蛋白质芯片技术介绍

——Thefutureisinyourhands!Theproteinchip2021/5/91Nowadays,thepoteomics(蛋白质的)researchisoneofageneraldirectionofthedevelopmentoflifescienceaftergenomics（基因组学的）research.Proteinstructureandfunctionhavethedirectimpactonthechangeoflifeactivity.Andthelevelofgenetranscription(转录)canonlyreflectsthechangeofgeneexpressionproductsinacertainextent.However,trulyfunctionalproteinprocessesafterthetranscription,translationregulationprocessing,controlingtoformandsoonsteps.Thusthedirectstudyofproteintotheinterpretation(翻译)ofthereallifephenomenonisofgreatimprotance.Butresearchonmeansandmethods(手段方法)oftheproteindoesn'thaveabigdevelopment,sothesearchforeffectiveandfastproteinanalysistechnologyhasbecomeoneofthemostimportantstep.2021/5/92Thencomes

theproteinchip2021/5/93Theproteinchip（蛋白芯片）technologyisquicklydevelopedinrecentyearsinchemicalbiologyasahighandnewtechnology.Itisthebasicprincipleismakevariousproteinsorderlyfixedonallkindsofmediumcarrier（介质载体）asatestchip.Andthen,usingthetagantibodies(抗体)thatarespecifiedbythefluorescent(荧光)substancematchwiththeprotein.Andthecorresponding(对应的)proteinwillmatchwiththechip.WhatisProteinmicrochip???2021/5/94Then,thefluorescentontheantibodywillindicatethecorresponding(对应的)numberofproteinanditsexpression.Alltheotherantibodiesthatdoesn'tmakecomplementarycombinationwillbewashedafterusingthefluorescentscanner(荧光扫描仪)orlaserscanningtechnology(激光扫描共聚技术).Throughanalysisthefluorescenceintensity(强度)ofeachpointonthechipandinteractionrelationshipbetweenproteinandprotein,thepurposeofachievingdeterminingallsortsoffunctionofgeneexpressionwillbedone.

2021/5/95Therearethreetypesofproteinmicroarraysthatarecurrentlyusedtostudythebiochemicalactivitiesofproteins.Alltypesofchips2021/5/96Analyticalmicroarraysarealsoknownascapturearrays.Inthistechnique,alibraryofantibodies,aptamersoraffibodiesisarrayedonthesupportsurface.Theseareusedascapturemoleculessinceeachbindsspecificallytoaparticularprotein.Thearrayisprobedwithacomplexproteinsolutionsuchasacelllysate.Analysisoftheresultingbindingreactionsusingvariousdetectionsystemscanprovideinformationaboutexpressionlevelsofparticularproteinsinthesampleaswellasmeasurementsofbindingaffinitiesandspecificities.Thistypeofmicroarrayisespeciallyusefulincomparingproteinexpressionindifferentsolutions.Forinstancetheresponseofthecellstoaparticularfactorcanbeidentifiedbycomparingthelysatesofcellstreatedwithspecificsubstancesorgrownundercertainconditionswiththelysatesofcontrolcells.Anotherapplicationisintheidentificationandprofilingofdiseasedtissues.2021/5/97Functionalproteinmicroarrays(alsoknownastargetproteinarrays)areconstructedbyimmobilisinglargenumbersofpurifiedproteinsandareusedtoideantifyprotein-protein,protein-DNA,protein-RNA,protein-phospholipid,andprotein-smallmoleculeinteractions,toassayenzymaticactivityandtodetectantibodiesanddemonstratetheirspecificity.Theydifferfromanalyticalarraysinthatfunctionalproteinarraysarecomposedofarrayscontainingfull-lengthfunctionalproteinsorproteindomains.Theseproteinchipsareusedtostudythebiochemicalactivitiesoftheentireproteomeinasingleexperiment.2021/5/98Reversephaseproteinmicroarray(RPPA)involvecomplexsamples,suchastissuelysates.Cellsareisolatedfromvarioustissuesofinterestandarelysed.Thelysateisarrayedontothemicroarrayandprobedwithantibodiesagainstthetargetproteinofinterest.Theseantibodiesaretypicallydetectedwithchemiluminescent,fluorescentorcolorimetricassays.Referencepeptidesareprintedontheslidestoallowforproteinquantificationofthesamplelysates.RPAsallowforthedeterminationofthepresenceofalteredproteinsorotheragentsthatmaybetheresultofdisease.Specifically,post-translationalmodifications,whicharetypicallyalteredasaresultofdiseasecanbedetectedusingRPAs.2021/5/99Theadvantagesofproteinchip1.Allofthecoarsebiologicalsamples(粗生物样品)canbedirectlyanalyzed(serum,urine,bodyfluids)直接用粗生物样品（血清、尿、体液）进行分析2.Itcanrapidlydetectmultiplebiomarkersatthesametime同时快速发现多个生物标记物3.Asmallamountofsample只需要小量样品（约2-10μL）4.Highfluxabilityofvalidation高通量的验证能力（一次性验证多个蛋白质分子）⒌foundlowabundanceproteins发现低丰度蛋白质（即在总蛋白提取物中含量很少的蛋白）2021/5/9106.Determinatethehydrophobicprotein测定疏水蛋白质(疏水蛋白质具有不同寻常的生化性质,很难用常规的蛋白分析方法检测到其存在)7.Inthesamesystem,makecimcdiscoveryanddetectionalltogether.Usingmonoclonalantibodychiptoidentifytheunknownantigen/protein,toreducethedeterminationofproteinsequenceofwork.在同一系统中集发现和检测为一体——利用单克隆抗体芯片，鉴定未知抗原/蛋白质，以减少测定蛋白质序列的工作量。8.Itcanquantitativelyusemonoclonalantibodychip,.Asaresultofthatthecombinationtochipantibodyisquantitative,itcanbeusedtomeasurequantityofantigen.可以定量利用单克隆抗体芯片，由于结合至芯片上的抗体是定量的，故可以测定抗原量.9.Usingmonoclonalantibodymicroarrayscancomplethefunctionofinsufficientcells,suchasdissolvecells,determinatingtheintracellularantigens.What'smore,itssensitivityismuchhigherthanthat.利用单克隆抗体芯片,可互补流式细胞仪不足的功能，如将细胞溶解，可测定细胞内的抗原，而且灵敏度远高于流式细胞仪。2021/5/911Therearefivemajorareaswhereproteinchipsarebeingapplied:diagnostics(诊断),proteomics,proteinfunctionalanalysis,antibodycharacterization,andtreatmentdevelopment.Diagnosticsinvolvesthedetectionofantigensandantibodiesinbloodsamples;theprofilingofseratodiscovernewdiseasebiomarkers;themonitoringofdiseasestatesandresponsestotherapyinpersonalizedmedicine;themonitoringofenvironmentandfood.Proteomicspertainstoproteinexpressionprofilingi.e.whichproteinsareexpressedinthelysateofaparticularcell.Applications2021/5/912Proteinfunctionalanalysisistheidentificationofprotein-proteininteractions(e.g.identificationofmembersofaproteincomplex),protein-phospholipidinteractions,smallmoleculetargets,enzymaticsubstrates(particularlythesubstratesofkinases)andreceptorligands.Antibodycharacterizationischaracterizingcross-reactivity,specificityandmappingepitopes.Treatmentdevelopmentinvolvesthedevelopmentofantigen-specifictherapiesforautoimmunity,cancerandallergies;theidentificationofsmallmoleculetargetsthatcouldpotentiallybeusedasnewdrugs.2021/5/913(1)Establishingafast,cheap,highproteinexpressionandpurificationmethodofflux,highfluxanddefiningeachantibodypreparationofantibodyaffinityspecificity;Thefirstgenerationofproteindetectionchipwillrelymainlyonantibodiesandothermacromolecules,clearly,withthesematerialpreparationcomplexchips,scaleproductionwillexistalotofpracticalproblems,inparticular,theidealsolution