyx Web Interface Manual 5 phenyx desktop Job menu A list of functions that can be performed on the selected job appears on the left side of the page Click on a job s check box or ID to open th
39. b reaches the point at which the input file is compressed you are able to Resubmit or view the Parameters for that job Simply click on its ID in the Job Report on the Phenyx Desktop in order to open the Job Menu and perform these operations Phenyx Web Interface Manual 37 parameters Parameters page Updated May 10 2008 The Parameters page contains a concise restatement of the main submission specifications including the unique job identification number specified title peak list file names databases accession numbers scoring model modifications thresholds etc You can also use the Parameters option on the Phenyx Desktop in order to see a comparison of submission settings for different jobs Select the desired job s by checking their boxes in the Job Report Choose Parameters from the Actions on Selected drop down menu The selected job s are inserted in a table on the Parameters page Note that currently Parameters are not accessible for imported job from other search engines Phenyx Web Interface Manual 38 proteins overview Proteins Overview page Updated May 10 2008 This first page of the results gives an overview of the identified proteins Parameters Click on the Parameters link to go to a concise restatement of the main submission points including the specified title scoring model modifications thresholds etc Compounds Overview Click on the Compounds Overview link to go to a listing of the mass spect
40. ce are found here Database s Available protein and or nucleotide sequence databases are given in the drop down list Information such as the release number and sequence types amino acid or nucleotide are displayed in brackets Select the sequence database s for the search Use the Shift or Ctrl keys to make multiple selections If you choose a highly annotated database such as UniProt_Swiss Prot many of the Phenyx Web Interface Manual 8 submission annotations are considered during the search For example Phenyx processes the entries and generates separate entities for each splicing variant or searches for post translationally modified amino acids if the corresponding annotations exist Licensed users of Phenyx are able to create their own repository of FASTA sequences with the Private Databank tool in the Management Console In special cases other databases can be added to the public server Please contact GeneBio at support phenyx ms com for further information e UniProt The Universal Protein Resource UniProt is the world s most comprehensive catalog of protein sequence and function data that was created by the consolidation of the Swiss Prot and TrEMBL databases Go to http beta uniprot org to learn more e UniProt_SwissProt named uniprot sprot in the pwi Database developed by the Swiss Institute of Bioinformatics SIB and the European Bioinformatics Institute EBI It is a curated database meaning that the en
41. ched peptide and the observed m z of the parent ion z Score The distribution of calculated scores is compared to that of random peptide sequences in order to find the mean and variance The z Score is then a measure of how far and in what direction the score deviates from the distribution s mean AC List of all proteins that correspond to the compound Click on an AC number to go to the Protein Match Details page Valid peptides can also be not associated to any protein if the protein is not validated that Phenyx Web Interface Manual compounds overview means the minimal AC score value for the protein as defined in the Submission page is not reached Functions Hide User Invalidated Entries Select the Hide User Invalidated Entries box to remove any unchecked peptide matches in the User column from the table Deselect the box to redisplay all peptide matches Display Following AC Specify a list of accession numbers to display in the table More than one AC number can be entered separated by a space Incomplete AC numbers are accepted such as POO no wildcard is necessary Select the Display Following AC box to update the table Deselect the box to redisplay all peptide matches The Hide User Invalidated Entries and Display Following AC filters are used in combination when both boxes are checked Select All Click on the Select All button to check all the boxes in the User column Deselect All Click on the Deselect All bu
42. ction of the match validity e Charge state No plus sign 1 charge 2 charges or 3 charges e Functional group lost NH3 loss of ammonia or H20 loss of water The asterisk indicates that a variable number of molecules is lost dependent upon the amino acids in the peptide For example y H20 refers to the doubly charged y fragments with potential losses of water 1 2 or 3 molecules can be subtracted because only the amino acids S and T can lose water Phenyx Web Interface Manual 57 peptide match details 58 In order to interpret the rest of the table several representations are used e Empty box No experimental m z matches the corresponding theoretical fragment e Gray box Impossible fragment For example it is not possible to have a N terminal fragment VFSN with a loss of NH3 as none of these amino acids have an ammonia group e Colored box Match made between the theoretical and experimental m z The different colors signify the relative intensity of the fragment calculated as a percentage of the most intense peaks in the mass spectrum Color codes show the percentage of most intense peaks in the mass spectrum that derived from that type of fragment 2 Neutral losses immonium ions and precursor masses tables Below the main table additionnal tables may display ion matches specific to e fragments with potential neutral losses e parent ions with potential neutral losses e immonium ions
43. dapt these values to your own validation rules Turbo As a filtering factor this feature should be used in the first round While it rejects the interpretation of spectra with poor Phenyx Web Interface Manual understanding Phenyx matches it accelerates the first round The only drawback is that it may fail to propose interpretations for low quality spectra Activate conflict resolution Conflicts arise when the scoring algorithm can match more than one peptide with acceptable z Score and p Value to a given spectrum The available data m z intensity signal or scoring model is not specific enough to generate a unique peptide identification There are many reasons why a mass spectrum may be represented by multiple peptide sequences originating from different proteins Some of these reasons are e Peptides which interchange isobaric amino acids such as leucine L and isoleucine I or without sufficient mass to charge resolution to distinguish lysine K and glutamine Q e Sequences in which amino acid compositions are identical but arranged in a different order and there is not enough signal present in the spectrum to discriminate them e Peptides with similar m z and scores which derive from diverse sequences They share some of the same peaks This happens for spectra that cover only partial sequences When faced with an unresolved conflict you are the most qualified to make sound judgments based on your experimental experi
44. date peptide matches sssssssse He 54 Peptide Match Details page DD Mass SpectruW incienso ERU RN NER 56 Fragment ion tables oce an 56 Results Comparison page 59 Insert jobs in comparison table sseee 59 Define a new comparison eee 59 Remove jobs from a comparison table eessen 60 Results comparison table usssnnseneennnner nenn nnne rennen een 61 Protein detail list 2 2 ad ai 62 Export Options uns een in deve cos 63 Detailed Results Comparison page 65 Detailed results comparison table 65 EXPO OPtlonS i 5 ces a AA ASA AAE pena ce ERES 66 Understanding Phenyx the scoring system 67 The MS MS scoring model cccceeeeeeeeeee eee eeee nennen nennen 67 Phenyx Web Interface Manual iv contents How a score is computed 68 How a score is normalized ccc 68 How Phenyx retrieves amino acid modifications not specified by thelser si ROI HENRI T edge 69 Reproducibility of z Scores and p Values unsssnssneenennneeneenen nn 71 Avallable Scorings eine EE EIE 73 Understanding Phenyx the effect of submission pa rameters on result accuracy 74 Limit the search space size uusssnnssneennnnneeneennnnneenee nennen nenn Change the taxonomy or AC list Change the cleavage mode
45. e menu Resubmit Opens the Submission page with the parameters set as in the selected job You can however make changes The peak lists that were previously used will automatically be uploaded if you keep the Resubmitted Peak Lists box checked You can also add new peak list s with a format different from that of the resubmitted data Parameters See a concise restatement of the main submission parameters including the specified title taxonomy scoring model modifications thresholds etc Proteins Overview A comprehensive summary of the identification results is presented Compounds Overview All mass spectra that were matched during the search are listed with their corresponding peptide identifications and protein affiliations Export Excel Exports the results of the job as an Excel xls spreadsheet in either the Microsoft application or a new browser window Use the Save As option to store a copy ofthe data to your hard disk Export XML Exports the Phenyx result file in Extensible Markup Language format to a new browser window Use the Save As option to store a copy of the data to your hard disk Export Text Opens a new browser window with the Identification jobs file browser tool for compiling specified Phenyx results into a text format These text formatted reports take into account any manual validation done by the user Select a job number and template from the drop down lists Click on the Report button The tab delimi
46. e 2 3 Trust Parent Charge Medium Number of Rounds 1 Enzyme Trypsin_ KR_noP Missed Cleavage s 1 Turbo Tolerance 800 ppm Coverage 20 Series b b y y Conflict Resolution Yes Parent Error Tolerance 2 5 Da Min Peptide Length 6 Min Peptide z Score 5 0 Max Peptide p Value 1 0E 6 AC Score 6 0 Note that the scale of the z Score versus p Value plots are not equal in each of the examples The various parameters do not have the same impact on the curve fitting Phenyx Web Interface Manual 75 understanding Phenyx Change the taxonomy or AC list effects of taxonomy selection AS ot tv et ge uniprot_sprot Taxonomy root lg AA uniprot sprot Taxonomy Homo sapiens b er ES ee Fei 3 y v Le ey E Les a N r 0 0001 0 000001 0 0000000 1E 10 1E 12 1E 14 1E 16 1E 18 1 p Value FIGURE 1 This graph shows the effect of taxonomy on pairs of z Scores and p Values The Root selection means that the full Swiss Prot database was searched Selecting Homo sapiens reduced the size of the database by a factor of about 13 The curve slowly moves to the right part of the graph meaning the smaller the size of the search space the lower the p Value for a given z Score Figure 1 demonstrates how reducing the search space size by a taxonomy restriction can generate better p Values In other words a match is of higher quali
47. e next graph In addition to the settings described above the following parameters were tweaked effects of number of selected modifications x n LIA 4 search on uniprot sprot databas Los ap with 3 modifications M m search on uniprot sprot databas ya with 2 modifications ad search on uniprot_sprot databas without modification z Score ms T T T T T 0 0001 0 000001 0 00000001 1E 10 1E 12 1E 14 1E 16 1E 18 p Value Parameter Job 1 Job 2 Job 3 Variable Oxidation_M Oxidation_M None Modifications PHOS Cys_CAM Cys_CAM FIGURE 4 The number of variable modifications increases the set of sampled peptides thus increasing the size of the search space As the number of theoretically modified peptides increases the more up and left the curve is displaced In other words the p Value worsens for a given z Score The increase in search space from Job 2 to Job 3 i e searching for the oxidation of methionine and the carboxy methylation of cysteine is about a factor of two to three which has a relatively small impact on the p Values However when a variable modification is added such as phosphorylation that looks at the less common amino acids serine S threonine T and tyrosine Y the search space increases significantly Phenyx Web Interface Manual 79 understanding Phenyx 80 The overall level of confidence in matches decreases even for the unmodified peptides
48. e scoring to perform the search Each scoring takes into account a specific set of ion fragment series The fragment error tolerance is already included in the selected algorithm Scoring description as displayed into the Scoring Model top down menu refers to the fragmentation process Where the parent mass is detected is not considered For instance the scoring model CID_LTQ_scan_Orbitrap_6ppm refers to data that have been fragmented in LTQ and detected in Orbitrap with a fragment error tolerance as up as 6ppm Phenyx scoring list is in progress and regularly updated Please contact us support phenyx ms com if your type of instrument is missing Phenyx Web Interface Manual 73 understanding Phenyx Understanding Phenyx the effect of submission parameters on result accuracy Updated May 10 2008 The general expectation from Phenyx is that the identified proteins and peptides accurately describe your experimental sample There are unstable factors that impact the accuracy such as the quality of the mass spectra and databases used in a search It is also known that the specification of certain parameters by the user on the Submission page can influence the correctness of a search These submission parameters are described below in order to help you make informed decisions When evaluating the results of different searches the z Score and or the p Value are the best standards for separating correct matches true positives fr
49. e states listed are computed e 1 One charge also commonly referred to as singly charged or the 1 charge state e 2 Two charges also commonly referred to as doubly charged or the 2 charge state e 3 Three charges also commonly referred to as triply charged or the 3 charge state e 4 Four charges also commonly referred to as the 4 charge state Trust Parent Charge The peak picking software that comes with your instrument assigns charges to the parent and fragment ions based on Phenyx Web Interface Manual 11 submission the observed mass to charge ratios The file format generally includes this information Do you trust the peak picking software and the file format e Yes The supplied charges are most likely correct Only the assigned charges should be calculated by Phenyx e Medium There is some uncertainty about the assigned charges and therefore all combinations should be taken into account by Phenyx For example a precursor ion charged 1 is considered as one charge Two charges are considered to be 2 or 3 This increases the calculation time Number of Rounds One or two sets of calculations can be carried out on the data A second round of calculations is used to narrow or fine tune the results obtained from the first round of scoring because only the accession numbers that fulfilled the first round criteria are processed during the second round In other words the first round searches the
50. ence understanding of the concepts and level of confidence in the selected databases How Phenyx resolves conflicts When the conflict resolution functionality is activated on the Submission page 1 Resolving a conflict is considered only if the conflicting peptide matches are of good enough quality i e if they reach a minimum z Score and p Value These thresholds are a function of the parent charge and are set by Phenyx If the peptides z Scores and p Values are too low then the conflict is not resolved and the matches get a rejected status The level of confidence is therefore not considered as high enough Phenyx Web Interface Manual 83 understanding Phenyx 84 2 When peptide matches have sufficient scores to be resolved a discrimination rule is applied based on a z Score difference and a p Value ratio These values are a function of parent ion charge They are configurable values that can be set by a Phenyx administrator If the difference between the highest scoring peptide and the compared one s is larger than the discrimination values then the conflict is considered as solved A winner and a loser are defined A winner will keep its valid status therefore contributing to the protein score and a loser will get an invalid status and will not contribute to the protein score Sum of 5 scans in range 1509 rt 90 8875 fud i 306 to 1525 rt 91 7826 f 4 i 310 Sum of 5 scans in range 1509 rt 90 8875 f 4 i 306
51. enzymes my trypsin Residue modifications cie XML user s def Cleavage site XML total def f 3roup default defs Site O cleav at FYL adjacent tleavages enzymes Residue modifications tentus XMLuser s def ed N lobs Management Regular expression or Import archived job s Gain formula Jtilities l po Convert ms ms peak lists E Job suctenary x C term oH ee Crew delete save new MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAOYLQQCPF A EDHVKLVNEVTEF AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEP ERNECFLOHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKEYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKORLKCASLOQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGNFLYEYAR y 3 In order for the enzyme to disappear from the Submission page log out and re log in to the Phenyx Web Interface Residue Modifications Use this tool to create edit test or delete an amino acid modification rule A list of your modifications appears in the Phenyx Web Interface Manual 27 management console 28 left hand scrollable box Click on a modification name to select it and view the details Name An abbreviation that represents the modification on the Submission page Description An explanatory statement about the modification such as its official or commo
52. er to zoom out and view the full spectrum Fragment ion tables Different tables consolidate the fragmentation findings Phenyx Web Interface Manual peptide match details 1 Main table There is one column per peptide amino acid and one row per fragment series Bolded amino acids were modified N terminal fragments a b c etc and C terminal fragments x y z etc are the most common products from the dissociation and detection of protonated peptides in a mass spectrometer The two rows of numbers in the table indicate the position of bond cleavage in the ordered peptide sequence for N terminal fragments 1 2 n and C terminal fragments n 2 1 This standard nomenclature is based on the original proposal of Roepstorff P and Fohlman J Biomedical Mass Spectrometry 1984 11 11 601 Value Types Use the drop down list to change the information displayed in the table The possible views are e Delta Mass The m z deviations between the experimental and theoretical masses are provided for the related fragments e Theoretical Mass Theoretical m z are provided for the related fragments e Peak Mass Experimental m z are provided for the related fragments e Peak Intensity Spectral ion intensities are provided for the related fragments The first column of the table defines the fragment e Type of fragment The list changes as a function of the ion series considered during the scoring process and as a fun
53. erved parent on masses and the theoretical calculated masses Input the value and choose the corresponding unit in Daltons Da or parts per million ppm Acceptance parameters Click on the red arrow to view or edit all the acceptance parameters Minimum Peptide Length Peptides with less than the specified number of amino acids are reported in the peptide match results but they do not contribute to the protein score They are labeled as invalid Minimum Peptide z Score Otherwise known as the peptide or match score The distribution of calculated scores is compared to that of random peptide sequences in order to find the mean and variance The z Score is then a measure of how far and in what direction the score deviates from the distribution s mean Maximum Peptide p Value The probability of a peptide match in a database occurring by chance with this score or better A p Value has a maximum of 1 0 Default score is set to 1e 05 The lower the p Value the more significant the match AC Score Minimum significant value for a protein s accession number score The AC score is the sum of the best scores for validated peptide sequences Protein matches scoring lower than this value are rejected from the identified proteins Valid peptides from rejected proteins are however also listed in the Compounds Overview page but AC column is then empty Phenyx Web Interface Manual submission Peak lists File Format Only one file format
54. es Peptides Peptide numbers the number of valid peptide matches followed by the total number of peptide matches found for the given protein Cov Coverage the percent ratio of all amino acids from valid peptide matches to the total number of amino acids in the protein Description Official protein name followed by common synonyms in parentheses If a protein shares all of its validated peptides with another protein then it is considered to be a subset and will not appear in the best scoring list It appears in the Protein Details pane under Subset for the principal and better scoring protein Protein details The main points about the protein are supplied on the right hand side of the best scoring protein table The brief statement includes the identification code ID the accession number AC the database name and the official protein name followed by common synonyms in parentheses Click on the AC link to go to the actual database entry For example a protein identified in UniProt will open a page from the ExPASy Proteomics Server developed by the Swiss Institute of Bioinformatics SIB This link works for public databases such as UniProt Swiss Prot and TrEMBL but not for databases which are not widely accessible such as on the Web Please contact GeneBio at support phenyx ms com for further assistance Click on the Protein Details link to go to the Protein Match Details page Subset A list of all proteins with pep
55. etical and experimental peak list i e mass spectrum A match is thus a collection of observations deduced from this comparison The basic peptide score is ultimately transformed into a normalized z Score and a p Value A basic peptide score is the sum of raw scores for up to twelve physico chemical properties such as e Presence of specific fragment ion types a b y y b H20 etc e Co occurence of ion series modeled by Hidden Markov Models e Relative intensity versus the fragmentation type e Amino acid modifications PTMs chemical modifications etc e Peptide amino acid composition e Peptide and fragment mass error e Number of missed cleavages Phenyx Web Interface Manual 67 understanding Phenyx 68 Each of these properties are computed during the comparison between theoretical and experimental data The influence of each component depends on the parent ion charge Some of the properties are parameters that the user can define on the Submission page How a score is computed A score decides whether a match is correct or not The relative influence of each of the components is difficult to decide ex nihilo But Phenyx measures the probabilities of observing correct H1 and wrong HO matches and describes the score as a log likelihood ratio Score L P M s H1 P M s HO where P M s H1 is the probability to observe the match M under the hypothesis H1 that the peptide s is correct and P M s HO is the probab
56. f Phenyx XML tags is given below Most nested tags are not described XML Tag Description lt inSilicoDefinitions gt Definitions used in the Phenyx calculations lt elements gt Defines the chemical elements lt isotopes gt Defines a chemical element with the same atomic number but different atomic mass lt molecules gt Defines common molecules such as water Phenyx Web Interface Manual management console XML Tag Description lt codons gt Defines the standard nucleic acids lt aminoAcids gt Defines the standard amino acids lt mRNAcodons gt Defines a specific sequence of three consecutive nucleotides which are part of the genetic code Messenger RNA are produced by transcription and used during translation as a template for protein synthesis lt cleavEnzymes gt Defines the enzymes that cleave proteins lt fragTypeDescriptions gt Defines the dissociation of a protein into peptide fragments in a mass spectrometer lt series gt Defines the standard fragment ion series a b c x y and z lt losses gt Defines the standard loss of water or ammonia lt fragTypes gt Defines the different possible combinations of fragments where ion series charge state and loss of water and ammonia are the variables lt internalFragTypes gt Defines possible fragments formed when peptide bonds are cleaved on both the N and C termini
57. face Manual 1 introduction General workflow The usage of the Web site is intuitive and follows a step by step process 1 Log in Connect to the Phenyx Calculation Unit on the Login page phenyx vital it ch pwi and access the Phenyx Desktop Submit Specify the parameters on the Phenyx Submission page View results See different summaries of the identified proteins and peptides on the Proteins Overview Compounds Overview Protein Match Details Peptide Match Details or even the Results Comparison pages Generate reports Export Excel XML or text formatted files of your job results Helpful tips There are several practical ways to get the most out of the Phenyx Web Interface Allow cookie handling in your browser Get a paper copy of a page using your browser s Print option Read Phenyx documents that are available at www phenyx ms com documentation documentation html Access the User Documentation page at http phenyx vital it ch docs index html or click on the Documentation link at the top of the Phenyx Desktop The manual is available online or as a PDF file Further knowledge of Phenyx including facts about the Phenyx Calculation Unit is supplied in the FAQ and Troubleshooting sections Contact GeneBio at any time with questions or comments by sending e mail to support phenyx ms com Phenyx Web Interface Manual phenyx desktop Phenyx Desktop page The Phenyx logo is included at the top
58. h ussessseensnennnnnnnnnnnnnnnnnnnnnnnnnennnnennnnnnsensnnennnn 66 Best scoring protein table ss sisisi econtra da de 39 BLAST ee a ERE be a3 wi di ceed nennen if va aes tea Ehen age 42 46 52 lige mE een 32 Cc Change passwotd eee neret eR x ee Pe ey xe en gt Charge State eR Cle v je MOMS id Aa FEAE 16 74 8 Cleavage Site a an e rh nee Do OC RARO 23 Cleavages enzymes 23 COMMEN des Mec MES KM EMPIRE rem 5 eorpore DE MT 4 59 Completed aoi ect ver enge re dehet pe dere ne ded ced ee 4 5 Compound nce prr I IMER E pU REM PATRRX ARYAR Pen C Ape 44 51 Compound description bio seen RR ements RO dde oc aan 55 Compounds Overview sn nn neni ran an hen 6 39 Conflict resolution uurunrornsnennannan ran iE aap nun nannnnnn nun 17 82 83 Convert MS MS peak lists sessssesuenenunnonannnnnnn nun ran nn ns nen nn na nennen 35 eo Ier EE 40 ecd po Tr Tuc mM 63 66 D Databanks utet a En EDS Databases ra Aa Ea Tra AINNE Si Doaa SETETE EIES Daten A A A ARE DB a A a d Defi ioiii een Default parent charge pcm Delete profile seen tor Dep rennen Era rl re E ER ye Paces s ree Praes 8 Delta Im 2z ii out eode sea IRR E rn are nee BICHSSLCCHAME ME Phenyx Web Interface Manual 86 index Description a HEMER 40 45 61 Deselect all anne a en ci aa nr sehen 4 53 Detailed results comparison csesseen mme 62 Detailed results comparison table cese 65 Discriminationrule
59. he Life Sciences Search Engine created by the National Center for Biotechnology Information NCBI Go to http www ncbi nlm nih gov gquery gquery fcgi for more information Taxonomy Classify a kingdom phylum class order or species This significantly narrows the search criteria A degree of certainty about the origin of the protein s is needed The main groups are given below By choosing one of the main groups all sub taxons are automatically selected Root Searches all taxons e Bacteria e Eukaryota e Archaea e Viruses e Other root Searches all other taxons except Bacteria Eukaryota Archaea and Viruses e NO TAXONOMY Only searches accession numbers input by the user in the AC List Licensed users of Phenyx have the possibility to set their own taxonomy tree as defined by the NCBI Taxonomy center http www ncbi nlm nih gov sites entrez db taxonomy please contact use at support phenyx ms com if you need help or if you want to have a trial setting on the Public server AC List Specify a list of accession numbers to search in the specified database s More than one AC number can be entered by using a comma to separate them If you input AC numbers then an OR operator is used to consider the Taxonomy and the AC List i e both the specified Taxonomy and AC numbers are searched If you wish to restrict your search to the list of AC numbers only then select NO TAXONOMY in the Taxonomy drop down list
60. hes for obligatory peptides such as unmodified ideally cleaved peptides or isotopically labeled peptides from quantification studies The second round looks to see if the identified proteins can be covered by other identified peptides Remember that the two round process only validates proteins found in the first round If your first round criteria are too tolerant the false positive rate will increase since the second round adds value to the proteins retrieved in the first round The appropriate use of a two round search should reduce the overall calculation time Specify stringent criteria to search in the first round and only query a small subset of a database with computation intense settings in the second round Phenyx Web Interface Manual 81 understanding Phenyx 82 Two round search parameters The most important parameters are listed below as a guideline for searches done on data from a classical protein identification prospective e g MudPIT or a gel based workflow or for the identification of a protein with minimal criteria to optimize sequence coverage Fixed Modification s If you specify a fixed modification in the first round it must also appear in the second round Variable Modification s It is generally better to keep variable modification s for the second round You will insure the retrieval of modified peptides for proteins accepted in the first round without increasing the search space size i e without int
61. ichiometric meaning that they can be either present or absent for a modifiable amino acid All permutations are considered Variable modifications should be used carefully Since the calculations are more complex they will increase both the computation time and the false positive rate Note that many modifications annotated in the UniProt_Swiss Prot database that alter an amino acid with a discrete mass are considered as variable modifications and are searched by default They are reported in the results even if a modification was not specified on the Submission page e Tolerance Influence the number of theoretical peptides produced by a modification This is the drop down list for Fixed Modifications Depending on your selection you can reduce the calculation time and the false positive rate All Each targeted amino acid is modified gt n 1 The number of modifiable amino acids per peptide is all or all except one gt n 2 The number of modifiable amino acids per peptide is all or all except one or two gt n 3 The number of modifiable amino acids per peptide is all or all except one two or three gt n 4 The number of modifiable amino acids per peptide is all or all except one two three or four This is the drop down list for Variable Modifications None Each targeted amino acid can potentially be modified lt 1 Up to one amino acid modification per peptide is considered lt 2 Up to two amino acid modificat
62. ility under the null hypothesis HO that the peptide s is random Score L log How a score is normalized The scores are normalized in order to be able to compare matches under various experimental conditions For each peak list Phenyx computes a score distribution for a search in a given database Then it computes a score distribution on a randomly sampled set of peptides to provide a random distribution Finally it normalizes the original scores to the random distribution Figure 1 The normalized peptide score is called the z Score peptide score distributions rm st 06 renormalized scores A B A random score distributions of 25 Esquire 3000 spectra B After normalization and fitted p value 0 Loser k Lota 0 1 i Gaussian distribution 40 20 0 0 40 4 2 0 2 1 FIGURE 1 Graph A represents the random score distributions for twenty five Esquire 3000 spectra Graph B is the product of normalization and fitting to a Gaussian distribution Phenyx Web Interface Manual understanding Phenyx The z Score therefore represents the distance from a random match However the normalized score is not always sufficient enough to assess the correctness of a match because the accuracy is dependent on the absolute size of the search space As a probabilistic algorithm Phenyx therefore associates a p Value to each match The p Value is the probability to obtain a score greater than or equal to a given score by chance The p Va
63. ion of the spectrum is repeated in the Compound column for each of the conflicting peptides Only peptides that respect the z Score threshold are reported A plus or minus sign is displayed in the Auto column depending on match s validity All peptide matches are also color coded in the Sequence column according to their definition Color Term Validity Code Definition Valid plus White The peptide match respects the p Value and minimal peptide length thresholds specified by the user on the Submission page Rejected minus Gray The peptide match does not respect the p Value and or minimal peptide length thresholds specified by the user on the Submission page Conflict plus Green A winner is a valid sequence winner that successfully satisfies the conflict resolution algorithm Multiple winners are possible for the same spectrum Phenyx Web Interface Manual 50 compounds overview gt Color ME Term Validity Code Definition Conflict minus Red A loser is a valid sequence loser that does not successfully satisfy the conflict resolution algorithm In comparison to the other conflict s its value is worse Multiple losers are possible for the same spectrum Multi The letter M denotes that a conflict exists between the given peptide and other potential matches that were found for the same compound Compound This is a label commonly attached to
64. ions per peptide are considered lt 3 Up to three amino acid modifications per peptide are considered lt 4 Up to four amino acid modifications per peptide are considered Enzyme Choose one of the commonly used reagents to digest proteins Select Do Not Cleave if no enzyme was used during sample preparation The term enzyme is defined as any proteolytic cleavage agent endo or 14 Phenyx Web Interface Manual submission exoprotease or chemical agent The cleavage rules of the listed enzymes are available in the Management Console Click on the Def Definitions link to go to the Management Console page Click on Cleavage Enzymes to view or edit the definitions Enzymes can be specific to the user My defs or common to all users Group defs The table below displays the basic rules for some default enzymes Name Cleaves where Exceptions ArgC C terminal side of R if Pis C term to R ChemDigest_and_Trypsin C terminal side of D orKorR and N terminal side of D ChymoTrypsin_ FYL C terminal side of F orYorL if P is C term to F orYorL ChymoTrypsin FYLW C terminal side of F or Y or Lor W if P is C term to F or Y or Lor W DoNotCleave means Does Not Cleave at AII GluC bicarbonate C terminal side of E if P or E is C term to E GluC_ phosphate C terminal side of D or E if P or Eis C term toDorE LysC C terminal side of K Pepsin pH
65. l chiarge ii ee a el ne 56 Theoretical Mass cionado ana nn anne Fan ae 55 57 TUES nes een nn en er 5 8 U IS uo iiie A AR nexo ee cere DE SRM AERR RE 9 UniProt Swiss Prot reversed zersssssennnennnnnnnonnnnnnnnnnnnnnnnnnnnnennnen une 9 unmatched fragment spectra list sse 34 UD a Cs da ni sra b EUER SEXE E EEUU ORO ER MARNE TX 4 Update eur ae een pelts penis Een an nme UERITAS 5 41 46 51 User Permissions eer eremo lemen Rhe rm nike Rena vies 7 Utilities a A enne nre mex FRE iren sine UP EY TN nen 35 V Validate peptide matches errem 54 NG ke 13 ere MAN PEEL HP 41 46 51 Mad 57 Varia Dility keia OLIO A tedtaaedds 71 Variable modifications us4sssssannenn anne nennen nennen 13 75 82 x X Tandem nn en RR RT 60 KMU total deb ss ans nen des va cag nee 30 ARS METRE 30 Z A 42 46 52 ZOOM TACO een ad eles EEEE Vi Selle Bean Pu ne ren 5 O 18 42 52 66 68 Phenyx Web Interface Manual
66. l number of peptide matches found for the given protein e Protein Score Effective score the protein score that is recalculated based on user validated peptides It is the sum of the best scores for validated peptide sequences Protein detail list A supplemental view of the comparison according to peptide matches can be obtained on the Detailed Results Comparison page Select check the protein s to be expanded upon in the Protein AC column The AC number is listed under the Protein Detail List on the left side of the page Deselect uncheck the proteins in the Protein AC column to remove them from the list Click the Add button to see the elaboration The first time this action is performed the selections are stored in memory Therefore more proteins can later be added to the original Detailed Results Comparison page by making selections and then clicking the Add button This buffer can only be cleared by clicking the Empty List button on the Detailed Results Comparison page A different additional Detailed Results Comparison page can be viewed by selecting or deselecting proteins in the Protein AC column Click the Open button to display the listed proteins in a new window Phenyx Web Interface Manual results comparison Export options Possibilities are offered to transfer the Phenyx comparison data to another software platform for further analysis or publication purposes CSV Comma Separated Value format Click the CSV link t
67. le page Click on Residue Modifications to view or edit the definitions Modifications can be specific to the user My defs or common to all users Group defs Use the Shift or Ctrl keys to make multiple selections If a modification is selected then it appears in the AA Modif Details table to the right Specify the Type and Tolerance for each modification To remove all of the modifications from the AA Modif Details table click anywhere but on the selected names To deselect one modification at a time hold down the Ctrl key and click on the name e Name The abbreviation for the amino acid modification e Type What combinations should be considered for the given modification Fixed This modification occurs for every instance of the modifiable amino acid in the protein sequence These modifications are practically stoichiometric meaning that they are supposed to be present for every modifiable amino acid All of the targeted amino acids in a peptide are modified and altered by the expected change in mass It is appropriate to select Fixed Modifications depending on the experimentally applied modifications For example you should select Cys_CAM as a Fixed Modification when your sample has been alkylated by iodoacetamide Variable This modification may or may not have occurred for every instance of the modifiable amino acid in the protein sequence These Phenyx Web Interface Manual 13 submission modifications are sub sto
68. level of confidence 2 You wish to identify proteins from a low complexity sample e g a 2D spot Broaden your query by searching large databases You should select parameters that are not too stringent assuming that you will have to manually check the results to retrieve putative proteins Phenyx Web Interface Manual understanding Phenyx 3 You wish to retrieve one or two known proteins i e you are able to specify the AC List for two proteins Since you know which proteins you are looking for you can specify the search parameters in a single round The lower the acceptance criteria the greater the peptide coverage The number of manual peptide validations needed to be done will also be higher in order to reduce the false positive rate Two round usage Two rounds are adapted to the following cases 1 You have a restricted sample e g a 2D spot and wish to raise the level of confidence in your results through increased sequence coverage You should select stringent parameters in the first round and apply looser parameters in the second round to fine tune your results Your aim is to find out what peptides can be generated from your protein mixture 2 You are looking for special or difficult peptides in your mixture You should be able to retrieve peptides with high combinatorial properties i e several variable modifications or peptides that resulted from nonspecific cleavage half cleaved The first round searc
69. lue is approximately 1 1 p N where p is the probability to get a given score as random and N is the number of peptides considered in the population to be matched The lower the p Value the more significant the match The p Value is influenced by the size of the search space A match is unlikely to be random for a small set of sequences For a given z Score a much better p Value is retrieved with a low value in the Swiss Prot database two hundred thousand entries than in the NCBI database several million entries How Phenyx retrieves amino acid modifications not specified by the user Phenyx takes full advantage of the annotated sequence information found in the Swiss Prot and TrEMBL databases such as post translational modifications PTMs binding sites variants alternative splicing of variants etc During a search Phenyx looks at the difference between an experimental peptide mass and a theoretical peptide mass in order to determine modifications to the protein sequence If a mass difference corresponds to a known PTM that is annotated in Swiss Prot even ifthe modification was not selected by the user on the Submission page then the peptide sequence is considered modified and reported in the results These modifications are always treated as variable by the scoring model An example of this functionality is shown in Figure 2 The MS MS data were collected from a human blood sample that had undergone proteolytic digestion The se
70. m the existing table deselect uncheck the AC number in the Protein AC List and click the Update button The protein s disappear from the table and the Protein AC List Empty List Clears the Protein AC List and table in order to start again Export options Possibilities are offered to transfer the Phenyx comparison data to another software platform for further analysis or publication purposes CSV Comma Separated Value format Click the CSV link to open or save the table as a delimited text file that uses a comma to separate the data Excel Exports the results of the comparison as an Excel xls spreadsheet in either the Microsoft application or a new browser window Use the Save As option to store a copy of the file to your hard 66 Phenyx Web Interface Manual understanding Phenyx Understanding Phenyx the scoring system Updated May 10 2008 This section is intended to assist you in gaining a greater facility and knowledge of Phenyx It contains more in depth explanations on important topics of interest It also encourages you to think critically about your job submissions and results The MS MS scoring model Journal papers and conference proceedings fully describing the MS MS scoring model used in Phenyx are available to the public The references can be found at http www phenyx ms com documentation documentation html In brief Phenyx computes a score to evaluate the quality of a match between a theor
71. match details 56 Match Delta m z The value is the difference between the theoretical m z of a matched peptide and the observed m z of the parent ion Match Score Otherwise known as the peptide z Score The distribution of calculated scores is compared to that of random peptide sequences in order to find the mean and variance The z Score is then a measure of how far and in what direction the score deviates from the distribution s mean Match Charge Theoretical z calculated charge state of the peptide Mass spectrum The plot of peak intensity versus m z is given Zoom Factor Input a number in order to multiply the magnification of x axis in the spectrum Then click on the area of the spectrum that you would like to become the center of the enlarged view Ion Series Selection Select one or more fragment ion types in the table and click on the Apply Changes button The related peaks are highlighted in the mass spectrum The peaks are annotated with the corresponding details The color legend appears under the graph For selected fragment series the colors are as follows e Red Unmatched mass spectral peak e Blue Fragment peaks with matching peptides The blue peaks are plotted using the theoretical m z and the intensity of the corresponding experimental m z Input a number in the Max Intensity box and click on the Apply Changes button in order to rescale the y axis Reset X amp Y axes Click on the Reset button in ord
72. n name Position Describes the type and location of the modified amino acid s Residue The standard one letter code for the targeted amino acid s Regular Expression Specifies the rules for a set of strings that you want to match to a pattern Peptide Terminus Indicates whether the modification occurs on the N terminal or C terminal side of the peptide Mass Modifications Describes the mass difference between the unmodified and modified amino acids Monoisotopic Delta Mass The monoisotopic mass that should be added subtracted due to the modification A plus sign or nothing is used to denote an addition A minus sign is used to denote a subtraction Average Delta Mass The average mass that should be added subtracted due to the modification A plus sign or nothing is used to denote an addition A minus sign is used to denote a subtraction Formula The molecular formula that gives the total number of atoms of each element that are added subtracted due to the modification For example CSH2 adds 1 C 1 S and 2 H to the targeted amino acid UniProt Annotations i e database annotation Describes known regions or sites of interest in a protein sequence If annotations are specified in the installed databases the FTMask corresponds to the term in the header of the database entries Any registered user can define their own amino acid modifications belonging exclusively to the individual Several online res
73. nd of the protein specifically and the other end nonspecifically either the C terminus or the N terminus Modif Modification s any amino acid undergone are cataloged The colon is a counter that surrounds each amino acid thus differentiaing the N and C terminuses from the side chains of the starting and ending amino acids For example a sequence of 8 amino acids will be denoted by 9 colons For a peptide MSGECACK the code Oxidation_M represents the oxidation of the side chain of methionine Find additional examples below The detailed description of what modification occurs is also shown using this particular nomenclature on the Protein Match Details page Scenario Nomenclature Sequence example Unmodified sequence AQQAADK Modified N terminus of peptide ACET_nterm AQQAADK Modified side chain of N terminal amino acid M Oxidation_M SGECACK Modified amino acid in an internal peptide sequence FSSC Cys_CAM GGSK Modified side chain of C terminal amino acid 117Oxidation_HW ATAGDTH Oxidation_HW Phenyx Web Interface Manual 43 proteins overview 44 Scenario Nomenclature Sequence example Modified C ir METH_cterm DFLMLYAR METH_cterm terminus of peptide Compound This is a label commonly attached to LC MS MS peak lists The description is used for identification purposes It can contain a Cmpd compound or
74. nnnnnnnnnnnnnnn nenn nun nun nennen nnn nnn 27 Resubmit 6 Resubmitted peak lists 19 Results comparison table oooococcccoconononnncorononanannnanannncrnnnanancnnnnnnnns 61 orbc 12 ROUNd Li ERR SR YR IA ee YARN ed ed REEL 12 A A A TEIEEDERETERER TIC 4 5 S ccu 53 Save aS Profile ive dene e erre ia sus van 8 eren 40 46 Score calculation rene eer nennt hh a nenne XR re Yen na 68 Score normalization ann e tree lA es ees Sates 68 SCOPING imode sss een eorr npe ti iant ia an rere n tore Re RP nn 11 67 73 Scoring Syste Mi escocia Re erre amer ien eben se Ree exce ge rx oe erai quio Ho 67 SEAN en ne A clin Fre REM ern dee 42 46 52 SEARCH NI ct An a is 11 SIS gola Space SIZE nee een 74 Select alla ann Ern 53 Sequence 41 46 52 55 65 SEQUENCE AUI RE 48 SEQUES Terrain atolas uie ve eue nes revente re Be ecu iu dele E exerce be ges PR ID 60 Server lOG emt np 19 Setas dela eM 8 Show invalid proteins ne ee HR ee 61 Stat Siia arenae Daa anin hapa a aE EEA N Aa EA EEE Ta aa PARAE EEEa Eaa Eaa 5 SUDMISSION ans nn ern EEEE neh 3 Phenyx Web Interface Manual 89 index 90 SUBMIE ARE HERNE nee nee 19 SUD nenn nee RER TE TER Eee ee 40 SU T TH PME 45 SWISS PrO a anona aiaa pocas Den die desidia eae aaa 9 Swiss Prot feature table ooonncronconcccncnncrancoracnaccanoncanracnnoranascanos 69 SI DLE tet de fate 45 T TAXONOMY wants rer 10 74 Theoretica
75. nuses from the side chains of the starting and ending amino acids For example a sequence of 6 amino acids will be denoted by 7 colons For a peptide MSGECACK the code Oxidation_M represents the oxidation of the side chain of methionine Phenyx Web Interface Manual 47 protein match details 48 Sequence views Two different graphical representations of the protein sequence and its coverage by identified peptide matches are given at the bottom of the page Figure 1 Peptide color code SHIN Valid Half Cleaved aa ox rx lt P ornrocroroz darrommod FETT a FaAd4mosrAa s Arnmnmrerne Search zo dmz 2 Score p value Pos emo Modif la 1 167 5 54 0 00159 379 393 half FIGURE 1 The first view A is a graphical representation of the protein sequence and the location of the identified peptides Information about the sequence coverage by the peptide matches can be visualized in the second view B The first view is the ordered arrangement of the amino acids in the protein The sequence is written from the N terminus at the left to the C terminus at the right using the one letter amino acid codes The start position of the first amino acid in each row is given on the left hand side The matched peptide sections are highlighted in colored boxes green for valid peptides and red for invalid peptides The second view is an illustration of the number of peptide matches that covered portions of the protein sequence
76. o open or save the table as a delimited text file that uses a comma to separate the data Excel Exports the results of the comparison as an Excel xls spreadsheet in either the Microsoft application or a new browser window Use the Save As option to store a copy of the file to your hard disk Phenyx Web Interface Manual 63 results comparison 64 Phenyx Web Interface Manual detailed results comparison Detailed Results Comparison page Updated May 10 2008 This page allows the user to view one or more job results in tabular format The table is organized according to identified peptide matches for those proteins and jobs currently selected on the Results Comparison page It is good habit to use the refresh function in your browser window in order to have any changes made to the selections on the Results Comparison page be reflected on the Detailed Results Comparison page Detailed results comparison table The peptide matches for selected proteins and jobs are compared in the table Listed peptides in the Sequence column are the reference for this table Protein AC is the default choice in the Group by menu on the left side of the page The table is then arranged by protein accession number Each job that was active in the table on the Results Comparison page is assigned an integer for the sake of clarity The Jobs List on the left side of the page is simply a legend explaining the representations The Proteins Overview
77. of peptide matches will lose their valid status when the Conflict Resolution function is activated e A conflict loser will never contribute to the respective protein score AC Score even if it has acceptable submission z Score and p Value criteria A protein can therefore become invalidated since its remaining peptide matches might not be sufficient to attain the minimum AC Score specified by the user on the Submission page e A rejected match can also be in conflict with another peptide s The definition of rejected takes precedence over conflict rulings e In queries performed using two rounds it is recommended to activate Conflict Resolution in the second round only Otherwise you run the risk of obtaining a disproportionate number of rejected matches or of losing protein identifications that do not have at least one valid match for lower quality spectra Phenyx Web Interface Manual 85 index Index A AR modif detalls s 1 i nee o od 13 A a ate 39 45 52 AC Isti eere O O IO 10 74 A A Eu 18 Acceptance ParameterS vico ee ae 18 Account settings aod Actions on selected en 59 Actions on selected job s vu ran 4 Add a ads Se 62 A following JODS iios inrer das 59 A a ure E oru Pte meiner 4 61 AE acid modifications a vested 13 Annotated amino acid modifications esessesnserernnnnnnnnnnn nennen nenn nn 69 Apply Changes a een a ae nette 56 pP MEET 41 46 51 B Best Peptide Matc
78. of this home page Navigation bar Logged As The username of the person currently logged in and the type of account privileges in parentheses Log Out Quit the Phenyx system A set of links to the major sections of the Phenyx Web Interface is given in the header row Submission Click on the Submission link to go to the Submission page where you can specify search parameters and peak lists Send a submission file to the Phenyx Calculation Unit for processing Result Comparison Compare the results of multiple jobs including output from SEQUEST Bioworks Mascot or X Tandem You can also use this functionality to open one job for a quick and easy visualization Management Console Specify user preferences and access the Phenyx functionalities You can define enzyme cleavage and amino acid modification rules You can import jobs from other third party software You can also transform peak lists from one format to another and produce various reports Documentation Access the online documentation section including the Phenyx user manual Job report A summary of your jobs is given in the center of the page Phenyx Web Interface Manual 3 phenyx desktop Completed Click on the Completed link to display only jobs that finished successfully in the report Running Click on the Running link to display only jobs that are currently being processed in the report Error Click on the Error link to display only jobs that failed to
79. om random matches false positives Limit the search space size The z Score is a normalized score and thus independent of the size of the search space It mainly depends on the random matches because the basic peptide score is normalized to a random distribution In contrast the p Value is dependent on the search space size The p Value deteriorates as the search space grows larger because the chance of a peptide being a random match increases The size of a search space can be defined by the following parameters e Taxonomy and or AC List The more specific the selection the smaller the database size e Cleavage Mode A half cleaved selection generates more theoretical peptides to be sampled thus expanding the search space e Number of Missed Cleavages Increasing the number of missed cleavages increases the number of peptides to be considered Phenyx Web Interface Manual 74 understanding Phenyx e Number of Variable Modifications The greater the number of modifications to look for the larger the sample set of theoretical peptides Examples of the effects of each of these parameters follow Figure 1 to Figure 4 The MS MS data were collected from a human blood sample that had undergone proteolytic digestion The set of submission parameters used to produce the data is given in the table below Parameter Setting Database Swiss Prot Instrument Type ESI QTOF Scoring Model Default Default Parent Charg
80. ore value However variability increases when searching a restricted set of Phenyx Web Interface Manual 71 understanding Phenyx proteins such as one accession AC number or a narrow taxonomy range Figure 3 A ldentification value variability four times performed on uniprot_sprot database Taxonomy Homo sapiens z Score o o E eo 15 20 25 30 p Value log B Identification value variability four times performed on one entry P02768 z Score 0 5 10 15 20 25 30 p Value log FIGURE 3 Searches A and B were submitted four consecutive times Each point in the graphs represents the average z Score and p Value for one compound i e a single peptide identification The variability is expressed by the error bars 72 Phenyx Web Interface Manual understanding Phenyx based on standard deviation displayed for both the x and y values All human protein entries in the Swiss Prot database were searched in A Only one protein entry was searched in B so the number of potential peptide sequences is relatively small Phenyx introduced random peptides to the set of theoretically digested peptides for this protein in order to reach an adequate number of peptides to perform scoring on The reproducibility of the results is observed to be dependent on the size of the sampling set worsening for limited searches Available scorings The type of instrument used to acquire the data determines the choice ofthe appropriat
81. oteins present in all the selected jobs are shown in the table e Show Invalid Proteins Check the box to display proteins that were invalidated automatically by Phenyx or manually by the user Group A grouping is assigned when jobs share a common AC and or have the same peptide matches The Group with the smallest number has the largest number of common denominators between the compared jobs Description Official protein name followed by common synonyms in parentheses In the job columns the number of valid peptides is given by default By clicking on the number link the Protein Match Details page opens White Phenyx Web Interface Manual 61 results comparison 62 boxes display information from subset proteins the same sub entries listed under Subset in the right pane of the Proteins Overview page By contrast blue boxes label main proteins the same listed in the best scoring protein table of the Proteins Overview page To change the information displayed in the job column go to the Display Options menu on the left side of the Results Comparison page Select check one or more of the following choices and click the Update button If more than one item is listed then they are separated by hyphens in the boxes of the table Deselect uncheck an item in the menu and click the Update button to remove it from the table e Valid Peptides Number of valid peptide matches found for the given protein e Peptides Tota
82. other one searching on the false database dbfalse argument You can also perform only one search with both databases selected in the search field The false database is a random database generated from the original database On the public server the random database corresponding to uniprot_sprot is named uniprot_sprot_rev database reverse sequences GeneBio prepares forward and random databases for the licensed users please contact GeneBio at support phenyx ms com to have personal database seting for a trial period 1 select false discovery rate pept matches excel in the drop down menu 2 select job IDs use the Shift or Ctrl keys to make multiple selections 3 paste the extra arguments for instance dbtrue uniprot_sprot dbfalse uniprot_sprot_rev 4 click on the export button to get the excel FDR report Phenyx Web Interface Manual 33 management console 34 unmatched fragment spectra list mgf format This export generates a file mgf format that only contains compounds for which there is no peptide interpretation Import Phenyx jobs or jobs from other search engines Import This tool converts data such as Mascot SEQUEST Bioworks X Tandem or X Hunter output as well as Phenyx archives into jobs with new identification ID numbers See also http gbwiki genebio com mediawiki index php CGI import e Select the relevant protein identification software in the Import from drop down menu
83. oup s Administrator Admin Default Local Group s Phenyx Web Interface Manual 22 management console A registered user has write access to the definitions of his her own cleavage rules and amino acid modifications My defs Every user has read access to the definitions of the default group and users with a locally installed version of Phenyx will also have read access to the definitions of their local group s Group defs My defs The My Definitions menu refers to enzymes and amino acid modifications that are specific to the individual logged in The tools and views are available to all except guest accounts Cleavages Enzymes Use this tool to create edit test or delete an enzyme rule A list of your enzymes appears in the left hand scrollable box Click on an enzyme name to select it and view the details The following information is applicable to cleavage rules e Name A description for the cleavage rule i e an official or common enzyme name that appears on the Submission page e Cleavage Site Describes the type and location of the peptide bonds to be cleaved Cleav At Lists the targeted amino acids For example RK refers to cleavage at arginine and lysine with no commas between listed amino acids Adjacent Lists the required amino acids that must be present or absent in order for a bond to be cleaved or not For example P means that proline must follow the targeted amino acid in order for
84. ources can assist you in creating these rules Phenyx Web Interface Manual management console FindMod Available through the Swiss Institute of Bioinformatics SIB at http www expasy org tools findmod RESID Available through the European Bioinformatics Institute EBI at http www ebi ac uk RESID UniMod Available through the UniMod public database web server at http www unimod org Delta Mass Available through the Association of Biomolecular Resource Facilities ABRF at http www abrf org index cfm dm home Define a new modification 1 D X e 0 10 11 12 Starting from the Phenyx Desktop click on the Management Console link The Management Console page opens in a new window Click on the Residue Modifications link under the My Defs menu A form appears in the right hand pane Click on the New button Fill in the Name field e g type DOPA Fill in the Description field e g type 3 4 Dihydroxy Phenylalanine Fill in the Residue field e g type F If many amino acids are targeted then simply list them without commas If needed then select the N term or C term to specify the position of the targeted amino acids on the peptide Enter the Monoisotopic Delta Mass e g 15 994 Enter the Average Delta Mass e g 16 Click on the Save button The new modification is added to the scrollable list of user defined modifications on the left hand side of the pane Try out the
85. peptide and the observed m z of the parent ion z Score The distribution of calculated scores is compared to that of random peptide sequences in order to find the mean and variance The z Score is then a measure of how far and in what direction the score deviates from the distribution s mean p Value The probability of a peptide match in a database occurring by chance with this score or better The lower the p Value the more significant the match Position The numerical start and end of the peptide in the main protein sequence MC The number of missed cleavages the allowed number of sites targeted amino acids per peptide that were not cut This is an error allowance for enzyme inefficiency partial cleavage e 0 Means that all sites were cleaved as theoretically expected Lessens the likelihood of random matches e 1 All combinations are computed for one uncleaved site including the case when zero missed cleavages occurred e 2 All combinations are computed for two uncleaved sites including the cases when zero and one missed cleavages occurred e 3 All combinations are computed for three uncleaved sites including the cases when zero one and two missed cleavages occurred Phenyx Web Interface Manual e 4 All combinations are computed for four uncleaved sites including proteins overview the cases when zero one two and three missed cleavages occurred e Half Enzyme digestion occurred at just one e
86. r after the completion of the submission process For example Job submission SUCCESSFUL 6024 on Mon Apr 24 16 55 16 CEST 2006 Phenyx Web Interface Manual 19 submission The message disappears and the page becomes available again for a new submission by clicking anywhere in the Submission Page 20 Phenyx Web Interface Manual submission Phenyx Web Interface Manual 21 management console Management Console page Updated May 10 2008 The Management Console is an interactive page where users can do the following e Create edit or view enzyme cleavage rules e Create edit or view amino acid modification rules e Create or edit a FASTA sequence database e Restore an archive of job files e Import job results from third party software Mascot SEQUEST Bioworks X Tandem e Access a peaklist format conversion and filtering tool e Produce various reports e Generate Phenyx files The options provided on the Management Console page are dependent upon the roles associated with the account a person is subscribed to Each account type has a related set of roles and groups For example the guest account has anonymous roles and gives non registered users basic rights An administrator can perform higher level tasks and generally manages a locally installed Phenyx system for a network of users i e a local group N Group User type Role possibilities possibilities Guest Anonymous Default User User Default Local Gr
87. r own repository of FASTA sequences Please contact GeneBio at support phenyx ms com to temporarily activate this feature for a trial period Jobs management Browse files or export job results browse files This tool generates a complete look at the Phenyx files associated with a job Select a job number Click on the Browse Files button The directory of all Phenyx files for the job appears at the bottom of the page By clicking on a file name you can either open or save the file to your hard disk With Mozilla Firefox you can save the file by right clicking and Phenyx Web Interface Manual management console choosing This Frame gt Save Frame As in the pop up menu With Internet Explorer you can copy and paste the file into a text editor by right clicking and choosing Select All in the pop up menu export This tool exports the specified Phenyx file for selected jobs use the Shift or Ctrl keys to make multiple job IDs selection when possible regarding the selected export feature Select the type of file from the drop down list Click on the Export button See also http gbwiki genebio com mediawiki index php CGI export Below are some details about two of the export features false discovery rate pept matches excel In order to get information about the FDR false discovery rate in your results you have to perform two jobs with the same data one job searching on the true database dbtrue argument the
88. ra and corresponding peptide matches The Proteins Overview is divided into three panes Best scoring Protein Table top left Protein Details top right and Peptide Match Table bottom With Mozilla Firefox two of the panes are resizable by clicking and dragging the Expand Contract boxes Horizontal and or vertical scroll bars appear if needed to view the information Best scoring protein table The proteins are listed according to their accession numbers and sorted in descending order according to score the protein with the highest score is the best match When you click on a row in the table it is highlighted By selecting a row more details about the protein are presented to the right of the table A summary of the matched peptides for the selected protein is given below the protein table AC Database accession number The suffix _WOSIGO denotes the protein sequence without the Signal sequence The suffix _WOPPO denotes the Peptide 1 in SwissProt The suffixes _CHAINO or _VAR1 denote Chain 1 from the original protein entry or Variant 2 from the original protein entry respectively ID Database protein identification code Swiss Prot refers to this as the entry name whereas other databases use the accession number Phenyx Web Interface Manual 39 proteins overview 40 Score Effective score the protein score that is recalculated based on user validated peptides It is the sum of the best scores for validated peptide sequenc
89. rameters eu ee ern eR Dean ee 4 6 38 39 Parent error tolerance musssnenaesussnene nn nenn nenne nn en nennen 18 Phenyx Web Interface Manual index Paitial cleavadge Aura a EE eere eus 16 Peak intensity eiie HERI cee Mr ERE eR re ai D SR deed een 57 Peak list cris ranas Per Resr Rr anA enses sets tuners esata a cas desing nes 19 Peak MaS Suresi d areae severe rege vo need een are iiie iin d 57 Pending cie ee reri e RUE MR LOb RE aee E i rA ERR E E OTI DD 4 5 Peptide match table eese eene hne nth th ndn 41 46 PEPtide leo PR T 67 POSILIOT MEE En iio 42 47 Precursor MASSOS Meca ota de aa ia a 58 Private databank nisin coven san rennen nen Profiles Em Protein AC csc ccnescevsuieneceaumensupeanerciacscieedesstiandeteSemstaseatarciag ears Protein AC list wi Protein detail list vis T Protein detallS o cues cele sa tesa da res sae Sexe E aes Protein information table ussessernennnnnnnnnnnnnnnnnnnn nennen nn nen 45 Protein SCONC N se E CE 62 Proteins nal J06S2 22 32 ss 61 Proteins OVervVie PEN 6 Mr MEE 13 PU PERS RIEF RER PER RR pere tear a ea a aaa aE E 42 47 69 R Refresh en a an 4 Remove jobs from a results comparison table uususeserenenunnnnn nn nn 60 Reproducibility 2 2 3 aeg cero nn nen a in 71 Reset to auto validation essessursenensennnnnnnnn nun nun nenne nennen nenn nen 53 RESCUE ZOOM 2 ER NER ca dai 56 Residue modifications ursusuunsnnn
90. roducing a higher random match rate or longer calculation times The oxidation of methionine is an exception This modification does not significantly impact the calculation time and might be contained in some single hit identifications Missed Cleavage s It is advised to limit the number of missed cleavages to O or 1 in the first round More missed cleavages are better suited to a second round because they are often not considered determinant in the identification of a protein Cleavage Mode It is best to use the Half cleaved option in the second round for the same reasons as Missed Cleavages Proteins are usually validated with peptides cleaved strictly according to the rules A protein is rarely validated based on nonspecifically cleaved peptides alone Conflict Resolution This feature should be selected in the second round Since solving conflicts reduces the number of peptides it is more appropriate to use it at the end of a search to avoid reducing the number of proteins that pass the first round Peptide z Score and p Value Looser parameters can be set in the second round Since a protein can be validated by a few good peptides it is more acceptable to associate peptides with lower scores to an already validated protein As the z Score threshold decreases the inclusion of peptides in a result and the p Value taken together with the outcome of conflict resolution determine which peptides contribute to a protein score You can a
91. rule on a model protein by clicking on the Test button The modified amino acids predicted to occur are highlighted in the sequence at the bottom of the pane In order for the modification to appear on the Submission page log out and re log in to the Phenyx Web Interface Phenyx Web Interface Manual 29 management console 30 Edit a user defined modification 1 Select a modification name in the scrollable list to view its rule e g DOPA Make the desired changes e g modify the Monoisotopic Delta Mass from 15 994 to 15 9949 Click on the Save button In order to implement the updated modification log out and re log in to the Phenyx Web Interface Delete a user defined modification des 2 Select a modification name in the scrollable list e g DOPA Click on the Delete button DOPA is removed from the list of user defined modifications In order for the modification to disappear from the Submission page log out and re log in to the Phenyx Web Interface XML User s Def The Extensible Markup Language file showing the statements used by Phenyx to define the user s enzyme and modification rules XML Total Def The Extensible Markup Language file showing all the statements used by Phenyx to define both the current user s and the affiliated group s enzyme and modification rules It also includes XML tags describing the elements molecules amino acids nucleic acids fragment ions etc A non exhaustive list o
92. t resolution if activated and therefore is not retained in the final scoring User The validity of the peptide match as determined by the user on the Compounds Overview page A plus sign means that the peptide match is considered valid by the user and computed in the final scoring of the protein A minus sign means that the peptide match is considered invalid by the user and therefore is not retained in the final scoring If the user chooses not to manually validate peptide matches then the validity assigned by Phenyx appears by default in the User column Sequence Amino acid sequence of the matched peptide The one letter amino acid codes are used Bolded amino acids were modified Click on the Sequence link to go to the Peptide Match Details page Search By clicking on the BLAST link the SIB Swiss Institute of Bioinformatics BLAST Network Service opens in a new browser window This tool enables you to find similar entries to the selected sequence in ExPASy s protein and nucleotide databases z Charge state of the theoretical peptide match Delta m z The value is the difference between the theoretical m z of a matched peptide and the observed m z of the parent ion Score Otherwise known as the peptide z Score The distribution of calculated scores is compared to that of random peptide sequences in order to find the mean and variance The z Score is then a measure of Phenyx Web Interface Manual protein match details
93. t of submission parameters used to produce the data is given in the table below Parameter Setting Database Swiss Prot Phenyx Web Interface Manual 69 understanding Phenyx Parameter Setting Instrument Type ESI Ion Trap Scoring Model HCTultra Default Parent Charge 1 2 3 Trust Parent Charge Yes Number of Rounds 1 Variable Modifications Oxidation_M Oxidation_HW Enzyme Trypsin_ KR_noP Missed Cleavage s 1 Cleavage Mode Normal Turbo Tolerance 800 0 ppm Coverage 20 Series b b y y Conflict Resolution Yes Parent Error Tolerance 2 0 Da Min Peptide Length 6 Min Peptide z Score 5 0 Max Peptide p Value 1 0E 6 AC Score 7 0 Phenyx Web Interface Manual understanding Phenyx 727 325 0 327 666 3 520 07 226 244 UniProtKB Swiss Prot entry P62258 1433E_HUMAN 14 3 3 protein epsilon Mozilla Firefox Fichier Edition Affichage Aler Marque pages Outils 2 o Barone Compadre E 14336 HuMAN 262256 In uniprot s 14 3 3 protein epsilon 14 3 36 Ac 10 Score WPep das cov Description 1 P62258 1433E HUMAN 43 9 10 12 24 14 3 3 protein epsilon 14 3 3E Subset Auto User Sequence z mie dme z Score pValue Pos ome Modit d R ONLTLWISDI 2 1090587 0387 128 5 870 34 226 244 R DNUTLWTSDI 2 1090496 040 119 1168 26 22
94. ted report appears at the bottom of the pane With Mozilla Firefox you can save the report and import it into Excel for example by right clicking and choosing This Frame gt Save Frame As in the pop up menu With 6 Phenyx Web Interface Manual phenyx desktop Internet Explorer you can copy and paste the report into a text editor or Excel by right clicking and choosing Select All in the pop up menu Click on the Report Details link for further explanations of the different templates If you are using a local installation of Phenyx you can define your own templates and make them available in the list As a user of a locally installed version you can change the name of your job in the title txt file However the new name is not updated in the reports i e only the original job title appears Note that you can use the Identification jobs file browser tool to extract information about TRAQ labeling reagents Select the job ID click on Export Text in the Job Menu and then choose the template called default ACPept_itraq The MS MS intensities of the reporter ions m z 114 115 116 and 117 are reported in the table of identified proteins and peptides Save the report in Excel as described above to analyze and quantify your samples User Permissions Opens a new browser window with the User Permissions tool for defining access to the given job By default the current user s account appears with all permissions Fill in the User Group N
95. the Management Console page Click on the Import link under the Jobs Management menu Select the relevant protein identification software in the Import from drop down list If working with Results data then click on the Browse button to locate the Local File or archive on your hard disk Otherwise copy and paste the Remote URL of a Mascot Peptide Summary Report a SEQUEST HTML Summary File or a SEQUEST Bioworks XML Summary file in the Add URL box If your Mascot or SEQUEST Bioworks data are only accessible using a login and password then type http login password result_url instead of a basic http result_url Click on the Import button to convert the file into a job with a new identification ID number Go to the Phenyx Desktop to start working with the job In the Title column of the Job Report an information about the origin of the imported job is given for example the original ID of an imported Phenyx job is referenced in brackets In case of an imported Mascot job the Title column displays MASCOT IMPORT and file title In the Comment column of the Job Report information about the imported file or URL is given Remove jobs from a comparison table To withdraw a particular job from an existing table deselect uncheck the ID number in the Jobs List and click the Update button The job s disappear from the table but not from the Jobs List Note that this action also directly effects the Detailed Results Comparison page Empty List
96. the number of missed cleavages the more complex the scheme to express the increased number of generated peptides Turbo A procedure that accelerates a search by pre processing the data before submitting it to the main scoring calculation A minimum percentage 20 by default of the peptide sequence coverage by b b b b y y or y y fragment series is looked for If this percentage is not attained the spectrum is not submitted for further scoring If the feature is activated click on the red arrow to view or edit the default settings Conflict Resolution A conflict can occur during scoring when a mass spectrum matches more than one peptide sequence in the selected protein databases A given spectrum should ideally correlate to a unique molecular structure except if a spectrum represents a mixture of peptides When more than one peptide reasonably matches a MS MS spectrum should the scoring algorithm deal with them Phenyx Web Interface Manual 17 submission 18 e Yes Phenyx can distinguish all possible matches and decide which is are the most probable e No Phenyx simply reports all sequences matching with an acceptable score Then when the results are available you deal personally with any questionable spectra having multiple matches Parent Error Tolerance There are invariably mistakes or uncontrollable factors that affect measurements You can allow a certain amount of deviation between the experimental obs
97. the user and therefore is not retained in the final scoring If the user chooses not to manually validate peptide matches then the validity assigned by Phenyx appears by default in the User column Sequence Amino acid sequence of the matched peptide The one letter amino acid codes are used Peptide sequences that include the original N or C terminus of the protein are denoted by a minus one 1 and forward slash For example K IIEEDDAYDFSTDYV 1 indicates that V is the C terminus of the protein from which the peptide originated Internal peptide sequences are identified by the use of forward slashes and the original protein amino acids that come before and after the peptide are given K KLVLILNK S is an internal peptide composed of the amino acids KLVLILNK K and S are the amino acids that appear before and after the peptide in the protein Bolded amino acids were modified Click on the Sequence link to go to the Peptide Match Details page Phenyx Web Interface Manual 41 proteins overview 42 Search By clicking on the BLAST link the SIB Swiss Institute of Bioinformatics BLAST Network Service opens in a new browser window This tool enables you to find similar entries to the selected sequence in ExPASy s protein and nucleotide databases z Charge state of the theoretical peptide m z Experimental mass to charge ratio d m z Delta mass to charge the value is the difference between the theoretical m z of a matched
98. tide matches that overlap with those of another identified protein The listed proteins have peptides that are included in the set of matches for a protein with a better score Only the better scoring proteins are considered as significant results and appear in the best scoring protein table Click on the AC link to go to the Phenyx Web Interface Manual proteins overview actual database entry For example a protein identified in UniProt will open a page from the ExPASy Proteomics Server developed by the Swiss Institute of Bioinformatics SIB Click on the Protein Details link to go to the Protein Match Details page Peptide match table A peptide match is the pairing of an experimental fragmentation spectrum to a theoretical segment of a protein A summary of all matched peptides for the selected protein is given Auto A plus sign means that the peptide match is considered valid by Phenyx and computed in the final scoring of the protein A minus sign means that the peptide match did not fulfill the specified p Value thresholds or has been rejected after conflict resolution if activated and therefore is not retained in the final scoring User The validity of the peptide match as determined by the user on the Compounds Overview page A plus sign means that the peptide match is considered valid by the user and computed in the final scoring of the protein A minus sign means that the peptide match is considered invalid by
99. tries post translational modifications splicing mutations etc are generated with relevant annotations from scientific publications or expert data Refer to http www expasy org sprot for additional information e UniProt_Swiss Prot reversed named uniprot sprot rev Reversed version of uniprot sprot All sequences have been reverted and each AC is followed by a rev e UniProt_TrEMBL The computer annotated supplement to Swiss Prot It contains protein translations for all DNA coding regions recorded in the European Molecular Biology Laboratory EMBL database as well as additional protein sequences extracted from the literature or submitted to UniProt which are not yet integrated into Swiss Prot TrEMBL is part of the International Sequence Database Collaboration between the DNA Data Bank of Japan DDBJ GenBank from the National Center for Biotechnology Information NCBI and the EMBL database Refer to http www expasy org sprot for further details e UniProtKB sptr A concatenated form of uniprot sprot and uniprot trembl e IPI The International Protein Index IPI is administrated by the EBI This database cross references the human rat and mouse proteomes found in Swiss Prot TrEMBL RefSeq and Ensembl Go to http www ebi ac uk IPI IPIhelp html for more information Phenyx Web Interface Manual 9 submission 10 e NCBInr The collected non redundant set of data from a global Entrez query Entrez is t
100. tton to uncheck all the boxes in the User column i e none of the peptide matches are selected Reset to Auto Validation Click on the Reset to Auto Validation button to restore the default selections made by Phenyx Any user validations in memory are lost Save After manually validating peptide matches click on the Save button to store the current selections The date and time of this action is logged at the top of the page Export Last Saved Validated Peptides After saving a selection of validations click on the Export Last Saved Validated Peptides link to export the Phenyx result file in Extensible Markup Language format to a new browser window Use the Save As option to store a copy of the data to your hard disk Phenyx Web Interface Manual 53 compounds overview Validate peptide matches User validations serve three main purposes to substantiate peptide identifications by filtering false positives from true positives to resolve conflicts and to generate scoring models fitted to your experiments 1 Evaluate a peptide s p Value and z Score on the Proteins Overview page i e a reliable match has a low p Value and high z Score 2 When a case is ambiguous look at the Peptide Match Details page to assess the quality of the mass spectrum and fragment ion table 3 On the Compounds Overview page check uncheck the box in the User column depending upon if the peptide match should be considered as valid or disregarded as inv
101. ty when there is less chance of being random Change the cleavage mode Three searches were performed in order to produce the next graph In addition to the settings described above the following parameters were tweaked Parameter Job 1 Job 2 Job 3 Taxonomy or AC List Homo sapiens Homo sapiens P02768 Cleavage Mode Half cleaved Normal Normal 76 Phenyx Web Interface Manual understanding Phenyx uniprot_sprot taxonomy Homo sapiens 44 Cleavage Mode half cleaved A P 42 ze uniprot_sprot taxonomy Homo sapiens Cleavage Mode normal e uniprot sprot NO_TAXONOMY AC list P02768 o Cleavage Mode normal Ss bi a N peptide VFDEFKPLVEEPQNLIK identification through the databases compound 041126AR_203 56 56 2 dta peptide RHPDYSVVLLLR identification through the databases 2 compound 041126AR 205 674 674 2 dta Fe T T T 1 0 0001 1E 08 1E 12 1E 16 1E 20 1E 24 1E 28 1E 32 1E 36 1E 40 p Value FIGURE 2 Peptide identifications show that the curve climbs toward the right part of the graph as the search size decreases For a given z Score the p Value gets better becomes smaller as the search size decreases The z Score p Value pairs for two peptide matches belonging to serum albumin P02768 are highlighted for the three different searches The cleavage mode has one of the most significant impacts on the results The number of identified peptides can improve
102. uted for three uncleaved sites including the cases when zero one and two missed cleavages occurred Cleavage Mode Did enzyme digestion occur according to the cleavage rules on one or both ends of the protein This is an error allowance for enzyme inefficiency e Normal Enzyme digestion occurred specifically on both termini of the protein as defined by the cleavage rules The cleavage rules defined in the Management Console are strictly applied e Half cleaved Enzyme digestion occurred at just one end specifically and the other end nonspecifically either the C or N terminus The enzyme cleavage rule defined in the Management Console is applied strictly to one terminus while a nonspecific component is applied to Phenyx Web Interface Manual submission the other terminus For every given peptide Phenyx will generate a set of peptides with identical N termini but different C termini and a set of peptides with identical C termini but different N termini Protein N terminus Protein C terminus 1 Normal cleavage mode peptide N terminus peptide C terminus 1 2 3 4 no missed cleavage MS SS Se 1 missed cleavage f 2 Half cleaved cleavage mode no missed cleavage RR gt 1 missed cleavage FIGURE 1 Schematic display of the normal cleavage mode 1 versus the half cleaved mode 2 Each block represents a peptide generated in silico from a given protein sequence The larger

Download Pdf Manuals

Related Search

Related Contents

Vous voulez apprendre sans peine un américain  Service Manual KT715-KT745  Valueline VLEB11040B20 power cable      "取扱説明書"    MANUAL DE INSTALACIÓN Y OPERACIÓN  MANUAL DE INSTRUÇÕES Monitoração de Válvula XNNN  Chelsea Service Manual - HY25-6442-M1/US  

Copyright © All rights reserved. Failed to retrieve file