Study On Quality And Quantity Analysis Methods For Oligosacchride

Glycosylation is considered to be the one of the most common types of post-translational modifications of proteins. Glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces where their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events. The development of the glycomics is far hinged since the lack of proper analytical methods. In the present dissertation, investigations of both quality and quantity analysis methods for glycomics were studied. The main contents of the present dissertation are as follows:1. A new approach for mass spectrometry detection method of glycan was developed based on on-line permethylation-SPE technique. Permethylation derivatization is preferred to stabilize the sialic acid residues and facilitate the determination of branching and interglycosidic linkages. Solid phase permethylation was carried out to fasten the derivatization procedure and to minimize the side reactions. For the first time carbon nanotubes were used as the absorbent for permethylated glycans at extremely harsh condition. This on-line SPE could be easily combined to either liquid or solid phase permethylation without changing permethylation reaction condition.2. A new strategy was investigated for the glycan analysis of proteins with the same peptide sequence based on the on-line permethylation coupling to mass spectrometry detection. N-glycans released from human Immune Globin G were used to evaluate the reliability of the present method. Then structures of N-glycans form different recombinant EPOs were identified. Different N-glycans were identified for the two recombinant EPOs tested, illustrating the different glycosylations of the two recombinant EPOs since they were from the different cell lines.3. Fast screening for anabolic steroids and glycosteroid in raw urine was achieved using reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. The ion/molecule reaction product and the oxime formed by its dehydration were observed using reactive DESI. The glycosteroid was ionized intact without hydrolysis from polytetrafluoroethylene. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; Suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect.4. An aerosol chemiluminescent (CL) detector coupled to CE for quantity analysis of saccharides analysis has been investigated. The CL emission was generated due to the catalyzing oxidization of saccharides on the surface of porous alumina. The saccharides such as sucrose,α-lactose, maltose, reffinose, galactose, xylose and glucose with only weak UV absorbance can be successfully detected with this CE detector. This detector offers advantage of direct detection of the analytes without involving in a derivatization procedure. In comparing with indirect UV detector, the addition of ionic chromophore into the electrophoresis buffer is therefore no longer needed. Aerosol CL detector is also a robust means over a relative wide range of pH, while high pH value was requested by using amperometric detection. The low baseline shift, low interference, relative wide linear range and long lifetime also make CE-aerosol chemiluminescent detector a potential means for routine analysis of saccharides.