PolyacrylamidegelelectrophoresisisawidelyusedtechniquetoseparateproteinsfromBIOLOGicalsamples.Moreover,thedevelopmentoftwo-dimensional(2D)gelelectrophoresishasprovidedatoolfordifferentialproteindisplay,whichallowsforthequantitativeanalysisofmany(~500-1000)ofproteinssimultaneously.Complexbiologicalquestionscannowbeapproachedbyanalyzingdifferencesinthe2Dgelpatternsbetweencontrolandexperimentalstates. Whilehighsensitivitystainingmethodscouldvisualizeproteinsatincreasinglylowlevels,originallyproteinidentificationwaslimitedbythesensitivityofEdmansequencing.However,inthelastfewyearsmassspectrometryhasbecomeestablishedasaviableandmoresensitivealternative,withthedevelopmentofESI-MSandMALDI-MS.PeptidemassfingerprintingusesthepeptidemassesobtainedbydigestiontosearchtheproteinandDNAdatabases,tofindproteinsthatshowasimilartheoreticaldigestpattern(seeeg.,Shevchenkoetal.1996aandb). Intheprotocolbelow,wedescribethein-geldigestionprocedure,asitisroutinelyperformedinourlaboratory,forpeptidemassmappingofpicomoletosubpicomolequantitiesofproteinderivedfromcoomassie-orsilverstainedpolyacrylamidegels.First,SDSisremovedfromthegelpriortodigestion,inseveralammoniumcarbonate/acetonitrilewashingsteps.Theexcisedgelpiecesaresubsequentlydriedandrehydratedwithenzymeinbuffer.Alternatively,thewashedgelpiecesmaybereducedandS-alkylatedpriortorehydrationwithenzyme.Afterdigestion,thepeptidesareextractedfromthegelandmassanalyzed. In-geldigestionprocedure Materials 25mMNH4HCO3in50%acetonitrile 25mMNH4HCO3,pH8 5%TFA/50%acetonitrile 0.1mg/mltrypsin(sequencegrade,Promega)in25mMNH4HCO3,pH8 10mMdithiotreitolin25mMNH4HCO3 55mMiodoacetamidein25mMNH4HCO3 Generally,trypsinistheproteaseofchoiceforpeptidemassfingerprinting,becauseofitsreliABIlityanditssubstratespecificity,yieldingpeptideswithC-terminalbasicresidues(ArgandLys),whichfacilitatesionizationandsubsequentmassspectrometricsequencing. Dissolvetrypsinjustbeforeuseinice-coldbuffer(toreduceauto-proteolysis). 1.Exciseproteinbands/spotsofinterestfromthegelandcuteachgelpieceintosmallparticles(~1mmx1mm)usingascalpelandplaceintoa0.65mlsiliconizedtube(PGCscientific).Alsocutoutagelpiecefromaprotein-freeregionofthegel,foraparallelcontroldigestiontoidentifytrypsinautoproteolysisproducts. AsmallgelparticlesizefacilitatestheremovalofSDS(andcoomassie)duringthewashes,andimprovesenzymeaccesstothegel. Forasilver-stainingprotocolcompatIBLewithmassspectrometry,seeShevchencoetal.(1996a). 2.Add~100ml25mMNH4HCO3in50%acetonitrile(orenoughtoimmersethegelparticles)andvortexfor10min.Usegel-loADIngpipettipstoremovethesolution(paleblueincaseofcoomassiestaining)anddiscard.Repeatthiswash/dehydrationstepupto~2-3times. Atthispoint,thegelslicesshrinkandbecomewhite.Thisvisualcriteriumshouldbeusedtodeterminewhetherornotadditionalwashesshouldbeperformed. 3.Drythegelparticlesfor~15mininavacuumcentrifuge. 4.Optionalreductionandalkylation.Add10mMdithiotreitolin25mMNH4HCO3,enoughtocoverthegelpiecesandreducefor1hrat56oC.CooltoroomtemperatureandreplacetheDTTsolutionbyroughlythesamevolume55mMiodoacetamidein25mMNH4HCO3.Incubatefor45minatroomtemperatureinthedarkwithoccasionalvortexing.Washthegelpieceswith~100ml25mMNH4HCO3for10minwhilevortexing,dehydratewith~100ml25mMNH4HCO3in50%acetonitrileandrehydrateagainwith~100ml25mMNH4HCO3anddehydrateagain.Removetheliquidphaseanddrythegelpiecesinavacuumcentrifuge. 5.Rehydratethegelparticlesin25mMNH4HCO3,pH8,containing0.05-0.1mg/mltrypsinbyvortexingfor5min.Donotaddmoresolutionthantheamountthatcanbeabsorbedbythegelparticles,otherwisealotoftrypsinautolysiswilloccur. Theenzyme-to-substrateratioemployedforin-geldigestionsisgreater(>1:10)thanforin-solutiondigestionsduetothehinderedenzymeaccesstotheproteinsubstrateinthegel.Moreover,therelativelowsaltconcentrationof25mMisusedtoreducethepossibilityofsubsequentsaltinterferencewithionizationinthemassspectrometer.Thisconcentrationmaybeincreasedifporosmicrotipsorziptipsaresubsequentlyusedfordesalting. 6.Ifneccessary,overlaytherehydratedgelparticleswithaminimumamountof25mMNH4HCO3,pH8,tokeepthemimmersedthroughoutdigestion. 7.Incubate12-16hoursat37oC. 8.Torecoverthepeptidesfromthegelparticles,perform~3extractions.Forthefirstextraction,add2volumesofwaterandvortexfor10min.Forsubsequentextractions,add5%formicacid/50%acetonitrile.Usegel-loadingtipstoremovethepeptidesolutionaftereachextractionandcollectinasiliconizedtube. Thesiliconizedmicrofugetubesandthehighformicacidconcentrationareusedtominimizeadsorptivesampleloss.TFA(5%)maybeusedasanalternativeforformicacidifMALDI-MSisused. 10.Storetherecoveredpeptidesat-20°Cinslicktubes. Fenselau,C.1997.MALDI-MSandstrategiesforproteinanalysis.Anal.Chem.661A-665A.Jungblut,P.andThiede,B.1997.Proteinidentificationfrom2-DEgelsbyMALDImassspectrometry.MassSpectrom.Rev.16:145-162.Patterson,S.D.andAebersold,R.1995.Massspectrometricapproachesfortheidentificationofgel-separatedproteins.Electrophoresis16:1791-1814.Shevchenko,A.,Wilm,M.,Vorm,O.andMann,M.1996a.Massspectrometricsequencingofproteinsfromsilver-stainedpolyacrylamidegels.Anal.Chem.68:850-858.Shevchenko,A.,Jensen,O.N.,Podtelejnikov,A.V.,Sagliocco,F.,Wilm,M.,Vorm,O.,Mortensen,P.,Shevchenco,A.,Boucherie,H.andMann,M.1996b.Linkinggenomeandproteomebymassspectrometry:Largescaleidentificationofyeastproteinsfromtwo-dimensionalgels.Proc.Natl.Acad.Sci.USA93:14440-14445.References
