This proposal describes continuation of research in developing highly sensitive and informative analytical methodologies for the benefits of glycobiology. Detailed structural information is being sought from minute quantities of complex glycoconjugates (glycoproteins and proteoglycans) through an effective combination of microisolation, microcolumn component separation and modern mass spectrometry. This information includes oligosaccharide sequencies, branching points and monosaccharide linkage forms, all to be achieved at femtomole (10-15 mol) levels. With the major methodological hurdles for submicrogram analyses of N-glycans solved under the present grant, the major emphasis is being placed on analyzing O-glycans at comparable levels. Analytical advances in this area are being sought through a combination of a substantially miniaturized beta-elimination procedure, separation of the formed alditols by microcolumn liquid chromatography and a novel capillary electrochromatographic (CEC) technique using macroporous polymers, and electrospray ionization/ion trap mass spectrometry coupled CEC. The potential of microscale separation and mass-spectrometry methodologies in structural elucidation will be demonstrated in four different research areas: (a) glycoproteins involved in transport and perception of pheromones in mammals; (b) clinically relevant glycosylation mechanisms in bile salt-stimulated lipase of human milk; (c) structural elucidation of glycans in a mate recognition marine pheromone and (d) structural elucidation of glycans in a sperm-binding protein of the egg vitelline envelope, with (b), (c), and (d) done through outside collaboration. The methodological problems of proteoglycan analysis reside primarily in the lack of resolution of their glycosaminoglycan components. The problems of high charge polydispersity will be addressed through the use of capillary electrophoresis in combination with a unique solute end-labeling technique that alters dramatically electromigration of highly charged oligomers and/or through highly selective charge complexation. The analytical advances proposed in the high-sensitivity glycoconjugate analysis should result in pushing the sensitivity of current methodologies by several orders of magnitude.
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