Characterization of protein-bound oligosaccharides using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

The first chapter provides an overview of oligosaccharide analysis by mass spectrometry. The significance and general properties of carbohydrates are briefly explained. The principles of matrix-assisted laser desorption/ionization (MALDI) and Fourier transform mass spectrometry (FTMS) are also presented.; The second chapter introduces signal suppression of sialylated by sulfated oligosaccharides in negative mode. This suppression factor is constant over a large concentration range. The method for solving this problem through the use of derivatization and anionic dopants is developed. A mechanism is also proposed to account for the observed suppression effect in MALDI.; Chapter three describes the method for the separation of structural isomers of oligosaccharides using capillary electrophoresis (CE) and mass spectrometry (MS). The use of benzylamine (N-quaternization) as a UV tag for CE separation and detection is investigated. The CE buffer system also is reexamined to improve separation and MS analyses. Off-line CE/MALDI is explored with micro-preparative CE collection. This technique provides a simple and rapid method to separate and analyze oligosaccharides.; Chapter four introduces an analytical approach for the concurrent characterization of N-glycosylation sites and site heterogeneity of oligosaccharides in glycoproteins. The method is based on a combination of non-specific proteolysis, deglycosylation, and MALDI-FTMS. This method is compared with the traditional trypsin approach to show the improved efficiency of our method. Several oligosaccharides and their N-glycosylation sites are studied.; In chapter five, an analytical method for the characterization of cell wall pectin-derived oligosaccharides (PDOs) obtained in tomato tissues infected by Botrytis cinerea is presented. Also, various desalting techniques for acidic oligosaccharides are studied to increase the MS sensitivity. The degree of polymerization of PDOs and the specificity of pectolytic enzymes is confirmed. These preliminary results are important to understand the structure-function relationship of pectin polymers.