糖蛋白和糖肽的化学合成.pptVIP

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 III.Glycomic AnalysisChromatography-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 III.Glycomic AnalysisChromatography-based Approaches Linkage Analysis of Total N-Glycansfrom ST6Gal-I Deficient Mice by GC-MS III. Glycomic AnalysisChromatography-based Approaches Nano-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 horizontal 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) 833. Hydrophilic Interaction Chromatography (HILIC) III. Glycomic AnalysisChromatography-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 chromato