| O-linked oligosaccharides in the egg jelly of South African toad, Xenopus laevis, play important roles in the fertilization process. They are involved in the protection of egg and embryo, recognition of sperm and block to polyspermy. Their structural characterization, therefore, is essential to understand the fertilization process. However, this is a difficult task due to the extremely high structural complexity and the low amounts of materials available. Mass spectrometry (MS) has become an important tool for elucidation of oligosaccharide structures. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) has been proven to be powerful, owing to its high sensitivity and capability of tandem MS. This dissertation first addresses the development and improvement of MS methods for structural elucidation of both neutral and acidic O-linked oligosaccharides using MALDI-FTMS. Exoglycosidase digestions monitored by MS analyses were used to validate neutral oligosaccharide structures determined through catalog-library approach, a method developed for structural elucidation based on characteristic fragments observed in the tandem MS experiments. Acidic oligosaccharides are structurally more complicated than neutral ones. Sulfated oligosaccharides were effectively isolated and separated. Unknown structures were determined based on the MS study of a small number of known ones. Fragmentation patterns of substructural motifs containing sulfate esters were revealed.; Jelly oligosaccharides are highly diversified, possibly being an evolutionary result for protection of eggs from pathogens. A rapid and sensitive approach, MS analysis coupled with liquid chromatography (LC), was demonstrated to rapidly profile the expression levels of oligosaccharides in egg jelly of individual organisms, and in individual eggs from same or different individuals. New and improved sample purification and separation techniques, solid phase extraction using porous graphitized carbon (PGC) cartridge and narrow-bore LC separation using columns of PGC and alkylarnine stationary phases, were successfully developed and applied to the studies of individual eggs. Sensitivity and efficiency were dramatically improved.; Improvement of current MS instrumentation was pursued. An Nd-YAG laser was installed to replace the original N2 laser to improve the detection sensitivity. Infrared multiphoton dissociation (IRMPD) was implemented as an alternative fragmentation method and provided structural information complementary to collision-induced dissociation. |
