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1、第四讲第四讲疾病蛋白质组学（一）疾病蛋白质组学（一）diseaseproteomics一、基本概念和总体研究概况一、基本概念和总体研究概况疾病蛋白质组学疾病蛋白质组学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，寻找置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋疾病诊断和预后的特异性蛋白质白质( (群群) )，包括特异性抗原及相关抗原、受体、酶等，以，包括特异性抗原及相关抗原、受体、
2、酶等，以及及药物治疗的靶标药物治疗的靶标等。通过深入了解这些疾病特异性蛋白等。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。治疗提供理论根据和解决途径。研究进展研究进展肿瘤蛋白质组：肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成研究细胞的增殖、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、肝细胞癌白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾
3、癌、肝细胞癌和神经母细胞瘤等和神经母细胞瘤等联合联合激光捕获微切割技术激光捕获微切割技术(Lasercapturemierodisseetion，LCM)，直接从肿，直接从肿瘤组织中提取纯肿瘤细胞，瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题以克服组织内异质性的问题，为肿瘤蛋白质组研，为肿瘤蛋白质组研究提供了技术上的保障。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。判断提供了重要依据。在心脏、肺部在心脏、肺部、内分泌系统、神经系统疾病、药物成瘾性、内分泌系统、神
4、经系统疾病、药物成瘾性、环境毒、环境毒理学理学、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、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。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等方面存在问题和发展趋势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。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
5、的蛋白质组相互作用网络的分析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；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；确地分析蛋白质的相互作用，界定相互作用连锁群；二、心血管疾病蛋白质组学二、心血管疾病蛋白质组学 Cardiovascular Proteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincluding
6、cardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwi
7、de.ResearchFocus1.Themyofilamentproteome.2.Redoxmodificationsinthecardiacproteome.3.Cardiacbiomarkers.4.Secretorymicrovesicles5.Proteomicsofthesecretome1.ThemyofilamentproteomeThemyofilament（肌丝）（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehe
8、arttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickf
9、ilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindica
10、tedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.肌球蛋白重链肌球蛋白重链(MHC):myosin heavy chain肌球蛋白轻链肌球蛋白轻链-1,2(MLC1,2): myosin light chain-1,2肌动蛋白：肌动蛋白：Actin肌球蛋白结合蛋白肌球蛋白结合蛋白C(MyBP-c): myosin binding protein C）肌钙蛋白肌钙蛋白(TnT, TnI, TnC):troponin T, I ，C
11、 -原肌球蛋白原肌球蛋白(Tm)： -tropomyosin肌联蛋白肌联蛋白: titinStructureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.Post-translational modifications of myofilament proteinsSamplepreparationTherea
12、retwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamoun
13、ts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,11371139.)DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylatio
14、nsignificantlydecreasesproteinpI values)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysisImmobilizedmetalaffinitycolumn(IMAC)Schematicofa
15、ffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.DetectionMethodsforProteinmodificationProteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandp
16、I values)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里叶转换离子回旋共振质谱）傅里叶转换离子回旋共振质谱）文献阅读文献阅读Proteomics Clin. Appl. (2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathoph
17、ysiology(p800-810)2.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidati
18、vePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.T
19、heycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifi
20、cations,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.(a)MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.(b)Listofamin
21、oacidsparticularlysusceptibletomodification.DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leads
22、totheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatironsulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthas
23、e;NR,nitratereductase;RSNOs,S-nitrosothiols.Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.亚磺酸亚磺酸磺酸磺酸次磺酸次磺酸亚砜亚砜 亚硝基硫醇亚硝基硫醇 羰基化羰基化 硝基化酪氨酸硝基化酪氨酸Com
24、monlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.ListofthemostutilizedmethodsinredoxproteomicsFrom:JournalofExperi
25、mentalBotany,Vol.59,No.14,pp.37813801,2008BiotinswitchmethodAhypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfree
26、thiolsarefinallyS-biotinylatedwithbiotin-HPDP.ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaf
27、finitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.Isotope Coded Affinity Tagging (ICAT) (a).Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(顺丁烯二酰亚胺顺丁烯二酰亚胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.Two
28、labelledformsofthereagentareused,theheavycontainingeightdeuteriums(氘氘)andthelightwithnone.(b)ProteinsfromtwodifferentcellstatesarelabelledwiththelightorheavyICATreagents.Thesamplesarethencombinedanddigested.TheICAT-labelledpeptidesareisolatedbyaffinitychromatographyusinganavidincolumnandthenanalysed
29、HPLC-MS(/MS)directlyorbyMALDIofthecollectedHPLCfractions.TheratioofthepeaksareasforspecificICAT-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstatesquantificationofproteincysteineoxidationListofcardiacproteinsdemonstratedtoundergooxidativemodificationRef:ProteomicsClin.
30、Appl.2008,2,8238363.CardiacbiomarkersDiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierint
31、erventiontosave“potentiallyviable”heartmuscle.proteomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.CurrentgoldstandardmarkersofCVdistress(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocard
32、iographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatin
33、ekinase(CK)Severalapproachescurrentlyusedtoquantitativelyprofileglobalproteomicexpressionpatternsfluorescence2-DDIGEcoupledtoMSanalysisProteinarraysin vitro andin vivo stableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardsla
34、belingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“label-free”differential(d)MS( (无标记的无标记的质谱定量方法质谱定量方法) )Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsand
35、MSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.4.SecretorymicrovesiclesVascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.Schematicfigureoftheuseofproteomicsforthecharacterizati
36、onofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellul
37、arcomponentsoftheatheroscleroticmicroenvironment.Thelimitationsofplasmaproteomicsplasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%ofthetota
38、lproteinmassinhumanplasma.prospectivebiomarkers:pgng/ml;albumin:3550mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteinsProteomicsofextracellularsecretoryvesicles(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsof
39、calcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.(1)Microparticlesarereleasedfromth
40、eplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponen
41、tsoftheendocyticpathway.Thecriticalpatho-physiologicalroleofmicroparticlesInthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease
42、,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamemb
43、rane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsvia directbindingofcellsurfacereceptorsProteomicsofmicroparticlesProteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(gree
44、nandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.characte
45、risationofmicroparticlesreleasedbyaparticularcelltypein vitro by proteomicsBesidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,proteomicscanbeappliedtothecharacterisationofmicroparticlesreleasedbyaparticularcelltypein vitro.plateletmicroparticles(J. Proteome Res. 2005;4:1516152
46、1)surface proteins typical of platelets, such as integrin aIIb, integrin b3 and P-selectin, and chemokines, such as CXCL4, CXCL7 and CCL5,380 proteins not previously identified in plateletsEndothelialcellsinresponsetostimulationwith(TNFa).(Proteomics 2005;5:44434455)cytoskeleton and cytoskeleton-bin
47、ding proteins (tubulin, actin, cofilin, vimentin, etc.)membrane-associated proteins that control transport and signalling (caveolin, annexins, dynein, etc.) foldingchaperones (calnexin, calreticulin, etc.)Adhesion molecules, such as ICAM-1 and integrins b1, a5 and a2TheroleofExosomesmodulateimmunere
48、sponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystemProteomicso
49、fexosomesdendriticcell-derivedexosomes(J.Immunol.2001,166,73097318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasann
50、exins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.mast-cellderivedexosomes(Arterioscler.Thromb.Vasc.Biol.2005,25,17441749)regulatethesecretionofplasminogenactivatorinhibitor-1byendothelialcellspossiblybyprothrombinasecomplex
51、TNFaangiotensinogenprecursors.5.Proteomicsofthesecretome“secretome”referredtothecomplexcollectionofproteinssecretedbyaparticulartypeofcell-oftenmaintainedin vitro.theanalysisofthesecretomeofdifferentbloodandvascularcelltypescouldbeofcriticalimportanceintheclarificationofheterogeneouscell-cellinterac
52、tionsandtheirregulationbyautocrineandparacrinefactors.ThelimitedcomplexityofthesecretomemakesitsuitablefortheapplicationofaproteomicapproachChallengestoProteomicsofthesecretomeItisdifficulttocompletelyavoidcross-contaminationwithproteinsoftheserumsupplementcommonlyusedincellcultures,althoughthecondi
53、tionedmediumisusuallysampledafterextensivewashingandduringincubationinserum-freemedium.Anyvariationinthecarry-overofserumproteinshasaprofoundimpactonquantitativecomparisonsCelldeathandcytoplasmicproteinreleaseintheculturemediumisasourceoffalsepositives,whichfurtherimpairsthereliabilityofproteomicana
54、lysisofthesecretome.Technicalinnovationstoimprovethecapabilityofsecretomeanalysisprotein-enrichmentbyprecipitation(e.g.carrier-assistedTCAprecipitation)Proteomics 2007,7:17571770high-abundanceserumproteindepletion(e.g.sodiumchloride/ethanolprecipitation)Proteomics 2005,5:26562664),LCfraction(e.g.RPt
55、C2Sorbent)J. Proteome Res. 2006,5:899906)dialysis/ultrafiltrationmethodsJ. Microbiol. Methods 2007,68:396402.SUMMARY一、疾病蛋白质组学（一、疾病蛋白质组学（diseaseproteomics）概念和总体研究）概念和总体研究概况概况二、心血管疾病蛋白质组学二、心血管疾病蛋白质组学1.Themyofilamentproteome.2.Redoxmodificationsinthecardiacproteome.3.Cardiacbiomarkers.4.Secretorymicrovesicles5.Proteomicsofthesecretome
