Comparative proteomics analysis reveals the mechanism of

ComparativeproteomicsanalysisrevealsthemechanismofComparativeproteomicsanalysisrevealsthemechanismofpre-harvestseeddeteriorationofsoybeanunderhightemperatureandhumiditystressLiqunWanga,HaoMaa,⁎,LiruSonga,YingjieShua,b,WeihongGucaStateKeyLaboratoryofCropGeneticsandGermplasmEnhancement,SoybeanResearchInstitute,NanjingAgriculturalUniversity,Nanjing,JiangsuProvince210095,PRChinabPlantScienceCollege,AnhuiScienceandTechnologyUniversity,AnhuiProvince233100,PRChinacAnimalandPlantIntroductionandResearchCenter,ShanghaiAgriculturalAcademy,Shanghai201106,PRChinaARTICLEINFOABSTRACTArticlehistory:Received29November2011Accepted8January2012Availableonline16January2012Hightemperatureandhumidity(HTH)stressduringsoybeanseeddevelopmentandmaturityinthefieldeasilyleadsseedtopre-harvestdeterioration.However,howproteinsandtheirinvolvedpathwaysindevelopingsoybeanseedsystematicallycausedeteriorationisstillnotlargelyunderstood.Torevealit,wecomparedtheproteomecompositionofde-velopingseed(R7period)ofapre-harvestseeddeteriorationsensitivesoybeancultivaratdifferentHTHstresstimepoints(24,96and168h)withtheircorrespondingcontrolsby2-DE.42proteinspotswerefoundtobedifferentiallyexpressedandsuccessfullyidentifiedbyMALDI-TOFMStomatch31diverseproteinspecies.Theseproteinswereinvolvedin13cellularresponsesandmetabolicprocessesincludingcarbohydratemetabolism,signaltransduction,proteinbiosynthesis,photosynthesis,proteinfoldingandassembly,energypathway,cellrescueanddefense,cellcycle,nitrogenmetabolism,lipidmetabolism,aminoacidmetabolism,transcriptionregulation,andsecondarymetabolitebiosynthesis.Basedontheseproteins'functionsandinvolvedpathways,togetherwithultrastructural,physicalandchemical,andmetabolomicdata,apre-harvestseeddeteriorationmechanismwasproposed.Suchamechanismallowsustofurtherunderstandthepossiblemanage-mentstrategyofcellularactivitiesoccurringintheHTH-stresseddevelopingseedsandpro-videsnewinsightsintotheHTHstressresponsesincropdevelopingseeds.©2012ElsevierB.V.Allrightsreserved.Keywords:SoybeanHightemperatureandhumiditystressMolecularmechanismPre-harvestseeddeteriorationProteomicsJOURNALOFPROTEOMICS75(2012)2109–2127Abbreviations:ω-6FAD,omega-6fattyaciddesaturase;AACPD,acyl-[acyl-carrier-protein]desaturase;ACCasesubunitbeta,acetyl-coenzymeAcarboxylasecarboxyltransferasesubunitbeta;CAT,catalase;CDPK,calcium-dependentproteinkinase;CHS-3,chalconesynthase3;CV.,cultivar;DAF,diaminofluorescein;DCF,dichlorofluorescein;DGD2,digalactosyldiacylglycerolsynthase2;EF-1-alpha,elongationfactor1-alpha;EF-Tu,elongationfactorTu;EST,expressedsequencetag;FBPase,fructose-1,6-bisphosphatase;Glnsynthetase,glutaminesynthetase;GmSTI,heatshockproteinSTI;GP,guaninenucleotide-bindingprotein;Hsp70,heatshock70kDaprotein;HTH,hightemperatureandhumidity;INR2,induciblenitratereductase[NADH]2;MALDI-TOFMS,matrix-assistedlaserdesorption/ionizationtime-of-flightmassspectrometry;MgC,magnesium-chelatase;MDA,malondialdehyde;MDH,malatedehydrogenase;NAD(P)H-DDS,NAD(P)H-dependent6′-deoxychalconesynthase;NiR,nitritereductase;NO,nitricoxide;PARP-3,poly[ADP-ribose]polymerase3;PEP-Case,phosphoenolpyruvatecarboxylase;PAL1,phenylalanineammonialyase1;PMF,peptidemassfingerprinting;PPP,pentosephos-phatepathway;qPCR,quantitativereal-timePCR;RH,relativehumidity;ROS,reactiveoxygenspecies;RuBisCOlargesubunit-P,ribulosebisphosphatecarboxylase/oxygenaselargechainprecursor;RuBP,ribulose-1,5-bisphosphate;TFIIB,transcriptioninitiationfac-torIIB;UDP-GlcDH,UDP-glucose6-dehydrogenase;Unipro,uniqueprotein;VPE,vacuolar-processingenzyme.⁎Correspondingauthorat:StateKeyLaboratoryofCropGeneticsandGermplasmEnhancement,SoybeanResearchInstitute,NanjingAgriculturalUniversity,Nanjing210095,PRChina.Tel.:+862584395324;fax:+862584395324.E-mailaddress:Lq-ncsi@njau.edu.cn(H.Ma).1874-3919/$–seefrontmatter©2012ElsevierB.V.Allrightsreserved.doi:10.1016/j.jprot.2012.01.007Availableonlineatwww.sciencedirect.comwww.elsevier.com/locate/jprot1.IntroductionUnfavorableenvironmentalconditions(hightemperature,highmoisture,drought,freezing,salt,etc.)duringseedgrowthanddevelopmentinthefieldandseedstorageinstorehousecanreducegermination,vigorandprocessingqualityofseedoffieldcrops[1,2].Thisphenomenoniscalledseeddeteriora-tion.Duetothedifferenceofperiodinwhichittakesplace,seeddeteriorationisoftenclassifiedintothreecategories:oneispre-harvestdeterioration(takeplaceinfield),anotherisdeteriorationduringharvest,andthethirdispost-harvestdeterioration(takeplaceduringseedprocessing,transporta-tionandstorage,etc.)[1,3,4].Soybean(Glycinemax(L.)Merrill)isoneofthemostimportantfoodandoilseedcropsintheworld[5].Duetoitshighproteinandoilcontent,soybeanseedisveryapttodeterioratebeforeharvestandduringprocessingandstorage.Manypreviousref-erenceshavereportedthathightemperatureandhumidity(HTH)stressduringsoybeanseeddevelopmentandmaturityinthefield,growthchamberandphytotronexperimentscanleadtopre-harvestseeddeterioration.Thisdeteriorationin-cludesproducingabnormalseeds,reducingseedgerminationrate,vigorandstorability,anddeterioratingseednutritionalquality,etc.[1,3,6–12].Thecaseofpre-harvestseeddeteriora-tionoftenoccursinmanysoybeanproductionareasaroundtheworld[1,11,13,14].Soybeanseedgenerallystartstodeterioratefromitsphys-iologicalmaturity(R7period)whenitbeginstopossesstheabilityofgermination.Pre-harvestseeddeteriorationhasbeenfoundplayingamoreimportantrolethanpost-harvestoneinsoybeanseeddeterioration[4,11,15].However,moststudiesonthemechanismsofsoybeanseeddeteriorationtodatehavemainlyfocusedonpost-harvestdeteriorationonesandindicatedthatpost-harvestseeddeteriorationisinvolvedinmanybiological,physiologicalandbiochemicalchanges,suchasnucleicacidmetabolism,membranestructureandpermeability,enzymeactivities,respirationintensity,lipidperoxidation,reactiveoxygenspecies(ROS)accumulation,andrehabilitationmechanism,etc.[12,13,15–19].Forpre-harvestseeddeterioration,amajorityofstudieshavejustfo-cusedontheeffectsofHTHstressonseedyield,germination,vigorandstorability,etc.[1,3,5–7,9–12].Usingmaturesoybeanseedsdevelopedunderhightemperature,arecentproteomicanalysisdetected20proteinidentitieswhoseaccumulationswerechangedbythehightemperature[14].Ofthem,14spotswereidentifiedas7subunitsofseedstorageproteins,andtheremaining6proteinswereidentifiedasthoserespondingtoabioticstressesorhavingafunctioninrespira-tion.Theirresultsindicatedthathightemperatureduringseeddevelopmentnotonlychangessoybeanseedcomposi-tionanddecreasesseedvigor,butalsochangesseedproteinexpressionprofiles.Sincewestilldonotcompletelyunder-standhowproteinsandtheirinvolvedpathwaysindevelop-ingsoybeanseedsystematicallycausedeterioration,itisnecessarytouseproteomicsstrategytorevealit.Theresultswillhelpustobreedorengineersoybeancultivarswithresis-tancetoHTHstress.Inthepresentstudy,weinitiatedthefirstfunctionalproteo-micinvestigationofproteinsthatareresponsivetoHTHstressindevelopingseedsofapre-harvestseeddeterioration-sensitivesoybeancultivar(cv.),NingzhenNo.1.Themainobjec-tivewastounderstandhowproteinsandtheirinvolvedpath-waysinthedevelopingsoybeanseedsunderHTHstresssystematicallycauseddeterioration.2.Materialsandmethods2.1.MajorchemicalsCHAPS,IPGDryStrip(pH3–10,17cm,non-linear),IPGbuffer,andiodoacetamidewerepurchasedfromBio-Rad(Hercules,CA,USA),thoiureaandn-octylglucopyranosidefromSigma(St.Louis,MO,USA),andtrypsin(Promega,Madison,WI,USA),ureaandacrylamidefromPromega(Madison,WI,USA).Deionizedwater(Millipore,Bedford,MA,USA)withresistancegreaterthan18MΩcmwasusedthroughout.Allotherchemi-calsandreagentswereofanalyticalgradeandpurchasedfromNanjingChemicalandRegentsCo.(Nanjing,China).2.2.PlantmaterialandstresstreatmentPre-harvestseeddeterioration-sensitivesoybeancv.NingzhenNo.1,whichwaspreviouslyscreenedoutof189landracesandcultivarsfromSouthChinabyincubatorweatheringfollowedbystandardgerminationtest,wasusedinthisstudy.ItwasonceapopularcultivarinthedownstreamregionofYangtzeRiverofChina.Theseedlingsofcv.NingzhenNo.1weregrowninpotsinfield,andtheagronomicmanagementprac-ticesforthemwerenormal.ForthesimulationofHTHstress,incubatorweatheringmethodwasusedinthisstudy.ItwasconductedaccordingtoKeigleyandMullen[6]withsomemod-ifications.Whenthepottedplantsofcv.NingzhenNo.1devel-opedtophysiologicalperiod(R7),theyweregroupedintotwogroups.Onegroupwastransferredtoagrowthchambermain-tainedat40°C/30°C,100%/70%humidity(RH),and10h/14hcycle(light/dark)for7days.Theotherwiththesamedevelop-mentalprogressionwasusedascontrolundernormalcondi-tion[30°C/20°C,70%RH,and10h/14h(light/dark)].Theseedsfromplantmiddleportionofthetreatmentandcontrolwerecollectedat0,24,96,and168h,respectively,andfrozeninliquidnitrogenandthenstoredat−80°Cforproteinextrac-tionorassay.Tominimizeenvironmentalerrors,threesetsofparallelsamplescollectedoverthreedifferentdayswereusedtoextractproteinandRNA.Forstatisticalpurposes,theserepresentedthreebiologicalreplicates.2.3.ROSandnitricoxide(NO)detectionROSproductioninepidermalcellwasdetectedusingdichloro-fluorescein(DCF)accordingtoPeietal.[20].SoybeanseedsfromHTHtreatmentornon-HTHtreatmentplantsweresam-pledat0,6,12,24,96,and168h,respectively.Thesoybeanseedepidermalpeelswerefloatedinincubationbuffer(10mMHepes-NaOH,0.2mMCaCl2,pH5.7)for30minunderavoidinglightand25°Cwithadding10mMH2DCFDA,andthenwashedfor30mininanincubationbuffer.Epidermalpeelswereobservedforfluorescenceusingaconfocalmicro-scope(Leica),withexcitationat488nmandemissionat525nm.NOlevelinsoybeanseedcellswasanalyzedusinganNO-sensitivedye,4,5-diaminofluoresceindiacetate(DAF-2DA)(Sigma-Aldrich).Epidermalpeelswerefloatedinanincubationbuffer(10mMHepes-NaOH,0.2mMCaCl2,pH5.7)andloadedwith5mMDAF-2DAunderavoidinglightand25°Cfor30minbeforewashedinincubationbufferfor30min.Theim-ageswereobservedunderaconfocalmicroscope(Leica)withexcitationat488nmandemissionat535nm.DCFandDAFfluorescenceintensitywasmeasuredbyLeicaconfocalsoftware(version2.5).Datawerequantifiedasmeanpixelintensities.2.4.DeterminationenzymeactivityandelectrolyteleakageTheleveloflipidperoxidationinseedsampleswasdeterminedintermsofmalondialdehyde(MDA)contentaccordingtoMadhavaandSresty[21].ContentofMDA,whichisanendprod-uctoflipidperoxidation,wasdeterminedbythethiobarbituricacidreactionandcalculatedfromtheabsorbanceat532nm.Themeasurementwascorrectedfornon-specificturbiditybysubtractingtheabsorbanceat600nm.Anextinctioncoefficientof155mM−1cm−1wasusedincalculation.Catalase(CAT)activ-itywasestimatedaccordingtoBergmeyer[22],whichmeasurestheinitialrateofdisappearanceofH2O2at240nm.Thereactionmixturecontained0.05MNa-phosphatebuffer(pH7.0)with0.1mMEDTAand3%H2O2.Thedecreaseintheabsorptionwasfollowedfor3minandμmolH2O2destroyedperminutewasdefinedasoneunitofCAT.ElectrolyteleakagewasassayedbyestimatingtheionsleachingfromtheseedsintoMilli-Qwater.Seedmaterialswereplacedin20mlofMilli-Qintwosets.ThefirstsetthatwassufferedfromtheHTHstresswaskeptatroomtemperaturefor4handitsconductivity(C1)wasrecordedusingaconductivitymeter.Thesecondsetwasauto-claved,itsconductivitywasalsorecorded(C2),andelectrolyteleakage[1−(C1/C2)]×100wascalculated.Theexperimentswerecarriedoutintriplicates.2.5.TransmissionelectronmicroscopeanalysisTransmissionelectronmicroscopeanalysiswasconductedaccordingtoSuzukietal.[23]withsomemodifications.Soy-beanseedsfromHTHstressandcontrolwerecutinto1mm×3mmandfixedinamixtureof2.5%glutaraldehydein0.1Mphosphatebuffer(pH6.8)foratleast2hat4°C.Afterwashedwithrinsingbuffer(0.2Mphosphatebuffer,pH6.8),thesampleswerepostfixedin1%osmiumtetraoxidein0.1Mphosphatebufferat4°Cfor2h.Theyweredehydratedinagradedalcoholseriesandembeddedinepoxyresin.Ultrathinsectionswerepreparedwithdiamondknivesonthemicrotome.Thesectionsongridswerestainedwith2%aqueousuranylacetatefor20minfollowedbyaleadelectronstainingsolution(Katayama,Osaka)for5min.ThesectionswereobservedusinganH-7000electronmicroscope.2.6.Determinationofsolubleprotein,totalnitrogen,andfattyacidcontentsThesolubleproteinconcentrationofdevelopingsoybeanseedswasmeasuredaccordingtoBradford[24],usingbovineserumalbumin(BSA)asstandard.Totalnitrogencontentwasdeterminedbythemicro-Kjeldahlmethod[25].ThefattyacidcontentofdevelopingsoybeanseedwasdeterminedusingGasChromatography(GC)accordingtoCasalandOliveira[26].2.7.Determinationofammonium(NHþ4),nitrate(NOÀ3)andnitrite(NOÀ2)concentrationsThesoybeanseedswereimmediatelyfrozeninliquidnitrogen.2gofthefrozentissuewashomogenizedtoafinepowderandtakenin50mlpolyethyleneintheplasticvial.Afteraddingof15mlofdistilledwater,thevialwascappedandshakeninwaterincubationat45°Cfor60min.Thesolutionwasfiltered.NitrateandnitriteconcentrationwasdeterminedaccordingtoUwahetal.[27]withsomemodifications.ThedeterminationofnitrateineachofsamplesolutionswasperformedbyMicro-platereader(TECANInfinite200,Untersbergstr,Austria).Theabsorbanceat540nmwasmeasuredandnitriteconcentrationsweredeterminedbyreferencetoastandardcurve.Theconcen-trationlevelsofnitrateinthesampleswerecalculatedfrom:A¼CÂV1ðÞ=V2ÂWðÞWhereAisnitrate(NOÀ3)content(μg/g);CistheconcentrationofNOÀ3bythestandardcurve;Wisweightofsamples;V1isthetotalvolumeofthesamplesolution;V2isthevolumeofthesamplesolutionaddedtothereactionsolution.Nitritelevelsinthesamplesolutionsweresimilarlydeter-minedexceptthatinthiscase,differentreagentswereused.Theconcentrationlevelsofnitriteinthesampleswerecalcu-latedfromtheaboveformula.Ammonium(NHþ4)concentrationwasmeasuredaccordingtoEglietal.[28]andScheiner[29].AmmoniumwasanalyzedbyMicroplatereader(TECANInfinite200,Untersbergstr,Austria)asdescribed.Phenolreagent(0.4ml)andhypochloritereagent(0.6ml)wereaddedtoa1mlsample,followedbyincu-bationatroomtemperatureinthedarkforatleast2h.Theabsorbancewasmeasuredat635nm.Theammoniumconcen-trationsweredeterminedbyreferencetoastandardcurve.2.8.MetabolomicsanalysisMetaboliteextractionwascarriedoutaccordingtoLisecetal.[30]andRoessneretal.[31]withsomemodifications,whileGC–MSanalysiswasconductedaccordingtoQiuetal.[32]withsomemodifications.SeedsweresampledseparatelyatdifferentHTHstresstimepoints(0,24,96,and168h)andimmediatelyfrozeninliquidnitrogenandstoredat−80°Cuntilfurtheranalysis.Seedsfromdifferentnon-HTHstresstimepoints(0,24,96,and168h)wereusedascontrol.Sixindependentsamplingswereconductedforeachtimepoint.Frozensamplewasgroundintofinepowderwithmortarandpestleinliquidnitrogen.Onehun-dredmgpowderofeachsamplewastransferredintoa10mlcentrifugetubeandmixedwith1.4ml100%methanol(pre-cooledat−20°C).Thehomogenatewasshookfor10minat70°Cinathermomixerat950rpm,and100μlofribitolsolution(2mg/mlstockindH2O)wasaddedasaninternalstandard.Afterwards,theextractwascentrifugedat11,000gfor30minandthesupernatantwasdecantedintoafreshculturetube.1.4mldH2Oand750μlofchloroformwereaddedintothetube.Themixturewasvortexedthoroughlyandcentrifugedat2200gfor15min.150μlofthepolarphase(methanol/dH2O)wasdec-antedintoafresh1.5-mltubeanddriedinaCentrivapbenchtopcentrifugalvacuumconcentrator(LabconcoCorporation,KansasCity,MO,USA).Thedriedresiduewasredissolvedandderiva-tizedfor90minat37°Cin80μlof20mg/mlmethoxyaminehy-drochloride,andthentrimethylsilylatedfor30minat37°Cwith40μlN-methyl-N-(trimetylsilyl)trifluoroacetamide(with1%trimethylchlorosilane).ThederivatizedextractwasanalyzedbyGC–MS(Thermo-FinniganTRACEGC2000gaschromatographlinkedtoaFinni-ganTraceGCDSQmass-selectivedetector,ThermoFinnigan,Austin,TX,USA).1μlaliquotoftheextractwasinjectedintoaDB-5MScapillarycolumn(30m×0.25mm×0.25μm,AgilentJ&WScientific,Folsom,CA).Theinlettemperaturewassetat250°C.Aftera5-minsolventdelay,initialGCoventemper-aturewassetat70°C;2minafterinjection,theGCoventem-peraturewasraisedto280°Cwitha5°C/minrate,andfinallyheldat280°Cfor13min.Thedetectorinjectiontemperaturewassetto280°Candtheionsourcetemperaturewasadjustedto200°C.Heliumwasusedasthecarriergaswithaconstantflowratesetat1.5ml/min.Themeasurementsweremadewithelectronimpactionization(70eV)inthefullscanmode(m/z50–650).WithineachGC–MSchromatogram,thepeakareaofeachcompound(Xi)wasnormalizedthroughbeingdi-videdbythepeakareaoftheinternalstandard(ribitol)(Xc)togetaresponseratio.Theresponseratiowasfurtherconvertedtotherelativemetabolitecontent(Ni)throughbeingdividedbyfreshweightofthesample(WFW).Ni¼Xi=XcÂWFWðÞWhereXisPeakarea,equivalentstowhatwecallsfragmentresponse(Xioffragmenti).Thisprocedurecorrectspipetteer-rorsandslightdifferencesinsampleamount.Inafurtherstep,therelativeresponseofafragment(Ri)isthatNiisdivid-edbytheaverageoftherelativemetabolitecontentofsixin-dependentexperiments(Navg)tofacilitatecomparisonsbetweensamples.Ri¼Ni=NavgThemetabolitesdetectedwereidentifiedbyXcalibur2.0soft-ware(ThermoFinnigan,Austin,TX,USA)coupledwithcommer-ciallyavailablemassspectrumlibraries:NIST2005(Fisons,Manchest,UK)andWiley7.0(PalisadeCooperation,Yonkers,NY).2.9.Totalsolubleproteinextractionfor2-DEanalysisProteinextractionwasconductedaccordingtoParkeretal.[33]withsomemodifications.Soybeanseeds(0.5g)weregroundinliquidnitrogentoafinepowder,andthepowderwassuspendedcompletelyin10mlof10%(w/v)TCAinace-tonewith1%(w/v)DTTat−20°Covernight.Aftercentrifuga-tionat14,000gfor30minat4°C,thepelletswerewashedthreetimeswith10mlofice-coldacetone.Thecollectedpro-teinpelletsweredriedwithvacuumdryingtoremoveanyremainingacetone.Thedriedpowderwasresuspendedcompletelyin2mloflysisbuffer(7Murea,4%[w/v]CHAPS,and0.2%ampholytes,pH3–10).Afterincubationatroomtem-perature(25°C)for1.5h,thesuspensionwascentrifugedat16,000gfor30minat25°Ctoremovetheinsolublematerial.Theresultingsupernatantwithpredominantlysolublepro-teinswasreducedbyadding5mMTris-(β-carboxyethyl)-phosphinehydrochloride.Thereductioncontinuedfor1hatroomtemperature.Sampleswerethenalkylatedbytreatmentwith16mMiodoacetamidefor1.5hatroomtemperature.Thisreactionwasquenchedbyadditionof65mMDTT.Thesampleswereimmediatelyfrozeninliquidnitrogenandthenstoredat−80°Cinaliquots.ProteinconcentrationwasdeterminedusingtheEZQProteinQuantitationKit(MolecularProbes,Inc.,Eugene,OR)permanufacturer'sinstructions2.10.2-DE,gelstaining,andimageanalysisFirst-dimensionalelectrophoresiswasconductedonanIPG-phorIEFsystem(Bio-Rad,Hercules,CA,USA).Theproteinex-tractwasdilutedtoafinalconcentrationof350μgofproteinsamplein400μloftwo-dimensionalrehydrationbuffer(2%[w/v]CHAPS,0.5%IPGbuffer,2Mthiourea,and6Murea).Aftercentrifugationfor10minat10,000g,400μlsupernatantwasloadedontoacommerciallyavailableprecastIPGstrip(17-cmnon-linear,pH3–10)andwasactivelyrehydratedat30Vfor18hat20°C.ThenfocusingwasperformedontheIPGphorapparatusunderthefollowingconditions:200Vfor40min,500Vfor40min,1000Vfor1h,2000Vfor1h,and8000Vfor5hachieving55,000VhT.BeforeSDS-PAGE,thestripswereequilibratedfor15minin10mlofreducingequilibrationbuffer(6Murea,37.5mMTris–HClatpH8.8,20%[v/v]glycerol,2%[w/v]SDS,atraceofbromphenolblueand1%[w/v]DTT)andforanother15mininalkylatingequil-ibrationbufferthatcontained2.5%(w/v)iodoacetamidein-steadof2%DTT.Thestripswereplacedonthetopofvertical12%SDS-PAGEselfcastgels.Theelectrophoresiswascarriedoutat20°Cand1.0W/gelfor2h,andthenat15W/geluntilthedyefrontreachedabout1cmfromthebottomofthegelusinganEttan™DALTSystem(GEHealthcare).GelswerestainedwithsilvernitrateasdescribedbyBlumetal.[34].Thesliverstained2-DEgelsweredigitizedwithaBio-RadFluorSsystemequippedwitha12-bitcamera.ThedatawereanalyzedwithPDQuestsoftware(version7.3,Bio-Rad,Hercules,CA,USA).Proteinspotswerematchedauto-maticallyandthensubjectedtocarefullymanualeditingandconfirmation.Eachspotincludedonthestandardgelmetsev-eralcriteria:itwaspresentinatleasttwoofthethreegelsandwasqualitativelyconsistentinsizeandshapeinthereplicategels.Therelativevolumeofeachspotwasassumedtorepre-sentitsexpressionlevel.Tofinddifferentiallyexpressedpro-teinspots,weusedseparatecontrols(24,96,and168h)foreachtreatmenttimepoint(24,96,and168h)andthenonlycomparedtheabundancedifferencesbetweentreatedandcontrolsamplesateachparticulartimepoint.ThestatisticalsignificanceofquantitativedatawasdeterminedusingStu-dent'sttest(n=3).Aspotabundanceratioofgreaterthan1.40orlessthan1.40(P<0.05)wasusedasathresholdtoiden-tifydifferentiallyexpressedproteinsinsubsequentstudies.2.11.ProteinidentificationanddatabasesearchProteinspotsshowingsignificantchangesinabundanceunderthestresswereselectedandexcisedmanuallyforproteinidentification.In-geldigestionofproteinspotswasperformedaccordingtoYaoetal.[35].AllMALDI-TOFmassspectrawerecollectedwithanUltrafle