系统生物学II - 先进测量平台 (2)学习资料

1.AdvancedplatformforSB?HighthroughputAutomaticLowcostHighspeedHighaccuracyBestgainofbioinformationinquantityandqualitywithunitresourceorcost.1Genomesequence：capillaryarrayelectrophoresisGeneexpressionprofiling基因表达谱:

biochip,RTPCRProteinsequence:separation&MSProteinstructure:x-raycrystallization结晶,NMRInteraction:twohybrid双酵母杂交,TAP,biochipProteinlocalization蛋白质定位:molecularimagingMetabolite：separation&MS,NMR22.Fundamentalinstrumentmethod1、Fluorescence荧光性2、Chromatography色谱分析3、Electrophoresis电泳法4、Biochip生物芯片5、BioMassspectrometry生物质谱分析6、x-raycrystallizationX射线结晶7、NMR核磁共振8、Molecularimaging分子成像3Microdeviceforhighspeed,parallelprocessandanalysisofbiomolecules对生物分子进行快速、并行处理和分析的微型器件。2.4.Biochip42.4.1.Biochipclassificationmicro-array微阵列Microfluidics微流体“Microsystem”“Micro(Miniaturized)TotalAnalysisSystem(TAS)”

“Lab-on-a-Chip”“Biochip”5Comparison

Microarray

Microfluidics

Theory

Biointeraction

Microfluidiccontrol

Nature

Array

Network

Fabrication制造

MEMS

MEMS

Usingtime

Once

Reusable

Application

Biomedicine

Biomedicine,chemistry,environment

Industry

developed

developing

62.4.2Fabrication制造ofgenechipIn-situsynthesisPhotolithgraphicsynthesisMolecularstampMicrodottingDotcontactSprayprintPiezoelectricprint7基因芯片的制作原位合成:光刻合成技术;分子印章技术;压电打印技术微量点样:点接触法;喷墨法2.4.3.Cellchip–multimicroelectrodearray82.4.3.Cellchip–chippatchclamp2.3.4.Tissuechip在基质表面固定大量的、可寻址的微小组织样本，用于高通量检测不同组织中DNA、RNA和蛋白质等分子的变化，称为组织芯片。Fabricationofmicrofluidicchip:Method:

MEMS－MicroelectromechanicalSystemSi硅orglass-basedmethodPolymer聚合物-basedmethod:PDMSSpincoatSU-8Pre-exposurebakingFixmaskandwafertoholderExposurePost-exposurebakeMolddevelop,rinse,dryandclear9PourPDMS10SpincoatPDMSlayerBakePDMS

CutPDMSedgePeeloffPDMSDrillholesChannelCheckWashPlasmatreatmentAttachmentChipbakingPolymer-basedmethod:PMMA11SoftlithographyMicrofluidiccapillaryelectrophoresis微流体毛细管电泳Conventionalcapillaryelectrophoresis常见的122.1、进样13PinchingPinchfactor:p=Ib/IsDispensefactor:d=Ibw/IsAlarieJ.P.etal.Electrophoresis2001,22,312-317.Electrokineticpinching14Is+Isw+Ib+Ibw=0

Is=IbwIsw=Ibg=Ib/Is(gatingfactor)Electrokineticgating15CZEseparationofflavinMetabolitese.g.16Applications1.DNA:genomics2.Protein:proteomics3.Metabolite:metabolomicsormetabonomics4.Clinicdiagnosis临床诊断5.Drugscreening药物筛选6.Singlecellanalysis7.Interactionanalysis交互分析8.Anti-bioterrorism9.Forensicanalysis法医鉴定……17QuadrupoleMSTOFMS182.5.Bio-MSMALDI19ESI500600700800900100011001200130014001500160017001800m/z0100%998.2942.7893.2848.6808.3616.2771.5738.0617.21060.5999.61131.11061.81211.81132.51137.51305.01413.71541.91696.3160001700018000m/z0100%16951.420ESI-Q-TOFMS21Bragg

diffractionequation：

DB=BF=dsin

n=2dsin

222.6.X-raycrystallization1H：2.7.NMR1924:Pauliraisedtheidea;1952:NobelPrizetoBloch(Stanford)&Purcell(Harvard)23ApplicationMolecularstructureidentificationMetabolomicsMedicaldiagnosis242.8.MolecularimagingOpticalimaging光学成像3.-Omicmeasurementplatform1.Genomicplatform2.Transcriptomicplatform3.Proteomicplatform4.Metabolomicplatform25GenomicsTranscriptomicsProteomicsMetabolomicsDNARNAProteinMetaboliteOMICS2627GenomeTranscriptomeProteomeMetabolome~30,000genes~100,000transcripts~1,000,000proteinforms?~2000to5,000metabolitesThe‘omic’s28283.1.Genomicplatform3.1.1.DNAsequencing1.Sangersequencing2.Cyclic-arraysequencing3.1.2.SNP(singlenucleotidepolymorphism,SNP)

3.Nanoporesequencing4.Hybridization-basedsequencing29Whatisgenomics?Genomicsis“thestudyoffunctions

andinteractionsofallthegenesin

thegenome,includingtheirinteractionswithenvironmentalfactors.Collins,FrancisandAlanGuttmacherNEJM,Vol.347:1512-1520.Agenomeis“alltheDNAcontained

inanorganismoracell,whichincludes

boththechromosomeswithinthe

nucleusandtheDNAinmitochondria…allourgenestogether.”30Genomics101:AnIntroductionTraditionalPublichealth

GeneticsRarediseasesSinglegenedisorders单基因失调PublichealthactivitiesNewbornscreening新生儿筛查Reproductivehealth生殖健康Geneticservices31ContemporaryGenetics

当代基因组学CommondiseasesMultiplegenes多基因Gene/environmentinteractionsPublichealthactivities/implicationsChronicdiseases慢性病Infectiousdiseases传染病Environmentalhealth环境健康学Epidemiology流行病学32GeneticMutationsAllofusmayhaveatleastone

geneticmutation.Someareinherited.遗传Othersoccurrandomlyorasaresultofenvironmentalfactors,suchasdiet,drugs,andinfections.Mostdiseaseshavemultifactorialcausation(geneticandenvironmental).Geneticvariationsputindividualsatincreasedriskfordevelopingcertaindiseases,buttheydonotmakeitcertainthatthosediseaseswilloccur.Geneticmutationshavebeen

identifiedthatplayarolein:Chronicdiseases慢性病CancerCardiovasculardisease心血管疾病Occupationaldiseases职业病Bladdercancer膀胱癌InfectiousdiseasesHIV/AIDS33Genesand10U.S.Killers:HeartdiseaseMalignantneoplasms恶性肿瘤Cerebrovasculardiseases脑血管疾病Chroniclower

respiratorydiseases慢性下呼吸道疾病Accidents意外事故

(unintentionalinjuries)Diabetesmellitus糖尿Influenzaandpneumonia流感和肺炎Alzheimer’sdisease阿尔茨海默氏病Nephritis,nephroticsyndrome,andnephrosis(kidneydisease)肾炎、肾病综合征等Septicemia败血症34What’snewingenomics?GenetictestingTodetectmutations检测突变Fordiseasediagnosisandprognosis预知Forthepredictionofdiseaseriskin

individualsorfamiliesSeveralhundredgenetictestsareinuse.Raregeneticdisorders家族遗传疾病

(musculardystrophies,cysticfibrosis,Huntington’sdisease)Complexconditions(breast,ovarian,

andcoloncancers)Pharmacogenomics药物基因组学Thedevelopmentofdrugstailored定做

tospecificsubpopulations亚种群basedongenesPharmacogenomicshasthepotentialto:DecreasesideeffectsofdrugsIncreasedrugeffectivenessMakedrugdevelopmentfasterandlesscostly35What’snewingenomics?(cont.)Recentresearchingenomicsincludes:Learningmoreaboutthegenetic

underpinnings基础

ofchronicdiseasesDevelopingmousemodelsofhumangenesDevelopinggeneticfingerprinting基因指纹鉴别法

forchildhoodcancerConductingstemcellresearch指导干细胞研究Identifyingtumorsuppressorgenes识别肿瘤抑制基因Policydevelopments政策演变relatedto

genomicsinclude:Activitiesrelatedtoanti-discrimination反歧视andethics伦理学ExpandednewbornscreeningNewfundingforresearchonrarediseases36ThemainaimofgeneticsistounderstandthisrelationshipTalkalittlebitaboutwhereIamcomingfromGenomicMedicine:

Predictive,personalized,andpre-emptive预测、个性化、先发制人371.SangersequencingPNAS,74(1977)560.Maxim-GilbertMethod38SangerMethodH2OXdNTPddNTP39Smithetal.Nature,321(1986)674.Couplingwithfluorescenttag荧光标记insteadof*PProberetal.Science,238(1987)336.40FrancisCollins

DirectorofHGPatNIHHumanGenomeProject(HGP)CollinsF.,GalasD.Science,262(1993)43.4142GenomicsTimelines43CapillaryElectrophoresis毛细管电泳444546Laserinducedfluorescencedetector激光激发荧光成像47ABI3700DNASequencerbyDr.Dovichi’sGroupatUA(UW)CapillaryArrayElectrophoresis毛细管阵列电泳48FourKeystonesforSangerSequencingddNTPchainterminationstrategy双脱氧核苷酸链终止2.Fluorescentlabel

荧光标记3.Capillaryelectrophoresisseparation

毛细管电泳分离4.Laserinducedfluorescencedetection

激光激发荧光成像检测49ProtocolofSangerSequencing方案1.GenomicDNAisfragmentedwithshotgunstrategy;2.Clonedtoaplasmid质粒vectorandusedtotransformE.coli;3.BacterialcolonyispickedandplasmidDNAisolated;4.Eachcyclesequencingreactiontakesplacewithinamicroliter-scalevolume,generatingaladderofddNTP-terminated,dye-labeledproducts;5.High-resolutionelectrophoreticseparationwithfourchannellaserinducedfluorescencedetection;6.Dataanalysis(bioinformatics):base-callingalignmentofsequencereads

denovoassembly(e.g.,BLASTorBLAT)genomebrowsingandannotationdatamining……50Advantage1.Longread-lengths(1000bp)2.Highrawaccuracy(99.999%)Disadvantage1.Biologicalbias偏离2.Relativehighcost

0.5$/1kb

5M$for1GB(10-foldcoverage)TheSangersequencingissuitableforsmall-scaleprojectsinthekilobase-to-megabaserange.Thisisaconsequenceofitsgreater‘granularity’(thatis,theabilitytoefficientlyoperateateithersmallorlargeproductionscales)relativetothesecondgenerationtechnologies.51Dr.Mathies’groupatU.C.BerkeleyNewdevelopment:MicrofluidicCapillaryArrayElectrophoresis微流体毛细管阵列电泳OneandHalfgenerationDNASequencer522.Cyclic-arraysequencingThesecondgenerationDNAsequencing:sequencing-by-synthesisDefinition定义:Theconceptofcyclic-arraysequencingcanbedefinedasthesequencingofadense稠密arrayofDNAfeaturesbyiterativecycles迭代循环ofenzymaticmanipulationandimaging-baseddatacollection.GeneralProtocol:1.LibrarypreparationbyrandomfragmentationofDNA;2.invitroligationofcommonadaptorsequences;3.Generationofclonally无性繁殖clustered聚集成群amplicons扩增(insitupolonies,emulsionPCRorbridgePCR);4.Cyclicarraysequencing;5.Dataanalysis.Category:1.454sequencing(RocheAppliedScience,Basel);2.Solexa(Illumina,SanDiego);3.SOLiD(AppliedBiosystems,FosterCity)4.Polonator(Dover,Harvard)5.SingleMoleculeSequencer(Helicos,Cambridge…)531.454sequencingStep1:54Step2:AninvitroconstructedadaptorflankedshotgunlibraryisPCRamplifiedinthecontextofawater-in-oilemulsion.OneofthePCRprimersistetheredtothesurface(5’-attached)ofmicron-scalebeadsthatarealsoincludedinthereaction.Alowtemplateconcentrationresultsinmostbead-containingcompartmentshavingeitherzerooronetemplatemoleculepresent.PCRampliconsarecapturedtothesurfaceofthebead.Afterbreakingtheemulsion,beadsbearingamplificationproductscanbeselectivelyenriched.EachclonallyamplifiedbeadwillbearonitssurfacePCRproductscorrespondingtoamplificationofasinglemoleculefromthetemplatelibrary.55Clonallyamplified

28-

mbeadsgeneratedbyemulsionPCRserveas

sequencing

features,arepre-incubatedwithBacillus

stearothermophilus(Bst)polymeraseandsingle-strandedbindingprotein,andarefurtherrandomlydeposited

toamicrofabricatedarrayofpicoliter-scalewells

(withdimensionssuchthatonlyonebeadwillfitperwell).

Withpyrosequencing,eachcycleconsistsof

theintroductionofasinglenucleotidespecies,

followedbyadditionofsubstrate(luciferin,

adenosine5’-phosphosulphate)todrivelight

productionatwellswherepolymerase-driven

incorporationofthatnucleotidetookplace.

Thisisfollowedbyanapyrasewashtoremove

unincorporatednucleotide.Step3:Strategyforcyclicarraysequencing56PPPPPPPPPOHPPATPATPATP+luciferin+luciferin+luciferinPPPPPP57PPPPPPPPPOHPPATPATPATP+luciferin+luciferin+luciferinPPPPPP581-mer2-mer3-mer4-merBrightnessofflashisproportionaltonumberofnucleotidesaddedFlashbrightnessTCACTTCAAGGGT…Flashistoobright59ATGCReadlength350-400bp200cycles~0.5Gb/run603.Advantage&Disadvantage:

Thekeyadvantageofthe454platformis

read-length.Forexample,the454FLXinstrumentgenerates~400,000

readsperinstrument-runatlengthsof200to300bp.itmaybethemethodofchoiceforcertainapplicationswherelongread-lengthsarecritical(e.g.,denovoassemblyandmetagenomics)

Themajorlimitationofthe454technologyrelatestohomopolymers(thatis,consecutiveinstancesofthesamebase,suchasAAAorGGG).Becausethereisnoterminatingmoietypreventingmultipleconsecutiveincorporationsatagivencycle.Asaconsequence,thedominanterrortypeforthe454platformisinsertion-deletion,ratherthansubstitution.612.Solexa62Step1:GenomicDNALibraryPreparation63Step2:

ClusterGeneration(BridgePCR)hybridizetothelawnofprimersextendedbypolymerases12345Double-strandedmoleculeisdenaturedOriginaltemplateshouldbewashedawayNewlysynthesizedcovalentlyattachedadjacentprimerstoformabridgeHybridizedprimerisextendedbypolymerasesDouble-strandedbridgeisformed6467891011Double-strandedbridgeisdenaturedResult:twocopiessingle-strandedtemplatesadjacentprimerstoformbridgesextendedbypolymerasesmultiplebridgesareformeddsDNAbridgesdenaturedReversestrandscleavedandwashedawayLeavingaclusterwithforwardstrandsonlySequencingprimerishybridizedtoadapter65Step3:SequencingEachsequencingcycleincludesthesimultaneousadditionofamixtureoffourmodifieddeoxynucleotidespecies,eachbearingoneoffourfluorescentlabelsandareversiblyterminatingmoietyatthe3’hydroxylposition.AmodifiedDNApolymerasedrivessynchronousextensionofprimedsequencingfeatures.Thisisfollowedbyimaginginfourchannelsandthencleavageofboththefluorescentlabelsandtheterminatingmoiety.66Automationisimportant!67Nebulizer~400bpIllumina68PPPPPPPPPPSTOPSTOPSTOPSTOPPP69MetzgerM(2009)NatureReviewsGenetics11:31-4670GCTGA…71Flowcell8lanesForpicturetaking:Eachlaneisbrokenupinto100tiles拼贴,eachfluorisimagedseparately–2400picturestakenpercycleCameratimeisthelimitingstep!72PPPPPPPPPPSTOPChemistryproblem1:terminatorisretained保留outofphase73PPPPPPPPPPPSTOPChemistryproblem2:fluor荧光剂isretained74Readlength30–120bp~3–30Gb/runGAIIIllumina>100Gb/runHiSeq90x106reads/lane*102bp/read=9x109bp/lane*16lanes/run=144Gb/run753.Features:Read-lengthsupto36bparecurrentlyroutine.Longerreadsarepossiblebutmayincurahighererrorrate.Read-lengthsarelimitedbymultiplefactors多因素thatcausesignaldecayanddephasing移相,suchasincompletecleavageoffluorescentlabelsorterminatingmoieties.Thedominant显性的errortypeissubstitution代替,rather

thaninsertionsordeletions(Homopolymers均聚物arecertainlylessof

anissuethan454).Averagerawerrorratesareontheorderof1–1.5%.Higheraccuracybaseswitherrorratesof0.1%orlesscanbeidentifiedthroughqualitymetrics质量度量学associatedwitheachbase-call.763.SOLiDSimilarto454platform,inSOLiDstrategygDNAwasfragmentedbyshotgun,andthenamplified放大withEmulsionPCR.微乳液PCR法77Clonallyamplifiedbeadsareusedtogenerateadisordered,densearrayofsequencingfeatures.Sequencingisperformedwithaligase,ratherthanapolymerase,eachsequencingcycleintroducesapartiallydegeneratepopulationoffluorescentlylabeledoctamers.Thepopulationisstructuredsuchthatthelabelcorrelateswiththeidentityofthecentral2bpintheoctamer(thecorrelationwith2bp,ratherthan1bp,isthebasisoftwobaseencoding).Afterligationandimaginginfourchannels,thelabeledportionoftheoctamer(thatis,‘zzz’)iscleavedviaamodifiedlinkagebetweenbases5and6,leavingafreeendforanothercycleofligation.Severalsuchcycleswilliterativelyinterrogateanevenlyspaced,discontiguoussetofbases.Thesystemisthenreset(bydenaturationoftheextendedprimer),andtheprocessisrepeatedwithadifferentoffset(aprimersetbackfromtheoriginalpositionbyoneorseveralbases)suchthatadifferentsetofdiscontiguousbasesisinterrogatedonthenextround.787980Features:814.SingleMoleculeSequencerThereisnoclonalamplificationsteprequired.Poly-A–tailedtemplatemoleculesarecapturedbyhybridizationtosurface-tetheredpoly-Toligomerstoyieldadisorderedarrayofprimedsinglemoleculesequencingtemplates.TemplatesarelabeledwithCy3,suchthatimagingcanidentifythesubsetofarraycoordinateswhereasequencingreadisexpected.Eachcycleconsistsofthepolymerase-drivenincorporationofasinglespeciesoffluorescentlylabelednucleotideatasubsetoftemplates,followedbyfluorescenceimagingofthefullarrayandchemicalcleavageofthelabel.Helicos82MetzgerM(2009)NatureReviewsGenetics11:31-4683105to140Megabasesperhour~35bpaveragereadlength84(2009)Volume27:847Helicos,28Xcoverage,84Gb752CNVs2.8MSNPs85IonTorrentSingle-moleculesequencing86GuptaPK.(2008)Single-moleculeDNAsequencingtechnologiesforfuturegenomicsresearch.TrendsBiotechnol.26:602-11+-Single-moleculesequencing87GuptaPK.(2008)Single-moleculeDNAsequencingtechnologiesforfuturegenomicsresearch.TrendsBiotechnol.26:602-11+-88GuptaPK.(2008)Single-moleculeDNAsequencingtechnologiesforfuturegenomicsresearch.TrendsBiotechnol.26:602-1189PacificBiosciences

Single-moleculesequencing90ZMW:ahole,tensofnanometersindiameter,fabricatedina100nmmetalfilmdepositedonasilicondioxidesubstrate detectionvolume20zeptoliters(10-21liters).excitationemissionemission91PacBiotechnologybackgrounder:/index.php?q=technology-introduction92WhentheDNApolymeraseencountersthenucleotidecomplementarytothenextbaseinthetemplate,itisincorporatedintothegrowingDNAchain.Duringincorporation,theenzymeholdsthenucleotideintheZMWsdetectionvolumefortensofmilliseconds,ordersofmagnitudelongerthantheaveragediffusingnucleotide.Thesystemdetectsthisasaflashofbrightlightbecausethebackgroundisverylow.ThepolymeraseadvancestothenextbaseandtheprocesscontinuestorepeatPacBiotechnologybackgrounder:/index.php?q=technology-introduction93multiplereadsofthesamemolecule94EidJetal.(2009)MoleculesReal-TimeDNASequencingfromSinglePolymerase

Molecules.Science323,133PMID:1902304495Doesitwork?150bpcirculartemplate~93%rawaccuracy15xcoverage99.3%accuracy96PacBioclaimsthat,by2013,thetechnologywillbeabletogivea‘raw’humangenomesequenceinlessthan3min,andacompletehigh-qualitysequencein15min.GuptaPK.(2008)Single-moleculeDNAsequencingtechnologiesforfuturegenomicsresearch.TrendsBiotechnol.26:602-11~2-5bp/sec97Comparison98IonTorrent99Nature475:348(2011)~100bpreads30Mb/run

100101IonTorrentreadquality102Applicationsofnext-generationsequencing1033.Nanoporesequencing1041054.Hybridization-basedsequencing杂交MicroarrayDNAbiochipIntermsofsequencing,limitationsofmicroarraysincludethefollowing:(i)sequencesthatarerepetitiveorsubjecttocrosshybridizationcannoteasilybeinterrogated被询问;(ii)itremainsunclearhowdenovosequencingcanbeachievedwithhybridization;(iii)withoutverycarefuldataanalysis,falsepositivesposeanimportantproblem,anditisnotclearhowtoobtaintheequivalentofredundant多余的coveragethatispossiblewithconventional常用的andcyclic-arraysequencing.Ithaditsgreatestimpactinthecontextofgenome-wideassociationstudies,whichrelyonarray-basedinterrogation(thatis,genotypingbyhybridization)ofahighlydefinedsetofdiscontiguousgenomiccoordinates.106SNP1073.2.TranscriptomicplatformNowthatmoreandmoregenomesequencesarebeingcompleted,newquestionsariselikewhatarethefunctionalrolesofdifferentgenesandinwhatcellularprocessesdotheyparticipate.Howaregenesregulatedandhowdogenesandgeneproductsinteract.Howdoesgeneexpressionlevelsdifferinvariouscelltypesandstatesandhowisgeneexpressionchangedbyvariousdiseasesortreatments.AlthoughmRNAisnottheultimateproductofagene,transcriptionisthefirststepingeneregulationandinformationaboutthetranscriptlevelsisneededforunderstandinggeneregulatorynetworks.Thus,thenewchallengeistoidentifyallgenes,theirexpressionpatternsandtheirfunction.

Transcriptomics

orglobalanalysisofgeneexpression,alsocalledgenome-wideexpressionprofiling,isoneofthetoolsthatisusedtogetanunderstandingofgenesandpathwaysinvolvedinbiologicalprocesses.Theideaunderlyingthisapproachiscalled“guiltbyassociation”,whichmeansthatgenesshowingsimilarityinexpressionpatternmaybefunctionallyrelatedandunderthesamegeneticcontrolmechanism.108ESTsequencing

SAGE:SerialanalysisofgeneexpressioncDNAmicroarraysandoligo-microarrays109

TheSAGEmethod

isbasedoncountingsequencetagsof10-15bpfromcDNAlibraries.Thesetagsarelinkedtogetherinachainandclonedintoavectorfromwhichtheyaresequenced.IncontrasttoESTsequencing,SAGEisbestappliedtoorganismswhosegenomicsequencesareknownorthathaveasubstantialcDNAsequencedatabaseinordertoidentifythegenesthataresequenced.Becauseonlyshortpiecesofsequencearesequencedpergene,theinsertswiththemultiplesequencetagshavetobesequencedseveraltimestoreducetheamountofsequencingmistakes.Inaddition,alargenumberoftagshavetobesequencedinordertoquantifytheexpressionofgenesthathavealowexpressionlevel.SAGE110DNAchip111Microarraydataandannotation112WhatisProteomics?Proteomics

includesnotonlytheidentificationandquantificationofproteins,butalsothedeterminationoftheirlocalizations,modifications,interactions,activities,and,ultimately,theirfunctions.

StanFieldsinScience,2001.1133.3.ProteomicplatformGenome

staticabletoamplify(PCR)homogeneousnovariabilityinamountProteome

dynamic–conditiondependentnoamplificationnon-homogenous(cell-specific)highvariabilityinamount(>106)为什么有蛋白质组学Comparison:Genome-Proteome1143.3.1、Identification&Quantitation3.1.1、From2DGEtoMS3.1.2、FromMDLCtoMS3.1.3、Quantative

Proteomics:SIT&ICAT3.1.4、Microarray-basedproteomics3.1.5、Singlecellproteomics115BLOTStain/BlotImage/DatabaseOrganTissueCells2-DArrayIPGSamplePrepFractionationSolubilizationSDSPAGEOOOOOOOOOOOOOOOOOOOOOOOOOSpotCutterMALDI-TOFMSNanoESI-MS/MSMassSpec2-DArrayProteinID

ImageDatabase

DatabaseSearchPROTEOMEBROWSERMassPrep3.1.1、From2DGEtoMS1162DGE:Snapshotsofacell细胞快照117118TheFirstDimension:IEF119TheSecondDimension:SDS1201211221231564Mouseliver肝脏proteinsIPG4-5IPG4-7IPG5-6IPG6-714912181429124IPG4-7IPG4-7MouseliverproteinsIPG5-6IPG6-7125GelStain着色剂CoomassieStainSilverStainFluorescentStain126GelcuttingGelPickerPickerheadRinsestationCameraandlightassemblyGeltrayMicrotitre-platesandracks127GelDigestionRinsestationSampleneedlesBuffervalvecontrolEnzymecontainerGelplug(sample)platesPeptideextractplates128SamplePreparationforMS

样品制备WashDryplugsCollectenzymeIncubateAddenzymeExtractpeptidesinnewplates129MALDI-TOF

基质辅助激光解吸电离飞行质谱130mPMF131mPMFSequencing132ProteinIDbyESI-MS/MS.表达序列标签(EST)133LCESI-MS/MSIsolateddigestedproteinsCapLCdataprocessinganddatabasesearchingresultsbrowserQ-TofautomatedESI-MS/MS134

.+++++++++SAMPLESOLUTIONNEBULISATIONATMOSPHERICPRESSURESKIMMERVACUUMIONEVAPORATION+++++++++++++ESI135ESI136Q-Tof(MS-MS)137EST138FindSequenceTag139ProteinIDbyLC/MS/MS从新测序(DeNovo)140DeNovosequencing16O/18Olabeledy’’ionsun-labeledbions(N-terminal)(C-terminal)141142Threelevelsofproteomicsequencing

Instrument仪器IdentificationPMF

MALDITOF

50％EST

LC/MS/MS

80％DeNovo LC/MS/MS

>80％143FlexibilityandVariationinthe

ProteomicsProcess

Labelling

and

Quantitation

Separation

Sample

Processing

IdentificationCharacterisation

Protein

Function

AssaysSamplePrepandFractionation

Samples:

Tissues

CellsSerum...Information:Proteins

Targets

Markers...144NumberofProteinsperGenomeHaemophilus 1742E.coli 4413Yeast 6600Caenorhabditis 18000Drosophila 13000Human 80000!

Evenwiththebest2Dgeltechnology,multipleproteinsaremigratingtogetherinE.coliandyeastsamples.145Laborintensive劳动密集Timeconsuming耗费时间Difficulttohandleandautomate难以自动化操作Samplesimbeddedingel样品嵌入Poorabilitytohandleproteinsthatare

hydrophobic疏水的acidicorbasic酸性的MWT<10kDaor>200kDaphosphorylated,glycosylated磷酸化和糖基化的membranebound薄膜束缚

LargeabundantproteinsmasksmalleronesSampleloading(sensitivity)limitedto<250ugand<500ulinvolumeHighresolutionseparationresolve>1,000proteinsIsoelectricpoint等电点andMWTinfoRunsinparallelHistoricalmethodofchoice2-DGelsforProteomics:

LimitationsAdvantages1463.1.2、FromMDLCtoMS147Prefractionation初步分离Removelargeabundantproteins(IgG,albumin)tohelpimprove2D-gelsensitivityEnrichlowabundantproteinstoboost醉翁急啊sensitivityRunGelIncompatible不相容的Samples(“gel-challenged”)Replace/augment2D-gelswithMDLCTechnologyReplace2D-gelswithcompleteMDLCPerformMDLCfollowedby1-DSDSPAGEUseMDLCwithICATtechnologyProteinTargetPurificationFunctionalProteomicsAnalyzeprotein-proteininteractionsWhereCanChromatography色谱技术

TechniquesHelpinProteomics?148ChromatographyforProteomicsBroadsamplerangeEasilyscaledforpreparativeConcentratesamplestoboostsensitivityHandlesanytypeofproteinStraightforwardautomation简单的自动化proteinsstayinliquidphaseEasytocollectfractionsFlexibilityinchemistryandexperimentdesignSamplepreptoMDLCCleanersamplesimprovequalityofMShits.NoPIorMWTinformationAdvantagesLimitations149MDLCScenarios情节

for