生物化學(xué) Exploring Proteins(講稿)

1§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins2§5.1樣品純化的基本流程TissueHomogenization(勻漿)Filtration(過(guò)濾）Crudeextract（粗提品）desalination(脫鹽)&Concentration(濃縮)

ColumnchromatographySaltingOut(鹽析）Centrifugation(離心)(Cultured)CellCelllysis可使用電泳分析每步的分離效果和最終樣品的純度3GeneralCentrifugation4Ultracentrifugation:separationandmassdeterminationm:themassoftheparticle;v:thepartialspecificvolumeρ:thedensityofthemedium;f:thefrictionalcoefficient(ameasureoftheshapeoftheparticle).5SaltingOutAmmoniumsulfateisthemostpopularsaltused6Dialysis7Concentration高分子量聚乙二醇（PEG）8§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins9Chromatography:separatingsubstancesbyallowingthemtopartitionbetweentwophases,onemobile,onestationary.Differencesincharge,size,Affinity,hydrophobicinteractions,chirality(smallmolecule)canbeexploitedforsubstanceseparationwithchromatography.§5.2層析（Chromatography）10Ion-exchangechromatographySize-exclusionchromatographyAffinitychromatographyHydrophobicinteractionchromatographyChromatography11Ion-exchangechromatography12線性鹽濃度梯度的形成13Size-exclusion(Gel-filtration)chromatographymassdetermination14Affinitychromatography15HydrophobicInteractionChromatography17§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins18Principleofelectrophoresis電泳原理與粒子大小、形狀有關(guān)的系數(shù)粒子所帶電荷大小帶電粒子的泳動(dòng)能力帶電粒子的移動(dòng)速度電場(chǎng)強(qiáng)度19MediaofelectrophoresisMedia:

polyacrylamidegel（聚丙烯酰胺凝膠）Socalled:polyacrylamidegelelectrophoresis,PAGE20丙烯酰胺亞甲基（甲叉）雙丙烯酰胺Formingofpolyacrylamidegel21Nativeelectrophoresis(非變性電泳）

Denaturationelectrophoresis

(變性電泳）

Isoelectricfocusingelectrophoresis(等電聚焦電泳，IEE）

Twodimensional(2D)electrophoresis(二維電泳）Variationofelectrophoresis22非變性電泳：電泳緩沖液、凝膠、上樣緩沖液（溶解樣品的緩沖液）中均不含蛋白質(zhì)的變性因素，電泳過(guò)程中盡量維持蛋白質(zhì)的構(gòu)象不變。變性電泳：除電泳緩沖液外、在凝膠中含有十二烷基磺酸鈉(SDS)，上樣緩沖液中含有SDS和二硫鍵還原劑(如巰基乙醇，二硫蘇糖醇[DTT]),電泳前樣品加熱煮沸9一般約5分鐘。該電泳英文縮寫：SDS23SDS的特點(diǎn)（1）SDS能夠與蛋白質(zhì)結(jié)合，導(dǎo)致蛋白質(zhì)變性。不同蛋白質(zhì)-SDS復(fù)合物形狀相似，都呈橢圓狀；（2）大量SDS的負(fù)電荷消除了不同種類蛋白質(zhì)間的電荷符號(hào)的差異，蛋白分子越大結(jié)合的SDS越多，各蛋白質(zhì)-SDS復(fù)合物的電荷密度（或荷質(zhì)比）趨于一致；（3）自由電泳時(shí)，它們的泳動(dòng)率基本相同，而在適宜濃度的聚丙烯酰胺凝膠介質(zhì)中電泳時(shí)，由于凝膠的分子篩效應(yīng)，電泳遷移率就取決于蛋白質(zhì)-SDS復(fù)合物的大小，也可以說(shuō)是取決于蛋白質(zhì)（亞基）分子量的大小。24SDS用來(lái)估測(cè)蛋白質(zhì)的分子量25Electrophoresis：檢測(cè)分離純化效果26IEF(等電聚焦電泳)separatesproteinsbypIThepHgradientinthegelisformedfirstbysubjectingamixtureofsmallmultichargedPolymersthathavemanypIvalues.27282DelectrophoresisIEE+SDS292Delectrophoresis30§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins31§5.4Sequencing&SynthesisInsulin32Sanger經(jīng)過(guò)10多年的努力，使用超過(guò)100g牛胰島素（bovineinsulin），于1953年公布測(cè)序結(jié)果。因該工作獲得1958年的諾貝爾化學(xué)獎(jiǎng)。首個(gè)蛋白質(zhì)的成功測(cè)序33StepsforsequencingStep1:AnalysisofaacompositionStep2:BreakingdisulfidebondsifexistingStep3:?CleavinglargepeptideintosmallStep4:Sequencingofthesmallpeptides Step5:OrderingthesmallpeptidesStep6:Locatingdisulfidebondsifexisting34Step1:AnalysisofaacompositionAla,2Gly,Asp,Phe,ArgPeptide6NHCl;~110°;~24hIon-exchangechromatography35Step2:BreakingdisulfidebondsifexistingTwomethods36Step3:?CleavinglargepeptideintosmallChemicalcleavagecyanogenbromideEnzymaticcleavage3738Step4:SequencingofthesmallpeptidesThisreagentscanbeusedinSanger’smethod39Step4:SequencingofthesmallpeptidesSanger’smethod40Step4:SequencingofthesmallpeptidesEdman’smethod41Step5:OrderingthesmallpeptidesThr－PheGly－LysThereareatleasttwocleavingways;TheFragmentsmustbeoverlap.42Step6:LocatingdisulfidebondsifexistingMethod1lane1lane3lane2Lane1：BreakingdisulfidebondsandCleavingpeptideintofragmentsLane2：OnlyCleavingpeptideintofragmentsLane1：Marker43Step6:LocatingdisulfidebondsifexistingMethod2：DiagonalelectrophoresisThemixtureofpeptidesissubjectedtoelectrophoresisinasinglelaneinonedirectionandthentreatedwithperformicacid,whichcleavesandoxidizesthedisulfidebonds.Thesampleisthensubjectedtoelectrophoresisintheperpendiculardirection.44測(cè)序例子45Theaasequencesofmanyproteinsarecurrently

deducedfromtheirgenesorcDNA（互補(bǔ)DNA）sequences46Peptidesofupto150residuescanbesynthesizedbyautomatedsolid-phasemethodsmainlyinventedbyR.BruceMerrifield(whowonthe1984NobelPrizeinChemistryforthisaccomplishment)§5.4Sequencing&Synthesis47484950§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA

§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins51IgG型抗體的特點(diǎn)H：heavychainL：lightchainV：variableregionC：constantregion—：二硫鍵抗原的特點(diǎn)52免疫印跡（Immunoblot）的原理53ELISA的原理Enzyme-LinkedImmunoSorbentAssay

54ELISA的種類55§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5免疫印跡與ELISA§5.6質(zhì)譜（MS）與蛋白質(zhì)研究§5.7蛋白質(zhì)3D結(jié)構(gòu)的測(cè)定§5ExploringProteins56帶電粒子在電場(chǎng)/磁場(chǎng)中的運(yùn)動(dòng)行為取決于其荷質(zhì)比(mass-to-chargeratio)，荷質(zhì)比不同的帶電粒子其運(yùn)動(dòng)行為不同，利用這種差異進(jìn)行測(cè)量分析的技術(shù)就是質(zhì)譜(Massspectrometry)。質(zhì)譜是目前測(cè)定分子量最準(zhǔn)確的方法，并且可用于測(cè)定分子結(jié)構(gòu)，蛋白質(zhì)核酸的測(cè)序。實(shí)現(xiàn)質(zhì)譜原理的具體設(shè)計(jì)方案有多種，在生命科學(xué)中應(yīng)用的質(zhì)譜主要是飛行時(shí)間質(zhì)譜(TimeofFlightMassSpectrometer，TOF)。質(zhì)譜（MS）的原理57Matrix-assistedlaserdesorption/ionizationmassspectrometry,MALDIMSMALDIMS（一種TOFMS）58MSpreciselymeasurestheratioofthemasstotheelectricchargeofaparticle(m/z)2is

nextto(m/z)1M:massofproteinX:massoftheaddedgroups(usuallytheH+)N:thenumberofcharges59TandemMS(MS/MS)cangivesequenceinformation60Phe–Pro–Gly–Gln–(Ile/Leu)–Asn–Ala–Asp–(Ile/Leu)–Arg61再示例62§5.1樣品純化的基本流程§5.2層析（Chromatography）§5.3電泳（Electrophoresis）§5.4肽的測(cè)序與合成

Sequencing&Synthesis§5.5