UsingMaxQuantforproteomicsAstepbystepguide

RongxiaoSa,DepartmentofChemistry

Pra@kJagtap,MinnesotaSupercompu@ngIns@tuteNov24,2009

Outline

StepbystepprocedureonusingMaxQuantforproteiniden@ca@on(salivaproteins,6rawles)

RawdataprocessingMascotsearchingproteingroupingandlegenera@ng

BriefprocedureonusingMaxQuantforSILACdata

WhychooseMaxQuant

Freeanduserfriendly,greatusersupportfromdiscussiongroup

Fasterthanotheralgorithms

Greatforlargedatasets(>5G)

Moreproteiniden@ca@on,ormoreMS/MSiden@ed

Crossreferencetovariousdatabases(GO,Swissprot,Ensembl,KEGG)

GreatforSILACquan@ca@on

SystemrequirementsforrunningMaxQuant

WindowsXPorvista32bitopera@ngsystem

Xcaliburinstalledonthesamecomputer

MascotDaemoninstalledoraccess

Atleastdualcoreprocessor(or2Duo)

Atleast2GRAM

Correctdataacquisi@onmodeonLTQorbitrap

MSscanshouldbeacquiredinprolemode,MS/MScentroidmode

Uselockmasscalibra@on

Login

Eitheronsdvlapp32oronpc1,pc2,pc3inWalterlibrary585withyouruseraccount

DoubleclickontheMaxQuantfoldershortcutonthedesktop

Tutorialleloca@on

Mycomputer\C:\Documentandsecngs\allusers\sharedtutorial

Foryourownpurposes,putyourlesinUdrive

Acknowledgements

EricSanfordinMSI

MadStoneinBMBB

Astepbystepguide

Loginview

Proteinandpep@degrouping,quan@ca@on

GenerateMSMlesformascotsearching

Generatedecoydatabases

ModulesofMaxQuant

Quant.exe

LoadingrawlesC:\Documentsandsecngs\allusers\shareddocuments\proteomicworkshop\rawles

Selectnumberofthreads=2

SelectParameters

SelectSingletsfornonlabeleddataSelectDoubletsforSILACdatawith2experimentsSelecttripletsforSILACdatawith3experiments

SelecthIPI_Maxquantfordatabaseforhumansamples

Selecttrypsinforenzyme

Star@ngQuant.exe

Processedlesarelocatedinthesamefolderasrawlesareandareinthefoldernamedcombined

Quant.exeDone

Quant.exegeneratedles

C:\documentsandsecngs\allusers\shareddocuments\proteomicsworkshop\rawles\combined

Allspectra.CID.iso.parAllspectra.CID.iso_0.msmAllspectra.CID.sil0.parAllspectra.CID.sil0_0.msmAllspectra.CID.sil1.parAllspectra.CID.sil1_0.msm

Mascotparameterles CorrespondingMSMles

Mascotdatabasesearching

SelectsearchparametersParameterleloca@on:C:\documentsandsecngs\allusers\shareddocuments\proteomicstutorial\processedles

Cau@on:Parameterlescannotbeonnetdrive(Udrive),ithastobesavedonC:orEdriveonSdvlapp32,pc1,pc2andpc3

Selecttaskeditorparameters

SelectMSMleforsearching

SearchFinished

FileLoca@on

Datledownload

Datledownload

Proteingroupingwithiden@fy.exe

Uploadrawles

Rawlesloca@on:C:\DocumentsandSecngs\allusers\shareddocuments\Proteomicsworkshop\Rawles

UploadDatles

Datlesloca@on:C:\DocumentsandSecngs\allusers\shareddocuments\Proteomicsworkshop\datles

UploadProteinDatabase

ProteinDatabaseloca@on:C:\DocumentsandSecngs\allusers\shareddocuments\Proteomicsworkshop\databases

UploadExperimentaldesignle

ProteinDatabaseloca@on:C:\DocumentsandSecngs\allusers\shareddocuments\Proteomicsworkshop\Processedles\combined

Changingparametersforproteinpep@degrouping

Proteinandpep@degroupinginprocess

Proteinandpep@degrouplesloca@on

Proteinandpep@degrouples