(BBB2002)technical improvement to 2D page of rice organelle membrance proteins

蛋白提取方法

Biosci.Biotechnol.Biochem.,66(5),1170–1173,

2002

Note

TechnicalImprovementto2D-PAGEofRiceOrganelleMembraneProteins

SatoshiMIKAMI,TadashiKISHIMOTO,HidetakaHORI,andToshiakiMITSUI?

LaboratoriesofMolecularLifeScience,GraduateSchoolofScienceandTechnology,NiigataUniversity,Niigata950-2181,Japan

ReceivedDecember3,2001;AcceptedJanuary15,2002

Cytosolicandmembrane-associatedproteinspre-paredfromricecellswereseparatedandcomparedbytwodi?erent2D-PAGEmethods,isoelectricfocusing

SDS-PAGEandnonequilibriumpHgradient(IEF)W

SDS-PAGE.AlthoughIEFelectrophoresis(NEPHGE)W

SDS-PAGEofthecytosolicproteinsshowedsu?cientW

resolution,somemitochondrialandbasicmicrosomalmembrane-associatedproteinswereweaklyorhardlydetectableonthe2Dgel.High-qualityand-quantityseparationoftheorganellemembrane-associatedpro-SDS-PAGE,theteinswasaccomplishedbyNEPHGEW

advantageofthismethodbeingmorecriticalintightlymembrane-boundproteinsthatwereunwashablewith

SDS-NaCl.TheseresultsindicatethatNEPHGEWPAGEisausefultoolfortheproteomicanalysisofricemembrane-associatedproteins.Keywords:

OryzasativaL.;membraneprotein;2D-PAGE;NEPHGE

otherwiseinterferewithisoelectricfocusing.Eachpreparationwasdissolvedandadjustedto1.0mlwithanextractionbu?erconsistingof100mMTris-HCl,10mMEDTA,100mMKCl,and2z(vWv)2-mercaptoethanol(pH7.5).Thesuspensionwasmix-edwithanequalvolumeofwater-saturatedphenolandshakenvigorouslyfor10minatroomtempera-ture.Aftercentrifugationat10,000×gfor10min,thelowerphenolphasewasshakenagainwithanequalvolumeoftheextractionbu?er.The?nalphenolphasewasmixedwith6volumesof0.1Mam-moniumacetateinmethanolandincubatedovernightat?209C.Theprecipitateobtainedbycentrifuga-tionat10,000×gfor10minwaswashedthreetimeswith0.1Mammoniumacetateinmethanolandoncewithacetone,beforebeingdried.Theextractionprocedurewascriticaltoobtainthehigh-qualityand-quantityseparationofricemembrane-associatedproteinsby2D-PAGE.Extractionwithacetoneor

SinceO'Farrell1)introducedtheusefultechniqueofhigh-resolutiontwo-dimensionalpolyacrylamidegelelectrophoresis(2D-PAGE),theprocedureof2D-PAGEhasbeenimprovedbyseveralresearchers2–6)andithasbecomeoneofthemostpowerfultoolsfortheseparationandquanti?cationofproteinsfromacomplexmixture.7–12)However,furthertechnicalim-provementforseparatingorganellemembranepro-teinsby2D-PAGEwasstillnecessary.Inthepresentcommunication,wereportasuitableprocedureby2D-PAGEfortheproteomicanalysisofriceor-ganellemembranes.

Suspension-culturedcellsderivedfromtheembryoofriceseed(OryzasativaL.cv.Nipponkai)werefractionatedbydi?erentialcentrifugationintothemitochondria(10,000×gpellet),microsomes(100,000×gpellet)andcytosol(100,000×gsuper-natant).13)Thesecell-fractionatedpreparationswereextractedwithphenolWchloroformWmethanolaccord-ingtotheprocedureofHurkmanandTanaka14)toremovethenonproteincomponentsthatwould

蛋白提取方法

2D-PAGEofRiceMembraneProteins1171

Fig.1.TwoDi?erent2D-PAGEPatternsoftheProteinsExtractedandPreparedDistinctCompartmentsofRiceCells.

Eachproteinsample(200mg)extractedfromthecytosol-(toppanel),mitochondria-(middlepanel),ormicrosome-enrichedfraction

SDS-PAGE(A)andNEPHGEWSDS-PAGE(B).Theseparatedproteinsinthe2Dgelswere(bottompanel)wasseparatedbyIEFWstainedwithCBB.

etheralonewasnotenoughtoachievegoodsepara-

tion(datanotshown).Thedriedprecipitatewas

dissolvedinalysisbu?erconsistingof9Murea,3z

(vWv)nonidetP-40(NP-40),2z(vWv)2-mercap-

toethanol,and2z(vWv)ampholine(pH3.5–10).

Finally,thesamplewascentrifugedat100,000×gfor

10mintoremoveanyinsolublematerials.

2D-PAGEwasperformedbytwodi?erentproce-

SDS-PAGE.Tomake12dures,IEFandNEPHGEW?rst-dimensionalIEForNEPHGEdiscgels(11.5cm

×q3mm),7.2gofurea,2mlofacrylamidestock

(30zacrylamideand1.6zbis-acrylamide),6.24ml

ofH2O,0.75mlof20z(wWv)NP-40,0.75mlof

v)ampholine,30mlof10z(wWv)ammoni-40z(wWumpersulfate(APS),and45mlofN,N,N?,N?-tetramethylethylenediamine(TEMED)weremixedandpouredintoglasstubes.Thecomponentsofam-pholineforIEFandNEPHGEwereinthepHrangeof3.5–10W5–8(1:1)and3.5–10W5–8W7–9(1:1:2),re-spectively.Theelectrodebu?ersandrunningcondi-tionsforIEFandNEPHGE,aswellasthoseofthegelcomponentsaresummarizedinTable1.Eightymlofeachsample(approximately200mgofproteins)wasloadedontoadiscgel.After?rst-dimensionalPAGE,thegelwasplacedinanequilibrationsolu-tionconsistingof0.06MTris-HCl(pH6.8),2.5z(wWv)SDS,5z(wWv)2-mercaptoetanol,and10z(vWv)glycerolfor10minwhilegentlyshakingtwice.

蛋白提取方法

1172S.MIKAMIetal.

Fig.2.SeparationPro?leoftheProteinsExtractedfromthe

NaCl-UnwashableFractioninRiceMicrosomesbyNEPHGEW

SDS-PAGE.

Microsomalmembranesweretreatedwith0.3MNaCland

centrifugedat100,000×gfor30min.Theproteins(200mg)in

theNaCl-unwashablefractionwereseparatedbyNEPHGEW

SDS-PAGE.

Thegelwasthenplacedonasecond-dimensional

SDS-slabgelandsealedwith0.5z(wWv)agarosedis-

solvedinanequilibrationsolutionwithout2-mercap-

toethanol.Theseparationgel(10×14cm,1mmin

thickness)contained0.1z(wWw)SDSand19z

(wWw)acrylamide(acrylamideWbisacrylamide,

30:0.135)ina0.375MTris-HClbu?er(pH8.8),

whilethestackinggelcontained0.1zSDSand

5z(wWw)acrylamide(acrylamideWbisacrylamide,

30:0.8)ina0.125MTris-HClbu?er(pH6.8).Elec-

trophoresiswascarriedoutaccordingtotheproce-

dureofLaemmli15)ataconstantcurrentof6–7mA

perplate.Theseparatedproteinsonthe2Dgelswere

visualizedbystainingwithCoomassiebrilliantblue

(CBB)R-250.Theisoelectricpointandmolecular

sizeofeachproteinwereevaluatedbyusingtheBio-

Rad2D-SDS-PAGEstandards.Theproteinspotsin

the2Dgelsweredetectedandcharacterizedbyanim-

aginganalyzerwithPDQuest2Dgelanalysis

software(Bio-Rad,Japan).

Acomparisonoftheseparationpro?lesofthe

proteinsextractedandpreparedfromdi?erentcom-

partmentsofricecellsbetweenIEFandNEPHGEWSDS-PAGEareshowninFig.1.InthecaseofIEFWSDS-PAGE,thecytosolicproteinsshowedgood

separationwithmanyproteinspotsspreadwidelyon

thegel(Fig.1A,toppanel).However,thetotal

CBB-stainedintensityofthemitochondrialand

microsomalmembrane-associatedproteinsonthe2D

gel(Fig.1A,middleandbottompanels)wasweak

comparedwiththatofthecytosolicproteins.The

stainintensitywasnotimprovedbyanychangesin

thegelandrunningconditions(datanotshown).Fig.3.SeparationPro?lesoftheProteinsExtractedfromMicrosomesintheShootandScutellarTissuesofRiceSeedlingsbyNEPHGEWSDS-PAGE.Riceseedsweregerminatedfor4daysinthedarkat309C.Themicrosomalmembraneproteinspreparedfromtheshootandscutellartissuesoftheseedlings(300mgand200mg,respec-tively)wereseparatedbyNEPHGEWSDS-PAGE.SomeproteinsperhapsaggregatedonthetopofgelduringIEF.AsshowninFig.1B(middleandbottompanels),manymoreproteins,particularlythebasicproteins,wereseparatedanddetectedbyNEPHGEWSDS-PAGEthanbyIEFWSDS-PAGE.Theimaginganalysisof2DgelsindicatedthatthetotalnumberandvolumeofproteinspotsdetectedontheNEPHGEWSDSgelswereapproximatelytwofoldthoseontheIEFWSDSgels(datanotshown).Itmustbestressedthattheintensityandnumberofspotsappearingintheacidicandneutralareaswasalsohigher,althoughitisknownthatNEPHGEWSDS-PAGEisusefulfortheseparationofbasicpro-teins.16)Theseparationofricemembrane-associatedproteinsbyNEPHGEWSDS-PAGEwashighly

蛋白提取方法

2D-PAGEofRiceMembraneProteins1173

reproducibleevenwhentheamountofproteins

loadedontothediskgelwasincreasedto350mg

(datanotshown).TheadvantageoftheNEPHGEWSDS-PAGEanalysiswasalsocriticalintightlymem-

brane-boundproteinsthatwereunwashablewith

NaCl(Fig.2).ThereasonwhyNEPHGEWSDS-

PAGEgavesuchgoodseparationofthemembrane-

residentproteinsmightbethatthebasicproteinsrap-

idlyenterthegelandseparatefromtheacidicpro-

teinsintheearlystageofelectrophoresis;therefore,

littleaggregationoftheacidicandbasicproteins

occursintheloadedsample.Incontrast,NEPHGEWSDS-PAGEwasinappropriateforricecytosolicpro-

teins(Fig.1B,toppanel).Microsomalmembrane

proteinspreparedfromtheshootandscutellartissues

ofriceseedlingsgerminatedfor4daysinthedarkat

309CwereseparatedbyNEPHGEWSDS-PAGEas

wellasthoseofsuspension-culturedcells(Fig.3).It

waseasytocompareandanalyzetheresidentpro-

teinsinmicrosomalmembranespreparedfromdi?er-

enttissuesbyNEPHGEWSDS-PAGE.Overallthese

resultsindicatethatNEPHGEWSDS-PAGEisause-

fultoolfortheproteomicanalysisofricemembrane-

associatedproteins.

Acknowledgment

Thisresearchwassupportedbygrant-aid(no.

10640628toT.M)fromtheJapanSocietyforthe

PromotionofScience,andbygrantaidforRice

GenomeProjectPR-1102(toT.M)formMAFF,

Japan.

References

1)O'Farrell,P.H.,High-resolutiontwo-dimensional

electrophoresisofproteins.J.Biol.Chem.,250,

4007–4021(1975).

2)Hirano,H.,Varietaldi?erencesofleafproteinpro-

?lesinmulberry.Phytochemistry,21,1513–1518

(1982).

3)Bjellqvist,B.,Ek,K.,Righetti,P.G.,Gianazza,E.,

Gorg,A.,Westermeier,R.,andPostel,W.,Isoelec-

tricfocusinginimmobilizedpHgradients:principle,

methodologyandsomeapplications.J.Biochem.

Biophys.Methods,6,317–339(1982).

4)Dunbar,B.S.,Kimura,H.,andTimmons,T.M.,

Proteinanalysisusinghigh-resolutiontwo-dimen-

sionalpolyacrylamidegelelectrophoresis.Methods

Enzymol.,182,441–459(1990).

5)Patton,W.F.,Pluskal,M.G.,Skea,W.M.,

Buecker,J.L.,Lopez,M.F.,Zimmermann,R.,

Belanger,L.M.,andHatch,P.D.,Developmentofa

dedicatedtwo-dimensionalgelelectrophoresissystemthatprovidesoptimalpatternreproducibilityandpolypeptideresolution.BioTechniques,8,518–527(1990).6)Herbert,B.R.,Mollyo,M.P.,Gooley,A.A.,Walsh,B.J.,Bryson,W.G.,andWilliams,K.L.,Improvedproteinsolubilityintwo-dimensionalelectrophoresisusingtributylphosphineasreducingagent.Electrophoresis,19,845–851(1998).7)Komatsu,S.,Kajiwara,H.,andHirano,H.,Ariceproteinlibrary:adata-?leofriceproteinsseparatedbytwo-dimensionalelectrophoresis.Theor.Appl.Genet.,86,935–942(1993).8)Kamo,M.,Kawakami,T.,Miyatake,N.,andTsugita,A.,SeparationandcharacterizationofArabidopsisthalianaproteinsbytwo-dimensionalgelelectrophoresis.Electrophoresis,16,423–430(1995).9)Santoni,V.,Rouquie,D.,Doumas,P.,Mansion,M.,Boutry,M.,Degand,H.,Dupree,P.,Packman,L.,Sherrier,J.,Prime,T.,Bauw,G.,Posada,P.,Rouze,P.,Dehais,P.,Sahnoun,I.,Barlier,I.,andRossingol,M.,Useofaproteomestrategyfortaggingproteinspresentattheplasmamembrane.PlantJ.,16,633–641(1998).Thiellement,H.,Bahrman,N.,Damerval,C.,Plomion,C.,Rossignol,M.,Santoni,V.,deVienne,D.,andZivy,M.,Proteomicsforgeneticandphysiologicalstudiesinplants.Electrophoresis,20,2013–2026(1999).Komatsu,S.,Rakwal,R.,andLiZ.,Separationandcharacterizationofproteinsinrice(Oryzasativa)suspensionculturedcells.PlantCellTissueOrg.Cul.,55,183–192(1999).Peltier,J.B.,Firso,G.,Kalume,D.E.,Roepsto?,P.,Nilson,F.,Adamska,I.,andvanWijl,K.J.,Proteomicsofthechroloplast:Systematicidenti?ca-tionandtargetinganalysisoflumenalandperipheralthylakoidproteins.PlantCell,12,319–342(2000).Mikami,S.,Hori,H.,andMitsui,T.,SeparationofdistinctcompartmentsofriceGolgicomplexbysu-crosedensitygradientcentrifugation.PlantSci.,161,665–675(2001).Hurkman,W.J.andTanaka,C.K.,Solubilizationofplantmembraneproteinsforanalysisbytwo-dimensionalgelelectrophoresis.PlantPhysiol.,81,802–806(http://wendang.chazidian.comemmli,U.K.,CleavageofstructuralproteinsduringtheassemblyoftheheadofbacteriophageT4.Nature,227,680–685(1970).Celis,J.E.,Rasmussen,H.H.,Olsen,E.,Madsen,P.,Le?ers,H.,Honnore,B.,Dejgaard,K.,Gromov,P.,Ho?mann,H.J.,Nielsen,M.,Vassilev,A.,Vintermyr,O.,Hao,J.,Celis,A.,Basse,B.,Lauridsen,J.B.,Ratz,G.P.,Andersen,A.H.,Kjaergaard,E.,Puype,M.,Damme,J.V.,andVandekerckhove,J.,Thehumankeratinocytetwo-dimensionalgelproteindatabase:Update1993.Elec-trophoresis,14,1091–1198(1993).10)11)12)13)14)15)16)