「糖蛋白和糖肽的化学合成【上课课件】」.ppt

糖蛋白和糖肽的化学合成 1 学校类 BiosynthesisofN glycoproteins SynthesizeofalipidboundoligosaccharideastheprecursorsforallthevariousoligosaccharidesexpressedinN glycoproteinsTransferprecursoroligosaccharidefromthelipid boundformtoanasparaginesidechainofapolypeptidechainTrimmingandprocessingoftheoligosaccharide N GlycosylationinEukaryotes 2 学校类 Step1 1ProductionofGlcNAc P P Dolichol MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 3 学校类 GlycosylationmutantsinYeast CHOcells obtainedbyplantlectinresistance andlymphomacellsmissingThy 1glycoprotein obtainedbyantibodykilling Wereusefulinelucidatingthepathway CDG CongenitalDisorderofGlycosylationinHumans Step1 2N GlycanPrecursorontheCytosolicLeafletoftheEndoplasmicReticulum ER MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 4 学校类 Step1 3BiosynthesisoftheN GlycanPrecursoronLumenalLeafletofER MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 5 学校类 Step1 4CompletionofBiosynthesisofN GlycanPrecursoronLumenalLeafletofER MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 6 学校类 Step2TransferPrecursorOligosaccharidetoProtein Oligosaccharyltransferasecomplex OST intheERmembranetransfersthedolicholN glycanprecursortoasparagineresiduesonnascentlytranslatedproteins YeastOSTcomplexcontainsninemembrane boundsubunits Target sequon forN glycosylationNecessarybutnotsufficientTransferco translationalbeforefolding 2 3ofproteinshavesequons 2 3sequonsactuallyoccupied somevariably 7 学校类 ER Golgi Step3 1InitialProcessingofN GlycansintheERandGolgi MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 8 学校类 Finalproductsoftenshow microheterogeneity ateachN Glycosylationsite Step3 2CompletionofProcessingofN GlycansintheERandGolgi MarquardtT DeneckeJ EurJPediatr 2003Jun 162 6 359 79 9 学校类 a N glycosidicasparagine 10 学校类 a O glycosidicserine 11 学校类 Lai XiWang的化学生物学合成方法 12 学校类 Lai XiWang的化学生物学合成方法 13 学校类 Lai XiWang的化学生物学合成方法 14 学校类 寡糖结构分析 15 学校类 概述 自然界的糖 人体中常见的单糖分子 D 葡萄糖Glc D 半乳糖Gal D 甘露糖Man L 岩藻糖FucL 6 脱氧半乳糖 D GlcNAc D GalNAc D ManNAc D 木糖Xyl D 核糖 D 脱氧核糖 L 唾液酸Neu5Ac D 果糖Fru 16 学校类 寡糖结构的要素 各个单糖的化学结构 什么糖 吡喃还是呋喃 糖苷键的链接顺序糖苷键的立体构型 寡糖结构分析的技术手段 质谱分析 需要样品量少 但自身的结构信息少 谱图分析难度大最易断裂处为糖苷键 特别是全甲基化修饰的寡糖HPLC分析 需要对照糖库分子的HPLC信息辅助核磁分析 能给出丰富的结构信息 但需要mg级的样品量和较高纯度化学以及酶法修饰 常作为必要的辅助手段 协助分析 17 学校类 寡糖结构分析中常用的酶法和化学法修饰策略 1 exoglycosidase配合质谱分析 以得到寡糖的结构2 蛋白的N glycan 使用PNGase EndoF或者EndoH处理3 蛋白的O glycan 使用还原消除法处理4 非还原端的荧光集团修饰 2 氨基吡啶等 提高灵敏度最常使用的耦合反应 还原氨化反应5 寡糖酸性水解 唾液酸是最敏感的常见单糖 岩藻糖次之 质谱分析中 类似 醋酸水溶液 唾液酸HF水溶液 Fuc0 5MHCl水溶液 全甲基化的Fuc6 全甲基修饰提高HPLC以及MS分析的灵敏度 更利于区分寡糖分析中的同分异构体对照标准 甲基化 糖库 可提供寡糖中单糖的组分 以及单糖连接顺序信息 18 学校类 EnzymesUsefulindetectingStepsinN glycanBiosynthesis Complex typeglycans 19 学校类 核磁的结构分析 核磁信息 H或者C的化学位移氢谱 糖环上多数的H 化学位移位于3 5 4 5之间Fuc的6位甲基 位于1 1左右唾液酸的3位次甲基 CH2 位于1 8 2 2之间anomeric位置 氢原子b H 一般位于4 5 5 a H一般位于5 5 5之间酰胺的甲基位于1 9左右碳谱 一般糖环上的碳原子一般位于60 80之间anomeric的C 一般位于100左右 a构型的碳原子化学位移数值常小于b构型C H 或者H H的耦合相关 COSY HSQC HMBC HMBC可以协助判断连接顺序HSQC可以协助判断6位C和氢原子信号耦合常数 phase sensitive的COSY二维谱 二面角越小 H H耦合越弱 多数糖 1 2的氢原子耦合常数为1 4 alpha 或者6 10hz beta 半乳糖的4位 和3位以及5位的耦合常数很小很弱NOE的空间效应 分子量接近2000左右 常使用ROSY谱图 可以协助判断连接顺序 以及糖苷键构型 20 学校类 21 学校类 J Am Chem Soc 2008 130 44 pp14420 14421 22 学校类 CarbohydrateChemistry Glycobiology 23 学校类 Importanceofglycoanalysis PharmaceuticalConcernsRegardingCarbohydratesPharmacokinetics Influenceonreceptorbinding Pharmacodynamics Distribution Clearance Defineproductas well characterized Lot to lotvariability DefinitionofintellectualpropertySomeGlycoconjugateDiseaseAssociationsimmunitytoinfectiousdiseases includingHIVrheumatoidarthritis alteredcompositionofIgGandlevelsoftheserummannose bindingprotein priondiseasescongenitaldisordersofglycosylation CDGs rare usuallyresultinginCNSimpairment oralpathologiescysticfibrosisheartpathologiescancer 24 学校类 A DellandH Morris Science291 2001 25 学校类 26 学校类 CarbohydrateAnalysisOffersUniqueChallenges StructuralanalysisofoligosaccharidesismorechallengingthanthatofoligonucleotidesorproteinsSynthesisisnot templatedriven Thisisbecauseofthecomplexitiesintroducedby BranchingLinkagesAnomericitySite specificglycosylation MicroheterogeneityHighSensitivityisessentialasthequantitiesofoligosaccharidesaregenerallylow g sub g 27 学校类 Workflowinglycoanalysis 28 学校类 Effectivemicroisolationofglycoproteins shortLCmicrocolumns lectin basedseparations gelsandblottingtechniques Sampletreatmentatmicroscale microscaleenzymaticandchemicalcleavages sampleclean up possiblyderivatization SeparationofglycanmixturesbyCZE CEC orcapillaryLCSensitiveMSmeasurementstodeterminesequences linkageforms branchingofglycans sitesofglycosylation etc PREREQUISITESFORHIGH SENSITIVITYMEASUREMENTSONGLYCANS 29 学校类 Glycananalysis Thisinvolves CompositionanalysissugarsreleaseformonosaccharidecompositionanalysisCompositionanalysisbyHPAE PADCompositionanalysisbyGC MSGlycanreleasefromglycoproteinforfurtheranalysis ChemicalEnzymaticGlycomicanalysisMS basedapproachesChromatography basedapproachesElectrokinetically drivenapproaches 30 学校类 I CompositionanalysisGlycanreleaseformonosaccharidecompositionanalysis ProvidesdirectevidencethatpolypeptideisglycosylatedProvidesabasisforstructuralelucidationofglycoproteinsMaysuggestclassesofoligosaccharidechainsMayserveasameasureofproductionconsistencyfortherapeuticrecombinantglycoproteinQuantitativereleaseofthemonosaccharidesisaccomplishedbyuseofacid 6MHClat100 C 31 学校类 I CompositionanalysisCompositionanalysisby Highperformanceanionicexchange pulsedamperometricdetection HPAE PAD 32 学校类 I CompositionanalysisCompositionanalysisbyGC MS PreparationofAlditolAcetatesPreparationofTrimethylsilyl TMS methylglycosides 33 学校类 I CompositionanalysisCompositionanalysisbyGC MS PreparationofAlditolAcetates 34 学校类 GLCProfileofAlditolAcetates SupelcoSP2330Column 35 学校类 I CompositionanalysisCompositionanalysisbyGC MS PreparationofTrimethylsilyl TMS MethylGlycosides 36 学校类 I CompositionanalysisExample 37 学校类 II GlycanReleaseEnzymatic N glycans N glycanscanbereleasedenzymaticallyfromglycoproteinsbyseveralcommerciallyavailableenzymes PeptideN glycosidaseF PNGaseF isthemostwidelyused Theenzymecleavesofftheintactglycansasglycosylamines whicharereadilyconvertedtoregularglycans TheenzymaticreleaseofglycansalsoresultsintheconversionofasparaginetoasparticacidattheN glycosylationsiteoftheprotein PNGaseFhasaverywidespecificity cleavingallN glycansexceptthosehaving 1 3 linkagestothereducing terminalGlcNAc TheircorrespondingglycoproteinsarecommonlyfoundinplantsandcanbeenzymaticallycleavedusingPNGaseA Otherendoglycosidasescanbemorespecific suchasEndoglycosidaseH EndoH knowntocleavehigh mannosestructuresandmosthybrids Moreover EndoHhydrolysesthebondbetweenthetwoGlcNAcresiduesofthechitobiosecore leavingaGlcNAcattachedtotheprotein Thisisdisadvantageous sincetheinformationrelatedtothepresenceoffucoseresiduesatthereducingendoftheglycansislost 38 学校类 II GlycanReleaseEnzymatic N glycans Glycoproteinoligosaccharide releasingenzymes GlycoamidaseAandFenzymesaremostcommonlyused arrows R A O NeilJ Chromatogr A720 1996 201 215l 39 学校类 II GlycanReleaseEnzymatic O glycans UnlikeN glycans noendoglycosidasesarereliablyavailableforthereleaseofO linkedoligosaccharides withthepartialexceptionofendo N acetylgalactosaminidase permittingthereleaseofunsubstitutedCore 1O glycans However thishighlyspecificenzymehasverylimiteduse asitdoesnotcovertheothercorestructures Atthistime chemicalreleasemethodsprovidetheonlyuniversalmeansforO linkedglycans 40 学校类 II GlycanReleaseChemical Hydrazinolysis N andO glycans TakasakiandKobata MethodsEnzymol 50 1978 50 improvedhydrazinolysis N andO glycans Pateletal Biochemistry 32 1993 679 Carlson elimination O glycans CarlsonandBlackwell J Biol Chem 243 1968 616 improved elimination O glycans Y Huang T Konse Y Mechref andM V Novotny RapidCommun MassSpectrom 16 2002 1199 1204 ammonia based elimination O glycans Y Huang Y MechrefandM V Novotny Anal Chem 73 2001 6063 41 学校类 II GlycanReleaseChemical Elimination Reductive EliminationofO GlycansStandardreagent 1MNaBH4 0 05MNaOH Drawbacks Oligosaccharidesarereducedtoalditols lackofareducingendnotfeasibleforchromatographyDesaltingisnecessary highconcentrationofsalts 42 学校类 II GlycanReleaseChemical EliminationwithBH3 NH3 Reductive EliminationofO glycanswithBH3 NH3 ammoniaboranecomplex Y Huang T Konse Y Mechref andM V Novotny RapidCommun MassSpectrom 16 2002 1199 1204 43 学校类 II GlycanReleaseChemical ammonia based elimination Y Huang Y MechrefandM V Novotny Anal Chem 73 2001 6063 44 学校类 III Glycomics Glycomics orglycobiologyisadisciplineofbiologythatdealswiththestructureandfunctionofoligosaccharides chainsofsugars Thetermglycomicsisderivedfromthechemicalprefixforsweetnessorasugar glyco andwasformedtofollowthenamingconventionestablishedbygenomics whichdealswithgenes andproteomics whichdealswithproteins Theidentityoftheentiretyofcarbohydratesinacell atissue oranorganismisthuscollectivelyreferredtoastheglycome 45 学校类 III GlycomicAnalysisGlycanpurificationforMS SincetheionyieldandcrystalformationinMALDI MSandESI MSanalysisareadverselyinfluencedbythepresenceofsaltsandbuffers theirpriorremovalbecomesdesirable Manymethodshavebeendevelopedfortheremovalofsaltsandbuffers dropdialysisNafion 117membranessyntheticmembranes polyethyleneandpolypropylene ion exchangeorhydrophobicresinspackedpipettetipsHydrophobicresins 46 学校类 III GlycomicAnalysisSamplepurificationexample SPE 47 学校类 III GlycomicAnalysisSamplepurificationforMALDIMS Positive ionMALDImassspectraoftheN linkedoligosaccharidesderivedfrom1mgofribonucleaseB a beforePNGaseFdigestion b enzymaticdigestwithoutpurification c enzymaticdigesttreatedwithC18packing and d enzymaticdigestedtreatedwiththeSP20SSresin 48 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS InMALDI MSthesampleismixedwithUVabsorbingmatrixe g DHB 2 5 dihydroxybenzoicacid andspottedonsampleplate Thenlaserisshinedonthespotandsubsequentevaporationintothetimeofflighttube 49 学校类 50 学校类 51 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS WhilemassdeterminationthroughMALDI MScanoftenleadtocompositionaldata intermsofisobaricmonosaccharides additionalinformationmustbesecuredthroughothermethodologies Monosaccharidesequences branchingand insomecases linkagescanbedeterminedthroughfragmentationthataglycanmayexperienceineitherapost sourcedecay PSD oracollision induceddissociation ThecombinationofMALDI MSandenzymaticsequencingusingexoglycosidasesprovidesthenecessaryinformationrelatedtosequence branchingandlinkageofaglycan 52 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofGlycansinMALDI MSFragmentationofglycansobservedinMALDI MSissimilartothatobservedinFAB MSandESI MSandisdependentonfactorssuchasionformation itschargestate theenergydepositedintoanion andthetimeavailableforfragment 53 学校类 SchemeofMALDI Q TOFMSconfiguration LinkageanalysisofGlycansinMALDI MS III GlycomicAnalysisMS basedApproaches MALDI MS 54 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS A GlycosidicFragmentation Domon B Costello C E GlycoconjugateJ 1988 5 397 409 LinkageanalysisofGlycansinMALDI MS 55 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS B Cross ringFragmentation Domon B Costello C E GlycoconjugateJ 1988 5 397 409 LinkageanalysisofGlycansinMALDI MS 56 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS Y Mechref A G Baker M V Novotny CarbohydrateRes 313 1998 145 155 GlycosidicFragmentation LinkageanalysisofGlycansinMALDI MS example 57 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS YehiaMechref MilosV NovotnyandCheniKirshnan Anal Chem Anal Chem 75 2003 4895 4903 Cross ringFragmentation LinkageanalysisofGlycansinMALDI MS example 58 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofglycansbyEnzymaticSequencing MALDI MS 59 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofglycansbyEnzymaticSequencing MALDI MS 60 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofglycansbyEnzymaticSequencing MALDI MS 61 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofglycansbyEnzymaticSequencing MALDI MS 62 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS LinkageanalysisofglycansbyEnzymaticSequencing MALDI MS 63 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS Enzymesusedinthesequencing 64 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS PermethylationofOligosaccharidesforMSAnalysisallowssimultaneousanalysisofneutralandsialylatedstructurespermitsreversed phaseLCseparationofpermethylatedstructuresenhancesMSMSSimplifiesMSMSinterpretationThisisaccomplishedbyMethylationofcarbohydratesindimethylsulfoxidebymixingwithpowderedsodiumhydroxideandmethyliodide CiucanuI KerekFCarbohydr Res 1984 131 209 217 Permethylation 65 学校类 III GlycomicAnalysisMS basedApproaches MALDI MS Permethylation example 66 学校类 III GlycomicAnalysisChromatography basedApproaches Methylateexposedhydroxylgroups Hydrolyzeglycosidicbonds Reducewithborohydride Acetylatenewlycreatedhydroxylgroups AnalyzebyGC MS LinkageanalysisbyGC MSofpartiallymethylatedalditolacetates PMAAs 67 学校类 PrimaryFragmentsforPMAAs 68 学校类 PrimaryandSecondaryFragmentsofPMAAsofTerminalGlucose 69 学校类 III GlycomicAnalysisChromatography basedApproaches LinkageAnalysisofTotalN GlycansfromST6Gal IDeficientMicebyGC MS 70 学校类 III GlycomicAnalysisChromatography basedApproaches Nano LC ESI MSofoligosaccharidesreleasedfromKLHbyPNGaseFtreatment A Basepeakchromatogram massrangem z700 2800 B O Massspectraobtainedforthetimewindowsindicatedbyhorizontalbarsin A Sodiumadductsarepresentedwithm zanddeducedmonosaccharidecomposition H hexose N N acetylhexosamine F fucose P pentose sodiumadduct Nom zvaluesaregivenforprotonandpotassiumadducts M Wuhrer C A M Koeleman D A M C H Hokke Anal Chem 76 2004 833 HydrophilicInteractionChromatography HILIC 71 学校类 III GlycomicAnalysisChromatography basedApproaches Comparisonofnegativeion a capillaryLC MSversus b nano LC MSanalysisofneutralO linkedoligosaccharides 5 5ng usinggraphitizedcarbonchromatography basepeakchromatograms CombinedMS1massspectraoftheregionwhereoligosaccharideswereelutedareshownasinserts G N Karlsson N L Wilson H J Wirth P Dawes H Joshi N H Packer RapidCommun MassSpectrom18 2004 2282 Nano LCwithGraphitizedCarbonPackings 72 学校类 III GlycomicAnalysisChromatography basedApproaches LC MALDI TOF TOFMSofon linepermethylatedglycansderivedfromamixtureofglycoproteins Reversed phaseLCanalysisofpermethylated 73 学校类 III GlycomicAnalysisChromatography basedApproaches sequencinglabeledglycanswithNP HPLC Ruddetal Currentopinioninbiotechnology 1997 74 学校类 III GlycomicAnalysisElectrokinetically drivenApproaches CE LabelingofGlyca