糖蛋白和糖肽化学合成

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,11/7/2009,#,单击此处编辑母版标题样式,1,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,糖蛋白和糖肽的化学合成,Biosynthesis of N-glycoproteins,Synthesize of a lipid bound oligosaccharide as the precursors for all the various oligosaccharides expressed in N-glycoproteins,Transfer precursor oligosaccharide from the lipid-bound form to an asparagine side chain of a polypeptide chain,Trimming and processing of the oligosaccharide,N-Glycosylation in Eukaryotes,Production of GlcNAc-P-P-Dolichol,Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,GlcNAc,Man,Gal,Sia,Fuc,Glc,Dolichol,GlcNAc,Man,Gal,Sia,Fuc,Glc,Glycosylation mutants in Yeast, CHO cells (obtained by plant lectin resistance),and lymphoma cells missing Thy-1 glycoprotein (obtained by antibody killing),Were useful in elucidating the pathway,CDG = Congenital Disorder of Glycosylation in Humans,N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER),Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ER,GlcNAc,Man,Gal,Sia,Fuc,Glc,Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,GlcNAc,Man,Gal,Sia,Fuc,Glc,Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ER,Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,GlcNAc,Man,Gal,Sia,Fuc,Glc,Step 2,Transfer Precursor Oligosaccharide to Protein,Oligosaccharyltransferase complex (,OST,) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins,Yeast OST complex contains nine membrane-bound subunits,Target “sequon” for N-glycosylation,Necessary but not sufficient,Transfer co-translational before folding,2/3 of proteins have sequons, 2/3 sequons actually occupied,(some variably),GlcNAc,Man,Gal,Sia,Fuc,Glc,ER,Golgi,Initial Processing of N-Glycans in theER and Golgi,Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,GlcNAc,Man,Gal,Sia,Fuc,Glc,Final products often show,“microheterogeneity” at each,N-Glycosylation site,Completion of Processing of N-Glycans in theER and Golgi,Marquardt T, Denecke J.,Eur J Pediatr,. 2003 Jun;162(6):359-79,a-,N,-glycosidic asparagine,a-,O,-glycosidic serine,Lai-Xi Wang的化学生物学合成方法,Lai-Xi Wang的化学生物学合成方法,Lai-Xi Wang的化学生物学合成方法,寡糖结构分析,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 Fuc,L-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,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,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：Fuc水溶液：全甲基化的Fuc,6）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,EndoA,EndoM,PNGF,Enzymes Useful in detecting Steps in N-glycan Biosynthesis,Complex-type glycans,Hybrid glycans,High mannose-type glycans,Peptide:N-glycosidase F (PNGase F),“N-glycanase”,Endo-beta-N-acetyl-,glucosaminidase H (Endo-H),Endo-F,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，化学位移位于之间,Fuc的6位甲基，位于左右,唾液酸的3位次甲基（CH2），位于之间,anomeric位置，氢原子,b,-H，一般位于4.5-5;,a,-H一般位于之间,酰胺的甲基位于左右,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,J. Am. Chem. Soc., 2008, 130 (44), pp 1442014421,Carbohydrate Chemistry & Glycobiology,Importance of glycoanalysis,Pharmaceutical Concerns Regarding Carbohydrates,Pharmacokinetics: Influence on receptor binding.,Pharmacodynamics: Distribution. Clearance.,Define product as “well-characterized”.,Lot-to-lot variability.,Definition of intellectual property,Some Glycoconjugate Disease Associations,immunity to infectious diseases, including HIV,rheumatoid arthritis (altered composition of IgG and levels of the serum mannose-binding protein),prion diseases,congenital disorders of glycosylation (CDGs) (rare, usually resulting in CNS impairment),oral pathologies,cystic fibrosis,heart pathologies,cancer,A. Dell and H. Morris,Science,291, 2001,Carbohydrate Analysis Offers Unique Challenges,Structural analysis of oligosaccharides is more challenging than that of oligonucleotides or proteins,Synthesis is not “template driven”.,This is because of the complexities introduced by:,Branching,Linkages,Anomericity,Site-specific glycosylation.,Microheterogeneity,High Sensitivity is essential as the quantities of oligosaccharides are generally low (g-sub g),Workflow in glycoanalysis,Effective microisolation of glycoproteins (short LC microcolumns; lectin-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 LC,Sensitive MS measurements to determine sequences, linkage forms, branching of glycans, sites of glycosylation, etc,PREREQUISITES FOR HIGH-SENSITIVITY,MEASUREMENTS ON GLYCANS,Glycan analysis,This involves:,Composition analysis,sugars release for monosaccharide composition analysis,Composition analysis by HPAE-PAD,Composition analysis by GC-MS,Glycan release from glycoprotein for further analysis:,Chemical,Enzymatic,Glycomic analysis,MS-based approaches,Chromatography-based approaches,Electrokinetically-driven approaches,I. Composition analysis,Glycan release for monosaccharide composition analysis,Provides direct evidence that polypeptide is glycosylated,Provides a basis for structural elucidation of glycoproteins,May suggest classes of oligosaccharide chains,May serve as a measure of production consistency for therapeutic recombinant glycoprotein,Quantitative release of the monosaccharides is accomplished by use of acid- 6M HCl at 100C,I. Composition analysis,Composition analysis by,(High performance anionic exchange pulsed amperometric detection),HPAE-PAD,I. Composition analysis,Composition analysis by GC-MS,Preparation of Alditol Acetates,Preparation of Trimethylsilyl(TMS) methyl glycosides,I. Composition analysis,Composition analysis by GC-MS,Preparation of Alditol Acetates,GLC Profile of Alditol Acetates(Supelco SP2330 Column),I. Composition analysis,Composition analysis by GC-MS,Preparation of Trimethylsilyl (TMS) Methyl Glycosides,analysis,Example,Release,Enzymatic (N-glycans),N-glycans can be released 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 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 enzymatically cleaved using PNGaseA.,Other endoglycosidases can be more specific, such as,Endoglycosidase 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. This is disadvantageous, since the information related to the presence of fucose residues at the reducing end of the glycans is lost.,Release,Enzymatic (N-glycans),Glycoprotein oligosaccharide-releasing enzymes.,Glycoamidase A and F enzymes are most commonly used (arrows),R.A. ONeil,J. Chromatogr.,A,720 (,1996,) 201-215l,Release,Enzymatic (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.,However, 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,Release,Chemical,Hydrazinolysis (N- and O-glycans),Takasaki and Kobata, Methods Enzymol., 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., 16 (,2002),1199-1204.,ammonia-based -elimination (O-glycans),Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 (,2001,) 6063.,Release,Chemical (-Elimination),Reductive,-Elimination of O-Glycans,Standard reagent: 1 M NaBH4, 0.05 M NaOH,Drawbacks:,Oligosaccharides are reduced to alditols: lack of a reducing end,not feasible for chromatography,Desalting is necessary: high concentration of salts,Release,Chemical (-Elimination with BH,3,.NH,3,),Reductive,-Elimination of O-glycans with BH3.NH3 (ammonia borane complex),Y. Huang, T. Konse, Y. Mechref, and M. V. Novotny,Rapid Commun. Mass Spectrom., 16 (,2002),1199-1204.,Release,Chemical (ammonia-based -elimination),Y. Huang, Y. Mechref and M.V. Novotny, Anal. Chem., 73 (,2001,) 6063.,Glycomics, or,glycobiology,is a discipline of biology that deals with the structure and function of oligosaccharides (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 cell, a tissue, or an organism is thus collectively referred to as the glycome.,Analysis,Glycan purification for MS,Since 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 desirable.,Many methods have been developed for the removal of salts and buffers.,drop dialysis,Nafion-117 membranes,synthetic membranes (polyethylene and polypropylene),ion-exchange or hydrophobic resins packed pipette tips,Hydrophobic resins,Analysis,Sample purification example SPE,Analysis,Sample purification for MALDI MS,Positive-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 treated with the SP20SS resin.,Analysis,MS-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.,III. Glycomic Analysis,MS-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 cases, 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 linkage of a glycan.,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of Glycans in MALDI-MS,Fragmentation of glycans observed in MALDI/MS is similar to that observed in FAB/MS and ESI/MS and is dependent on factors such as,ion formation,its charge state,the energy deposited into an ion, and,the time available for fragment,Scheme of MALDI-Q-TOF MS configuration,Linkage analysis of Glycans in MALDI-MS,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),A. Glycosidic Fragmentation,Domon, B.; Costello, C. E.,GlycoconjugateJ.,1988,5, 397-409.,Linkage analysis of Glycans in MALDI-MS,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-MS,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Y. Mechref, A. G. Baker, M. V. Novotny,Carbohydrate Res.,313 (,1998,): 145155.,Glycosidic Fragmentation,Linkage analysis of Glycans in MALDI-MS; example,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Yehia Mechref, Milos V. Novotny and Cheni Kirshnan,Anal. Chem.,Anal. Chem., 75 (,2003,), 4895-4903.,Cross-ring Fragmentation:,Linkage analysis of Glycans in MALDI-MS; example,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Linkage analysis of glycans by Enzymatic Sequencing (MALDI-MS),III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Enzymes used in the sequencing,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Permethylation of Oligosaccharides for MS Analysis,allows simultaneous analysis of neutral and sialylated structures,permits reversed-phase LC separation of permethylated structures,enhances MSMS,Simplifies MSMS interpretation,This is accomplished by Methylation of carbohydrates in dimethyl sulfoxide by mixing with powdered sodium hydroxide and methyl iodide (Ciucanu I, Kerek F,Carbohydr. Res.,1984,131, 209-217,),Permethylation,III. Glycomic Analysis,MS-based Approaches (MALDI-MS),Permethylation; example,Analysis,Chromatography-based Approaches,Methylate exposed hydroxyl groups.,Hydrolyze glycosidic bonds.,Reduce with borohydride.,Acetylate newly created hydroxyl groups.,Analyze by GC-MS.,Linkage analysis by GC-MS of partially methylated alditol acetates (PMAAs),Primary Fragments for PMAAs,Primary and Secondary Fragments of PMAAs of Terminal Glucose,Analysis,Chromatography-based Approaches,Linkage Analysis of Total N-Glycansfrom ST6Gal-I Deficient Mice by GC-MS,III. Glycomic Analysis,Chromatography-based Approaches,Nano-LC-ESI-MS of oligosaccharides released from KLH by PNGaseF treatment.,􀁺(A) Base peak chromatogram (mass range,m,/,z,700-2800).,􀁺(B-O) Mass spectra obtained for the time windows indicated by horizontal bars in (A). Sodium adducts are presented with,m,/,z,and deduced monosaccharide composition. H, hexose; N,N,-acetylhexosamine; F, fucose; P, pentose; , sodium adduct. No,m,/,z,values are given for proton and potassium adducts.,M. Wuhrer, C.A.M. Koeleman, D.A. M., C.H. Hokke,Anal. Chem.,76 (2004) 833.,Hydrophilic Interaction Chromatography (HILIC),III. Glycomic Analysis,Chromatography-based Approaches,Comparison 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). 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 Spectrom,18 (2004) 2282.,Nano-LC with Graphitized Carbon Packings,III. Glycomic Analysis,Chromatography-based Approaches,LC/MALDI/TOF-TOF MS of on-line permethylated glycans derived from a mixture of glycoproteins.,Reversed-phase LC analysis of permethylated,III. Glycomic Analysis,Chromatography-based Approaches,sequencing labeled glycans with NP-HPLC,Rudd etal. Current opinion in biotechnology, 1997,III. Glycomic Analysis,Electrokinetically-driven Approaches (CE),Labeling of Glycans by Reductive Amination,III. Glycomic Analysis,Electrokinetically-driven Approaches,III. Glycomic Analysis,Electrokinetically-driven Approaches,CE profile of APTS-labeled glycans derived from mAb. The upper trace represents 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 electroosmotic flow; lex488 nm, lem520.,Y. Mechref, J. Muzikar, M.V. Novotny,Electrophoresis, 26(,2005,)20342046,Capillary Electrophoresis,III. Glycomic Analysis,Chip-based Approaches (CE),III. Glycomic Analysis,Chip-based Approaches (CE),(A) ME profiling of serum samples from a noncirrhotic chronic hepatitis 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 Med.,10 (2004) 429.,III. Glycomic Analysis,NMR spectroscopy,Analysis of glycosaminoglycans,Structure of sulfated glycosaminoglycans,Analysis of glycosaminoglycans,2-Cyanoactamide reacts with reducing sugars under basic conditions at high temperature.,Used as post-column derivatization for GAG disaccharide analysis.,Applicable to any separation.,Post-column Labeleing with 2-Cyanoacetamide,Column,1%,2-CA,0.25 M,NaOH,Reaction,Coil,130,o,C,“Cooling,Bath”,UV Detector,Fluorescence,Detector,100 pMole,5 pMole,Analysis of glycosaminoglycans,IP-RP HPLC,IEC HPLC,Flow Path for Post-column Labeling,Analysis of glycosaminoglycans,