solubilization of delipidated macrophage membrane proteins for analysis by 2D electrophoresis

蛋白提取方法

Electrophoresis2000,21,641±644641AugustoJaimeSolubilizationAntonioM.Simðes-Barbosa

R.SantanaL.Teixeiramembraneofdelipidatedmacrophage

Chagas©two-dimensionalproteinselectrophoresisforanalysisbyMultidisciplinaryDisease

Laboratory,ResearchTwo-dimensionalelectrophoresisofproteinsisoftenprecludedduetothelackofsolu-Pathology,bilizationofcellmembraneextractsinanaqueousmedium.VariousadditivesandBrasília,Brasília,UniversityDepartmentof

Brazilofdetergentshavebeenusedtocircumventtheproblem,buttheirefficacymaynotbe

satisfactory.Inthisstudy,theremovaloflipidiccomponentsofthecellmembrane

extractwithchloroform-methanolwasusedtoachievesolubilization.Optimaldelipida-

tionwasobtainedwithacetonewashings.Thisprocedureincreasedsolubilizationof

membraneproteinsfromamurinemacrophagecellline,thusshowingasubstantialim-

provementingelresolution.Thetwo-dimensionalgelsloadedwithdelipidatedextract

provedtobefreeofsmearingandhorizontalstreaking.Inaddition,otherproteinspots

wererevealedthatwerenotdetectedinthegelsloadedwithundelipidatedcellmem-

braneextract.

Keywords:Chemicaldelipidation/Membraneproteinsolubilization/Two-dimensionalelectro-

phoresisEL3726

1IntroductionSomenewdetergentsoradditivesthatpromotesolubili-Physiologicandpathologicprocessesarecharacterizedzationofmembraneproteins,suchaszwitterionicdeter-bydifferencesingeneexpression.Inthelasttwentyyearsgents,arenotcommerciallyavailable[15±http://wendang.chazidian.comano-biochemistshaverefinedandusedtwo-dimensionalelec-chemicaldelipidationwithdifferentsolubilizingagentstrophoresistoanalyzesuchdifferencesinproteinextractsmayimproveseparationofmembraneproteinsbytwo-

[1±3].ThistechniqueallowsseparationofhundredsofdimensionalelectrophoresisinanimmobilizedpHgra-proteinspotssimultaneously,sinceitscapabilitywasfur-dientgel.Here,weproposedelipidationwithacetonetherimprovedbyalteringthepHgradient.Therefore,two-washingsasageneralmethodtoobtainsolubilizationdimensionalelectrophoresishasbeenusedtoverifypro-ofmembraneproteinsbydismountingsupramolecularteinsoverorunderexpressedduringsurrogatingproc-assembliesoflipids.TheresultsoftheseexperimentsessesofcellularstresssuchascancerandHIVinfectionsshowedthatdelipidationcanimproveresultsfor

[4±9].Moreover,computerizedanalysisoftwo-dimen-two-dimensionalelectrophoresisofmembraneproteinsionalrelativemassandpIhavecreateddatabasesofextractsofamurinemacrophagecellline.

proteinmaps[10±14].Despitebeingafrequentlyused

tool,lackofsolubilizationofmembraneproteinextracts2Materialandmethods

maypreventtwo-dimensionalanalysis.Hydrophobic

interactionsbetweenpolypeptidechainsandthelipidic2.1Material

componentofthemembranecanbeeasilydisruptedwith

SDS.However,thisanionicdetergentmayinterferewithThetwo-dimensionalelectrophoresissystem,gels,andthepIbynegativelychargingaprotein.ThislimitationhasCHAPSwerepurchasedfromLKB-Pharmacia(Uppsala,beenpartlysolvedbycombiningachaotropicagentandaSweden).Urea,SDS,DTT,silvernitrate,formaldehyde,nonionicdetergentsuchasureaandTritonX-100,re-chloroform,methanol,butanol,TCA,acetone,MEMcellspectively.Ureacanconsiderablydecreasehydrophobicculturemedium,andfetalbovineserum(FBS)werefrominteractions,butitinducesdenaturation,exposeshydro-Sigma(St.Louis,MO,USA).Deionizedwaterfromaphobicresidues,andincreasesthepotentialforhydro-Milli-ROandMilli-Qtandemsystem(Millipore,Bedford,phobicinteractions[15].MA,USA)wasusedinallsolutionsandbuffers.Correspondence:Dr.AntonioR.2.2Cellculture

tidisciplinaryology,FacultyResearchofHealthonSciences,Chagas©L.Disease,Teixeira,LaboratoryforMul-

UniversityDepartmentofPath-MurinemacrophagecelllineP388D1-IL1wascultivated

BoxinMEMmediumcontaining10%v/vFBSandsupple-E-mail:04536,ofBrasília,P.O.

mentedwithantibiotics.Theculturewasmaintainedina

Fax:+55-61-273-4645ateixeira@unb.br70.919-970Brasília,Brazil

greenhouse,at37oC,with5%CO2.

??WILEY-VCHVerlagGmbH,69451Weinheim,20000173-0835/00/0303-0641$17.50+.50/0ED-2dnascimoetorP

蛋白提取方法

642A.Simðes-Barbosa,J.M.SantanaandA.R.L.TeixeiraElectrophoresis2000,21,

641±644

2.3Membraneproteinpurification

6´10´7to108cellswereocollectedbycentrifugationat2000gfor15min,at4C,andwashedtwicewithPBS,pH7.5.Thecellpelletwasresuspendedin3mLbufferA(0.1MTris-HCl,pH7.5,0.25Msaccharose,0.5%v/vTri-tonX-100,20MEDTA,20mMNa-p-tosyl-L-lysinechloro-methylketone(TLCK),20mME-64,10mM(RAMC),8mMPMSF.Ruptureofthecellswasachievedafterfivecyclesofstirringat12000rpmfor15s,followedbya5minperiodofrest.Thiscellhomogenatewassubjectedtocentrifugationat10000´gfor15min.Thesupernatantwastransferredtonewtubesandultracentrifugedat100000´gfor2htopelletthecytoplasmicmembraneproteins.Thecellsamplesyielded1300±1800mgofmem-braneproteins.Theseprocedureswereconductedat4oC.

2.4Solubilizationofmembraneproteins

Urea/TritonX-100.Inatypicalexperiment,themembraneproteinswereresuspendedin9Murea,2%v/vTritonX-100,130mMDTT,8mMPMSF,2%v/vPharmalyte3±10;finalvolume,0.5mL.Urea/CHAPS.Themembraneproteinpelletwasresuspendedin2%w/vCHAPS,3Murea,130mMDTT,10mMTris,pH7.5,8mMPMSF;finalvolume,0.5mL.Inthisprocedure,theImmobilinedrystripgelwasrehydratedinthesamesolution,asrecom-mendedbythemanufacturer.

2.5Chemicalproteinextract

delipidationofcellmembrane

Themembraneproteinextractsolubilizedwiththeusualurea/TritonX-100solutionwassubjectedtodifferentpro-tocolsofdelipidation,asfollows.

2.5.1TCA/acetone

Themembraneproteinextractwasprecipitatedin10%w/vTCA,followedby30minincubationandcentrifugationat14000´gfor10min.Thepelletwaswashedtwicewithacetoneatroomtemperature.Duringthewashings,thepelletwasvigorouslyvortexedfor30sandrecentri-fugedat14000´gfor5min.Thepelletwasair-driedandthenresuspendedwiththeinitialvolumeoftheurea/TritonX-100solution.

2.5.2Mildorganicextractions

Butanolextraction.Onevolumeofwater-saturatedbutan-olwasusedtodelipidatethemembraneproteinextract.Theorganicandaqueousphasesweremixedbyvigorousvortexingfor5minatroomtemperature.Thedelipidated

aqueousphaseofthemembraneproteinswascollectedaftercentrifugationat14000´gfor5min.Chloroformextraction.Thefollowingmixturewaspreparedandusedtodelipidatethemembraneproteinextract:chloroform/methanol/water,18:9:1.Theprocedureusedfororganicextractionwasasdescribedabove.

2.6Two-dimensionalelectrophoresis

Thetwo-dimensionalelectrophoresiswasperformedwithImmobilinedry-striplinearpH3±10,11cm,forisoelectricfocusing,followedbyseparationinExcelGelSDS,gra-dient8±18.Atotalof80mLofmembraneproteinextract,correspondingto200±280mgbeforedelipidation,wereloadedintoeachgel.Thetwo-dimensionalelectrophore-sisprocedure,silverstainingdetection,andgeldryingwereperformedstrictlyaccordingtothemanufacturer©srecommendations.Finally,thegelswereoverlaidforcom-parativeanalysis.

2.71-DSDS-PAGE

Theorganicphasesofmembraneextractsweresecuredandsubjectedtorotaryevaporation,for30min,at37oC.Theresiduesresuspendedinsamplebufferwereana-lyzedin10%w/v1-DSDS-PAGEandsilver-stained.

3Resultsanddiscussion

Solubilizationhasbeendefinedasaprocessofbreakingtheinteractionsbetweenthesubstancestobeanalyzedandanyinterferingsubstances[15].Incellmembraneextracts,thelipidsarethemaininterferingsubstancewithhydrophobiccoresofproteins.Thelipidiccomponentofthecellmembraneextractrequiresasuitablesolubilizing

Figuredatedurea/Tritoncell1.Two-dimensionalmembraneproteins,electrophoresispartiallysolubilizedofundelipi-ing.ArrowshowsX-100.aNotesmearsmearingofinsolubleandproteins.

horizontalstreak-in

蛋白提取方法

Electrophoresis2000,21,641±644Figurebrane2.Two-dimensionalelectrophoresisofcellpendedprotein10%inurea/Tritonextract.Themembranepelletwasresus-mem-beingTCAanddelipidatedX-100withsolution,acetoneprecipitatedwashingsbeforewithinsolubleloadedandproteins,inthegel.Noteabsenceofsmearingofproteins.asignificantCirclesshowincreaseintensificationnewprotein

intheofresolutionsomeproteinspotsspots.

ofthebasicFigurebrane3.pendedproteinTwo-dimensionalextract.Themembraneelectrophoresisextractofwascellmem-a2.5.2).mixtureinurea/TritonofX-100solutionanddelipidatedresus-withdiscreteNoteabsencechloroform/methanol/waterofsmearing,minimal(seestreaking,Sectionbutshowspotslossnotofpresentresolutioninundelipidated

ofthebasicsamples.proteins.Circlesagenttoobtainproteindispersion.Inthisregard,deter-gentsandchaotropicagentsdisruptsuchnoncovalentinteractions,providingasmuchdispersionaspossibleinanaqueousmedium.Monomericlipidmoleculesmaybindproteins,thusalteringmolecularmassorpI.Suchlipidbindingproteinsaremainlylipidcarriersthatcanbeeasilysolubilizedbydetergentsandchaotropicagents.Aproblemariseswhenthelipidsassemblelargemicelles,

Delipidationofcellmembraneforelectrophoresis

643

membranes,andvesicles.Solubilizationofsuchsamplesmaynotbeachievedinthesmallvolumestobeloadedinthegel.

Wehaveobservedthatcompletesolubilizationofmem-braneproteincouldnotbeachievedintheurea/TritonX-100solution.Thepatternoftwo-dimensionalelectro-phoresisofthispartiallysolubilizedmembraneextractrevealedsmearingandhorizontalstreakingofproteinsassociatedwithlipids,whichdidnotattaincompleteiso-electricfocusinginthegel(Fig.1).However,chemicaldelipidationofmembraneextractofmurinemacrophagesincreasedwatersolubilityanddidnotinterferewiththeelectrophoreticseparationbypIandmolecularmassofproteins.

Inthisstudy,theexcesslipidsinthemembraneextractfromamurinecelllinewasremovedwithdifferentcombi-nationsoforganicsolvents.Thetwo-dimensionalelectro-phoreticpatternsoftheseproteinswerecomparedandrevealedspotsthatwereseeninthesamepositionsinchemicallydelipidatedsamples.Typically,smearingandhorizontalstreakingdisappearedaftersolubilizationofthemembraneproteinextractbydelipidation(Fig.2±4).Inaddition,thesolubilizationofmembraneextractbydelipi-dationintensifiedthespotsbyincreasingtheamountofproteininatleastsomespotsintheelectrophoresisgel.Furthermore,theproteinsthatwerepoorlyfocusedintheIEFwhenundelipidated,focusedwellafterdelipidation.Occasionally,thereweresomespotswithdecreasedabundanceafterdelipidation(Fig.4).ThezwitterionicdetergentCHAPScombinedwithureadidnotresultincompletesolubilizationofthemembraneproteinextractin

Figurebranependedprotein4.Two-dimensionalelectrophoresisofcellmem-saturatedinstreakingbutanol.urea/Tritonextract.Themembranepelletwassus-NoteX-100absenceanddelipidatedinwater-andinresolutionbutconsiderableofthebasiclossproteins.

inproteinofsmearing,contentminimalofspots

蛋白提取方法

644A.Simðes-Barbosa,J.M.SantanaandA.R.L.TeixeiraElectrophoresis2000,21,

641±644

Figuredatedurea/CHAPS.cell5.Two-dimensionalmembraneelectrophoresisofundelipi-FeaturesNotesmearingproteins,andpartiallysolubilizedincomparedofinsolubleproteins.

withmolecularthatofmassFig.1.andArrowphorizontalIwereshowsalteredstreaking.asmearwhenofourexperimentalconditions.Figure5showsasmearoflipidsandhydrophobicproteinsinasampleresuspendedinurea/Chaps.Inaddition,thissolubilizationprocedureinterferedwiththeelectrophoreticpatternofproteinmigrationinthetwo-dimensionalgel.

Interestingly,precipitationofmacrophagemembranepro-teinswithTCAfollowedbyacetonewashingsdidnotcauselossofproteins.Further,weobservedthatthisdeli-pidationprocedureimprovedspotformation,decreasedstreaking,http://wendang.chazidian.comparingelectrophoreticpatternsofcrudemembraneproteinextractswithdelipi-datedsamples,itwasshownthatsmearingandhorizontalstreakingresultingfrominsolubleproteinsandlipidsdis-appeared.Inaddition,theappearanceofnewproteinspotsandtheincreasingamountsofproteininsomespotsareadvantageousfeaturesofsolubilizationofcellmembraneextract,byacetonedelipidation.Acriticalfea-turemayberelatedtothepoorresolubilizationofsomeproteinsafterprecipitationandaputativelossofsomeothersthatsolubilizebetterinorganicratherthaninaque-ousmedium.Inthisregard,hydrophobicmembranepro-teinscouldbeextractedfromEscherichiacolibychloro-form-methanol,butsomeproteinswerereducedinabundance[19].Infact,delipidationofmurinemacro-phagemembraneextractwithchloroformorbutanolshowedsomelossofproteinsatthebasicendoftheIEFgels,butthisfeaturewasnotpresentingelsinwhichpro-teinsweredelipidatedwithacetone.Moreover,the

organicphaseofacetonewashingsdidnotshowproteinbandsinsilver-stained1-DSDS-PAGE.

Insummary,thelackofanyinterferenceinthepIandmo-lecularmassduringelectrophoreticseparation,absenceofsmearedinsolubleproteinsandlipids,consistentappearanceofnewproteinspots,inadditiontosignificantreductionofhorizontalstreakingandthebestbalanceofspotnumbersandstreaking,arefeaturesachievedthroughchemicaldelipidationofthemembraneproteinsbytheTCA/acetonemethod.

ReceivedJune10,1999

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