《糖蛋白和糖肽的化学合成#上课课件》由会员分享，可在线阅读，更多相关《糖蛋白和糖肽的化学合成#上课课件（85页珍藏版）》请在金锄头文库上搜索。
1、糖蛋白和糖肽的化学合成糖蛋白和糖肽的化学合成1学校类Biosynthesis of N-glycoproteins1.Synthesize of a lipid bound oligosaccharide as the precursors for all the various oligosaccharides expressed in N-glycoproteins2.Transfer precursor oligosaccharide from the lipid-bound form to an asparagine side chain of a polypeptide chain3
2、.Trimming and processing of the oligosaccharideN-Glycosylation in Eukaryotes2学校类Step 1.1Production of GlcNAc-P-P-DolicholMarquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79 GlcNAcManGalSiaFucGlcDolichol3学校类GlcNAcManGalSiaFucGlcGlycosylation mutants in Yeast, CHO cells (obtained by plant le
3、ctin resistance) and lymphoma cells missing Thy-1 glycoprotein (obtained by antibody killing)Were useful in elucidating the pathwayCDG = Congenital Disorder of Glycosylation in HumansStep 1.2N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER)Marquardt T, Denecke J. Eur J Pe
4、diatr. 2003 Jun;162(6):359-79 4学校类Step 1.3 Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ERGlcNAcManGalSiaFucGlcMarquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79 5学校类GlcNAcManGalSiaFucGlcStep 1.4Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ERMarqu
5、ardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79 6学校类GlcNAcManGalSiaFucGlcStep 2 Transfer Precursor Oligosaccharide to ProteinOligosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteinsYeast OST
6、complex contains nine membrane-bound subunitsTarget “sequon” for N-glycosylation Necessary but not sufficientTransfer co-translational before folding 2/3 of proteins have sequons 2/3 sequons actually occupied (some variably)7学校类GlcNAcManGalSiaFucGlcERGolgiStep 3.1 Initial Processing of N-Glycans in
7、theER and GolgiMarquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79 8学校类GlcNAcManGalSiaFucGlcFinal products often show“microheterogeneity” at eachN-Glycosylation siteStep 3.2 Completion of Processing of N-Glycans in theER and GolgiMarquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-7
8、9 9学校类a-a-N-glycosidic asparagine10学校类a-a-O-glycosidic serine11学校类Lai-Xi Wang的化学生物学合成方法的化学生物学合成方法12学校类Lai-Xi Wang的化学生物学合成方法的化学生物学合成方法13学校类Lai-Xi Wang的化学生物学合成方法的化学生物学合成方法14学校类寡糖结构分析寡糖结构分析15学校类概述概述自然界的糖：人体中常见的单糖分子自然界的糖：人体中常见的单糖分子D-葡萄糖葡萄糖 GlcD-半乳糖半乳糖 GalD-甘露糖甘露糖 ManL-岩藻糖岩藻糖 FucL-6-脱氧半乳糖脱氧半乳糖D-GlcNAcD-G
9、alNAcD-ManNAcD-木糖木糖 XylD-核糖核糖D-脱氧核糖脱氧核糖L-唾液酸唾液酸 Neu5AcD-果糖果糖 Fru16学校类寡糖结构的要素寡糖结构的要素: 各个单糖的化学结构（什么糖，吡喃还是呋喃）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链接顺序 糖苷键的立体构型糖苷键的立体构型寡糖结构分析的技术手段寡糖结构分析的技术手段: 质谱分析：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最易断裂处为糖苷键，特别是全甲基化修饰的寡糖 HPLC分析：分析：需要
10、对照糖库分子的需要对照糖库分子的HPLC信息辅助信息辅助 核磁分析：核磁分析：能给出丰富的结构信息，但需要能给出丰富的结构信息，但需要mg级的样品量和较高纯度级的样品量和较高纯度 化学以及酶法修饰：化学以及酶法修饰：常作为必要的辅助手段，协助分析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：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
11、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EndoAEndoMPNGF18学校类Enzymes Useful in detecting Steps in N-glycan BiosynthesisComplex-type glycansHybrid glycansHigh mannose-type gly
12、cansPeptide:N-glycosidase F (PNGase F)“N-glycanase”Endo-beta-N-acetyl-glucosaminidase H (Endo-H)Endo-F19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
13、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), pp 144201442122学校类Ca
14、rbohydrate Chemistry & Glycobiology23学校类Importance of glycoanalysisPharmaceutical Concerns Regarding CarbohydratesPharmacokinetics: Influence on receptor binding.Pharmacodynamics: Distribution. Clearance.Define product as “well-characterized”.Lot-to-lot variability.Definition of intellectual propert
15、y Some Glycoconjugate Disease Associationsimmunity to infectious diseases, including HIVrheumatoid arthritis (altered composition of IgG and levels of the serum mannose-binding protein)prion diseasescongenital disorders of glycosylation (CDGs) (rare, usually resulting in CNS impairment)oral patholog
16、iescystic fibrosisheart pathologiescancer24学校类A. Dell and H. Morris, Science291, 200125学校类26学校类Carbohydrate Analysis Offers Unique ChallengesStructural analysis of oligosaccharides is more challenging than that of oligonucleotides or proteinsSynthesis is not “template driven”.This is because of the
17、complexities introduced by:BranchingLinkagesAnomericitySite-specific glycosylation.Microheterogeneity High Sensitivity is essential as the quantities of oligosaccharides are generally low (g-sub g)27学校类Workflow in glycoanalysis28学校类Effective microisolation of glycoproteins (short LC microcolumns; le
18、ctin-based separations; gels and blotting techniques)Sample treatment at microscale (microscale enzymatic and chemical cleavages; sample clean-up; possibly derivatization)Separation of glycan mixtures by CZE, CEC, or capillary LCSensitive MS measurements to determine sequences, linkage forms, branch
19、ing of glycans, sites of glycosylation, etcPREREQUISITES FOR HIGH-SENSITIVITY MEASUREMENTS ON GLYCANS29学校类Glycan analysisThis involves:1.Composition analysissugars release for monosaccharide composition analysisComposition analysis by HPAE-PADComposition analysis by GC-MS2.Glycan release from glycop
20、rotein for further analysis:ChemicalEnzymatic3.Glycomic analysisMS-based approachesChromatography-based approachesElectrokinetically-driven approaches30学校类I. Composition analysis Glycan release for monosaccharide composition analysisProvides direct evidence that polypeptide is glycosylatedProvides a
21、 basis for structural elucidation of glycoproteinsMay suggest classes of oligosaccharide chainsMay serve as a measure of production consistency for therapeutic recombinant glycoproteinQuantitative release of the monosaccharides is accomplished by use of acid- 6M HCl at 100C31学校类I. Composition analys
22、isComposition analysis by (High performance anionic exchange pulsed amperometric detection) HPAE-PAD32学校类I. Composition analysis Composition analysis by GC-MSPreparation of Alditol AcetatesPreparation of Trimethylsilyl(TMS) methyl glycosides33学校类I. Composition analysis Composition analysis by GC-MSP
23、reparation of Alditol Acetates34学校类GLC Profile of Alditol Acetates(Supelco SP2330 Column)35学校类I. Composition analysis Composition analysis by GC-MSPreparation of Trimethylsilyl (TMS) Methyl Glycosides36学校类I.Composition analysisExample37学校类II.Glycan ReleaseEnzymatic (N-glycans)N-glycans can be releas
24、ed enzymatically from glycoproteins by several commercially available enzymes. Peptide N-glycosidase F (PNGaseF) is the most widely used. The enzyme cleaves off the intact glycans as glycosylamines, which are readily converted to regular glycans. The enzymatic release of glycans also results in the
25、conversion of asparagine to aspartic acid at the N-glycosylation site of the protein. PNGaseF has a very wide specificity, cleaving all N-glycans except those having (1-3) linkages to the reducing-terminal GlcNAc. Their corresponding glycoproteins are commonly found in plants and can be enzymaticall
26、y cleaved using PNGaseA. Other endoglycosidases can be more specific, such asEndoglycosidase H (Endo H) known to cleave high-mannose structures and most hybrids. Moreover, Endo H hydrolyses the bond between the two GlcNAc residues of the chitobiose core, leaving a GlcNAc attached to the protein. Thi
27、s is disadvantageous, since the information related to the presence of fucose residues at the reducing end of the glycans is lost. 38学校类II.Glycan ReleaseEnzymatic (N-glycans)Glycoprotein oligosaccharide-releasing enzymes.Glycoamidase A and F enzymes are most commonly used (arrows)R.A. ONeil J. Chrom
28、atogr. A720 (1996) 201-215l 39学校类II.Glycan ReleaseEnzymatic (O-glycans)Unlike N-glycans, no endoglycosidases are reliably available for the release of O-linked oligosaccharides, with the partial exception of endo-N-acetylgalactosaminidase, permitting the release of unsubstituted Core-1 O-glycans. Ho
29、wever, this highly specific enzyme has very limited use, as it does not cover the other core structures. At this time, chemical release methods provide the only universal means for O-linked glycans 40学校类II.Glycan ReleaseChemicalHydrazinolysis (N- and O-glycans)Takasaki and Kobata, Methods Enzymol.,
30、50 (1978) 50. improved hydrazinolysis (N- and O-glycans)Patel et al., Biochemistry, 32 (1993) 679. Carlson -elimination (O-glycans)Carlson and Blackwell, J. Biol. Chem., 243 (1968) 616. improved -elimination (O-glycans)Y. Huang, T. Konse, Y. Mechref, and M. V. Novotny, Rapid Commun. Mass Spectrom.,
31、16 (2002) 1199-1204. ammonia-based -elimination (O-glycans) Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 ( 2001) 6063. 41学校类II.Glycan ReleaseChemical (-Elimination)Reductive -Elimination of O-Glycans Standard reagent: 1 M NaBH4, 0.05 M NaOHDrawbacks:Oligosaccharides are reduced to alditols
32、: lack of a reducing endnot feasible for chromatography Desalting is necessary: high concentration of salts42学校类II.Glycan ReleaseChemical (-Elimination with BH3.NH3)Reductive -Elimination of O-glycans with BH3.NH3 (ammonia borane complex)Y. Huang, T. Konse, Y. Mechref, and M. V. Novotny, Rapid Commu
33、n. Mass Spectrom., 16 (2002) 1199-1204.43学校类II.Glycan ReleaseChemical (ammonia-based -elimination)Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 ( 2001) 6063.44学校类III.GlycomicsGlycomics, or glycobiology is a discipline of biology that deals with the structure and function of oligosaccharides
34、 (chains of sugars). The term glycomics is derived from the chemical prefix for sweetness or a sugar, glyco-, and was formed to follow the naming convention established by genomics (which deals with genes) and proteomics (which deals with proteins). The identity of the entirety of carbohydrates in a
35、 cell, a tissue, or an organism is thus collectively referred to as the glycome.45学校类III.Glycomic AnalysisGlycan purification for MSSince the ion yield and crystal formation in MALDI/MS and ESI-MS analysis are adversely influenced by the presence of salts and buffers, their prior removal becomes des
36、irable. Many methods have been developed for the removal of salts and buffers.drop dialysisNafion-117 membranessynthetic membranes (polyethylene and polypropylene)ion-exchange or hydrophobic resins packed pipette tipsHydrophobic resins 46学校类III.Glycomic AnalysisSample purification example SPE 47学校类I
37、II.Glycomic AnalysisSample purification for MALDI MSPositive-ion MALDI mass spectra of the N-linked oligosaccharides derived from 1 mg of ribonuclease B:(a) before PNGaseF digestion; (b) enzymatic digest without purification; (c) enzymatic digest treated with C18 packing; and (d) enzymatic digested
38、treated with the SP20SS resin.48学校类III.Glycomic AnalysisMS-based Approaches (MALDI-MS)In MALDI-MS the sample is mixed with UV absorbing matrix e.g. DHB (2,5-dihydroxybenzoic acid) and spotted on sample plate.Then laser is shined on the spot and subsequent evaporation into the time of flight tube.49学
39、校类50学校类51学校类III. Glycomic AnalysisMS-based Approaches (MALDI-MS)While mass determination through MALDI/MS can often lead to compositional data (in terms of isobaric monosaccharides) additional information must be secured through other methodologies.Monosaccharide sequences, branching and, in some ca
40、ses, linkages can be determined through fragmentation that a glycan may experience in either a post-source decay (PSD) or a collision-induced dissociation. The combination of MALDI/MS and enzymatic sequencing using exoglycosidases provides the necessary information related to sequence, branching and
41、 linkage of a glycan. 52学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS) Linkage analysis of Glycans in MALDI-MSFragmentation of glycans observed in MALDI/MS is similar to that observed in FAB/MS and ESI/MS and is dependent on factors such asion formation,its charge state,the energy deposite
42、d into an ion, and the time available for fragment 53学校类Scheme of MALDI-Q-TOF MS configurationLinkage analysis of Glycans in MALDI-MSIII. Glycomic Analysis MS-based Approaches (MALDI-MS)54学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)A. Glycosidic FragmentationDomon, B.; Costello, C. E. Gl
43、ycoconjugateJ.1988, 5, 397-409. Linkage analysis of Glycans in MALDI-MS55学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)B. Cross-ring Fragmentation Domon, B.; Costello, C. E. GlycoconjugateJ.1988, 5, 397-409. Linkage analysis of Glycans in MALDI-MS56学校类III. Glycomic Analysis MS-based Approa
44、ches (MALDI-MS)Y. Mechref, A. G. Baker, M. V. Novotny, Carbohydrate Res.,313 (1998): 145155.Glycosidic FragmentationLinkage analysis of Glycans in MALDI-MS; example57学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Yehia Mechref, Milos V. Novotny and Cheni Kirshnan, Anal. Chem., Anal. Chem.,
45、75 (2003), 4895-4903. Cross-ring Fragmentation:Linkage analysis of Glycans in MALDI-MS; example58学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS)59学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Linkage analysis of glycans
46、 by Enzymatic Sequencing (MALDI-MS)60学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS)61学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS)62学校类III. Glycomic Analys
47、is MS-based Approaches (MALDI-MS)Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS)63学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Enzymes used in the sequencing64学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Permethylation of Oligosaccharides for MS Analysis qallows
48、simultaneous analysis of neutral and sialylated structuresqpermits reversed-phase LC separation of permethylated structuresqenhances MSMSqSimplifies MSMS interpretationThis is accomplished by Methylation of carbohydrates in dimethyl sulfoxide by mixing with powdered sodium hydroxide and methyl iodid
49、e (Ciucanu I, Kerek F Carbohydr. Res. 1984,131, 209-217)Permethylation65学校类III. Glycomic Analysis MS-based Approaches (MALDI-MS)Permethylation; example66学校类III.Glycomic AnalysisChromatography-based ApproachesMethylate exposed hydroxyl groups.Hydrolyze glycosidic bonds.Reduce with borohydride.Acetyla
50、te newly created hydroxyl groups.Analyze by GC-MS.Linkage analysis by GC-MS of partially methylated alditol acetates (PMAAs)67学校类Primary Fragments for PMAAs68学校类Primary and Secondary Fragments of PMAAs of Terminal Glucose69学校类III.Glycomic AnalysisChromatography-based ApproachesLinkage Analysis of To
51、tal N-Glycansfrom ST6Gal-I Deficient Mice by GC-MS70学校类III. Glycomic AnalysisChromatography-based ApproachesNano-LC-ESI-MS of oligosaccharides released from KLH by PNGaseF treatment. (A) Base peak chromatogram (mass range m/z700-2800). (B-O) Mass spectra obtained for the time windows indicated by ho
52、rizontal bars in (A). Sodium adducts are presented with m/zand deduced monosaccharide composition. H, hexose; N, N-acetylhexosamine; F, fucose; P, pentose; , sodium adduct. No m/zvalues are given for proton and potassium adducts. M. Wuhrer, C.A.M. Koeleman, D.A. M., C.H. Hokke, Anal. Chem.76 (2004)
53、833. Hydrophilic Interaction Chromatography (HILIC)71学校类III. Glycomic AnalysisChromatography-based ApproachesComparison of negative ion (a) capillary LC/MS versus (b) nano-LC/MS analysis of neutral O-linked oligosaccharides (5.5 ng) using graphitized carbon chromatography (base peak chromatograms).
54、Combined MS1 mass spectra of the region where oligosaccharides were eluted are shown as inserts. G.N. Karlsson, N.L. Wilson, H.-J. Wirth, P. Dawes, H. Joshi, N.H. Packer, Rapid Commun. Mass Spectrom18 (2004) 2282.Nano-LC with Graphitized Carbon Packings72学校类III. Glycomic AnalysisChromatography-based
55、 ApproachesLC/MALDI/TOF-TOF MS of on-line permethylated glycans derived from a mixture of glycoproteins.Reversed-phase LC analysis of permethylated73学校类III. Glycomic AnalysisChromatography-based Approachessequencing labeled glycans with NP-HPLCRudd etal. Current opinion in biotechnology, 199774学校类II
56、I. Glycomic AnalysisElectrokinetically-driven Approaches (CE)Labeling of Glycans by Reductive Amination75学校类III. Glycomic AnalysisElectrokinetically-driven Approaches 76学校类III. Glycomic AnalysisElectrokinetically-driven ApproachesCE profile of APTS-labeled glycans derived from mAb. The upper trace r
57、epresents standard core-fucosylated biantennary/disialylated, monosialylated, and asialylated glycans. Conditions: column, polyacrylamide-coated 50/365 mm ID/OD; length, 50.5 cm total, 40.5 cm effective length; temperature, 257C; injection pressure, 0.5 psi for 5.0 s; voltage, 15 kV anodic electroos
58、motic flow; lex488 nm, lem520. Y. Mechref, J. Muzikar, M.V. Novotny, Electrophoresis, 26(2005)20342046Capillary Electrophoresis77学校类III. Glycomic AnalysisChip-based Approaches (CE)78学校类III. Glycomic AnalysisChip-based Approaches (CE)(A) ME profiling of serum samples from a noncirrhotic chronic hepat
59、itis patient (upper trace) and cirrhotic patient (lower trace). (B) Profiling of the same samples using the ABI377 gel-based DNA-sequencer. Symbols: , N-acetylglucosamine; , mannose; , galactose; , fucose. N. Callewaert, H. van Vlierberghe, A. van Hecke, W. Laroy, J. Delanghe, R. Contreas, Nature Me
60、d.10 (2004) 429.79学校类III. Glycomic Analysis NMR spectroscopy80学校类Analysis of glycosaminoglycans81学校类Structure of sulfated glycosaminoglycans82学校类Analysis of glycosaminoglycans 2-Cyanoactamide reacts with reducing sugars under basic conditions at high temperature. Used as post-column derivatization f
61、or GAG disaccharide analysis. Applicable to any separation.Post-column Labeleing with 2-Cyanoacetamide83学校类Column1%2-CA0.25 MNaOHReactionCoil130oC“CoolingBath”UV DetectorFluorescenceDetector100 pMole5 pMoleAnalysis of glycosaminoglycansIP-RP HPLCIEC HPLCFlow Path for Post-column Labeling84学校类Analysis of glycosaminoglycans85学校类
