疾病蛋白质组学培训课件

疾病蛋白质组学

diseaseproteomics运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。通过深入了解这些疾病特异性蛋白质的结构和功能，揭示疾病过程中细胞内全部蛋白质的活动规律，为多种疾病发生、发展机制的阐明和早期诊断及治疗提供理论根据和解决途径。疾病蛋白质组学

diseaseproteomics运用蛋白1研究进展肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞癌和神经母细胞瘤等联合激光捕获微切割技术(Lasercapturemierodisseetion，LCM)，直接从肿瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题，为肿瘤蛋白质组研究提供了技术上的保障。鉴定了一批肿瘤相关蛋白，为肿瘤的早期诊断、药靶的发现、疗效和预后的判断提供了重要依据。在心脏、肺部、内分泌系统、神经系统疾病、药物成瘾性、环境毒理学、传染病、内耳相关疾病等方面，蛋白质组研究成果也为其提供了新的诊疗方向。国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面研究进展肿瘤蛋白质组：2存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但仍停留在初级比较阶段。进一步鉴定、验证，发展成应用于临床的生物标志物开展全方位的蛋白质组相互作用网络的分析进一步提高蛋白分离和鉴定的通量、灵敏度和规模；提高生物信息学应用范围与准确率，进行信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群；存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩3二、心血管疾病蛋白质组学

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.二、心血管疾病蛋白质组学

Cardiovascular4ResearchFocusThemyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretomeResearchFocusThemyofilament5ThemyofilamentproteomeThemyofilament（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.ThemyofilamentproteomeThemy6Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickfilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindicatedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.肌球蛋白重链(MHC):myosinheavychain肌球蛋白轻链-1,2(MLC1,2):myosinlightchain-1,2肌动蛋白：Actin肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）肌钙蛋白(TnT,TnI,TnC):troponinT,I，C-原肌球蛋白(Tm)：-tropomyosin肌联蛋白:titinAsimplifiedillustrationoft7Oxidantscanreactwithproteinstocauseoneoftwobroadconsequences.国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面Inthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.二、心血管疾病蛋白质组学

CardiovascularProteomics(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.high-abundanceserumproteindepletion(e.2005,19,1137–1139.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.1-D-gelseparationfollowedbyWesternblotCardiacbiomarkers.(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).2-DE,2-DDIGE(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).1-D-gelseparationfollowedbyWesternblotCardiacbiomarkers.Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(顺丁烯二酰亚胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.Proteomicsofextracellularsecretory

vesiclesBiotinswitchmethodProteinarraysProteomicsofmicroparticlesStructureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.Oxidantscanreactwithprotei8Post-translationalmodificationsofmyofilamentproteinsPost-translationalmodificatio9疾病蛋白质组学培训课件10SamplepreparationTherearetwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamounts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978–987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,1137–1139.)SamplepreparationTherearetw11DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysisDetectionMethodsforProtein12Immobilizedmetalaffinitycolumn(IMAC)Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.Immobilizedmetalaffinitycol13DetectionMethodsforProteinmodificationProteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandpIvalues)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里叶转换离子回旋共振质谱）DetectionMethodsforProtein14文献阅读ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)

文献阅读ProteomicsClin.Appl.(20152.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.2.Redoxmodificationsinthe16OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.Outlineofpotentialconsequen17MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.MechanismsofROSgeneration.18DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatiron–sulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereductase;RSNOs,S-nitrosothiols.Diagramshowingtheproduction19Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.亚磺酸磺酸次磺酸亚砜

亚硝基硫醇

羰基化

硝基化酪氨酸Structureofcommonredoxmodi20Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.Commonlyobservedoxidativemo21TheroleofExosomesSeveralapproachescurrentlyusedtoquantitatively

profileglobalproteomicexpressionpatternsMSanalysis:Redoxmodificationsinthecardiacproteome.Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.1-D-gelseparationfollowedbyWesternblotimmobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysiscarrier-assistedTCAprecipitation)[Proteomics2007,7:1757–1770]3781–3801,20082-DE,2-DDIGEMoreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)（ref:Proteomics2002,2,978–987.Oxidantscanreactwithproteinstocauseoneoftwobroadconsequences.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).dialysis/ultrafiltrationmethods[J.ListofthemostutilizedmethodsinredoxproteomicsAtlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatiron–sulphurcentresinproteinsarethemaintargets.Outlineofpotentialconsequencesofoxidativestressincellsystemquantificationofproteincysteine(ref:FASEBJ.ListofthemostutilizedmethodsinredoxproteomicsFrom:JournalofExperimentalBotany,Vol.59,No.14,pp.3781–3801,2008TheroleofExosomesListofth22Biotinswitchmethod

Ahypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS-biotinylatedwithbiotin-HPDP.ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.BiotinswitchmethodThebiot23IsotopeCodedAffinityTagging(ICAT)

(a).Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(顺丁烯二酰亚胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.

Twolabelledformsofthereagentareused,theheavycontainingeightdeuteriums(氘)andthelightwithnone.

(b)ProteinsfromtwodifferentcellstatesarelabelledwiththelightorheavyICATreagents.

Thesamplesarethencombinedanddigested.

TheICAT-labelledpeptidesareisolatedbyaffinitychromatographyusinganavidincolumnandthenanalysedHPLC-MS(/MS)directlyorbyMALDIofthecollectedHPLCfractions.

TheratioofthepeaksareasforspecificICAT-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstatesquantificationofproteincysteineoxidationIsotopeCodedAffinityTagging24ListofcardiacproteinsdemonstratedtoundergooxidativemodificationRef:ProteomicsClin.Appl.2008,2,823–836Listofcardiacproteinsdemon253.CardiacbiomarkersDiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave“potentiallyviable”heartmuscle.proteomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.3.CardiacbiomarkersDiagnosis26CurrentgoldstandardmarkersofCVdistress(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T

（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatinekinase(CK)Currentgoldstandardmarkers27Severalapproachescurrentlyusedtoquantitatively

profileglobalproteomicexpressionpatternsfluorescence2-DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“label-free”differential(d)MS(无标记的质谱定量方法)Severalapproachescurrentlyu28ThelimitedcomplexityofthesecretomemakesitsuitablefortheapplicationofaproteomicapproachProteindegradation:thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%ofthetotalproteinmassinhumanplasma.ChallengestoProteomicsofthesecretomeaseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.ProteinarraysCurrentgoldstandardmarkersofCVdistress二、心血管疾病蛋白质组学

CardiovascularProteomics肌钙蛋白(TnT,TnI,TnC):Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.（ref:Proteomics2002,2,978–987.Proteomicsofextracellularsecretory

vesiclesAlltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.plateletmicroparticles(J.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)二、心血管疾病蛋白质组学

CardiovascularProteomicsMicroparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.Thelimitedcomplexityofthe29Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsandMSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.Workflowforlabel-freedMSan304.SecretorymicrovesiclesVascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.4.SecretorymicrovesiclesVasc31Thelimitationsofplasmaproteomicsplasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%ofthetotalproteinmassinhumanplasma.prospectivebiomarkers:pg~ng/ml;albumin:35–50mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteinsThelimitationsofplasmaprot32Proteomicsofextracellularsecretory

vesicles(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.(1)Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponentsoftheendocyticpathway.Proteomicsofextracellularse33Thecriticalpatho-physiologicalroleofmicroparticlesInthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsviadirectbindingofcellsurfacereceptorsThecriticalpatho-physiologic34Samplepreparation运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。RPtC2Sorbent)[J.foldingchaperones(calnexin,calreticulin,etc.研究细胞的增殖、分化、异常转化、肿瘤形成aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.ListofthemostutilizedmethodsinredoxproteomicsSeveralapproachescurrentlyusedtoquantitatively

profileglobalproteomicexpressionpatternsendocyticproteinswereabundantcomponentsoftheproteomeofexosomes.(A)Workflowchart.Immobilizedmetalaffinitycolumn(IMAC)myosinheavychainMicroparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.(A)Workflowchart.dialysis/ultrafiltrationmethods[J.Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereductase;RSNOs,S-nitrosothiols.ChallengestoProteomicsofthesecretome肌钙蛋白(TnT,TnI,TnC):肌钙蛋白(TnT,TnI,TnC):ProteomicsofmicroparticlesProteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.SamplepreparationProteomicso35characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomicsBesidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,proteomicscanbeappliedtothecharacterisationofmicroparticlesreleasedbyaparticularcelltypeinvitro.plateletmicroparticles(J.ProteomeRes.2005;4:1516–1521)surfaceproteinstypicalofplatelets,suchasintegrinaIIb,integrinb3andP-selectin,andchemokines,suchasCXCL4,CXCL7andCCL5,380proteinsnotpreviouslyidentifiedinplateletsEndothelialcellsinresponsetostimulationwith(TNFa).(Proteomics2005;5:4443–4455)cytoskeletonandcytoskeleton-bindingproteins(tubulin,actin,cofilin,vimentin,etc.)membrane-associatedproteinsthatcontroltransportandsignalling(caveolin,annexins,dynein,etc.)foldingchaperones(calnexin,calreticulin,etc.)Adhesionmolecules,suchasICAM-1andintegrinsb1,a5anda2characterisationofmicroparti36TheroleofExosomesmodulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystemTheroleofExosomesmodulatei37Proteomicsofexosomesdendriticcell-derivedexosomes

(J.Immunol.2001,166,7309–7318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasannexins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.mast-cellderivedexosomes

(Arterioscler.Thromb.Vasc.Biol.2005,25,1744–1749)regulatethesecretionofplasminogenactivatorinhibitor-1byendothelialcellspossiblybyprothrombinasecomplexTNFaangiotensinogenprecursors.Proteomicsofexosomesdendriti385.Proteomicsofthesecretome“secretome”referredtothecomplexcollectionofproteinssecretedbyaparticulartypeofcell-oftenmaintainedinvitro.theanalysisofthesecretomeofdifferentbloodandvascularcelltypescouldbeofcriticalimportanceintheclarificationofheterogeneouscell-cellinteractionsandtheirregulationbyautocrineandparacrinefactors.Thelimitedcomplexityofthesecretomemakesitsuitablefortheapplicationofaproteomicapproach5.Proteomicsofthesecretome39ChallengestoProteomicsofthesecretomeItisdifficulttocompletelyavoidcross-contaminationwithproteinsoftheserumsupplementcommonlyusedincellcultures,althoughtheconditionedmediumisusuallysampledafterextensivewashingandduringincubationinserum-freemedium.Anyvariationinthecarry-overofserumproteinshasaprofoundimpactonquantitativecomparisonsCelldeathandcytoplasmicproteinreleaseintheculturemediumisasourceoffalsepositives,whichfurtherimpairsthereliabilityofproteomicanalysisofthesecretome.ChallengestoProteomicsofth40Technicalinnovationstoimprovethecapabilityofsecretomeanalysisprotein-enrichmentbyprecipitation(e.g.carrier-assistedTCAprecipitation)[Proteomics2007,7:1757–1770]high-abundanceserumproteindepletion(e.g.sodiumchloride/ethanolprecipitation)[Proteomics2005,5:2656–2664]),LCfraction(e.g.RPtC2Sorbent)[J.ProteomeRes.2006,5:899–906])dialysis/ultrafiltrationmethods[J.Microbiol.Methods2007,68:396–402].Technicalinnovationstoimpro41SUMMARY一、疾病蛋白质组学（diseaseproteomics）概念和总体研究概况二、心血管疾病蛋白质组学Themyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretomeSUMMARY一、疾病蛋白质组学（diseaseprote42二、心血管疾病蛋白质组学

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.二、心血管疾病蛋白质组学

Cardiovascular43Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.Structureofaregionoftheo442-DE,2-DDIGEOutlineofpotentialconsequencesofoxidativestressincellsystemThefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.fluorescence2-DDIGEcoupledtoMSanalysis(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).Significantcostofusinglabeledreagentsinlarge-scalestudies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）肌联蛋白:titindendriticcell-derivedexosomes(J.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.ListofthemostutilizedmethodsinredoxproteomicsTodate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）2-DE,2-DDIG