This proposal outlines methodology to define all components of a primary carbohydrate sequence at the sensitivities characteristic of contemporary mass spectrometry. Two studies are detailed: (i) completion of a recently initiated sequencing strategy using chemically modified and derivatized glycans; and (ii) investigation of a significantly new and promising area for sequencing underivatized samples. Aspects of the derivatization method have been described, but components of structure remain unresolved. To cope with these details, Dr. Reinhold discusses glycan depolymerization, (to augment CID linkage information), releasing labile termini (to localize sialic acid and fucosyl termini), and studies to closely evaluate chromium trioxide oxidation (to determination anomeric configuration). These objectives can be subdivided into four separate subprojects: first, extend linkage and branching details to complex and hybrid glycans by preparing smaller, overlapping oligomer subsets via partial depolymerization; second, for multibranched (antennae) structures, evaluate methodology for locating labile non-reducing termini, by mild chemical release and CD(3)-remethylation, and stablizing the neuraminyl linkage through carboxyl modification; third, commence a detailed study into the chemistry and applicability of chromium trioxide oxidation (CTO) for the characterization of anomeric configuration in larger and more complex glycans; and fourth, initiate an investigation of negative ion CID to capitalize on the extensive fragmentation observed for chloride adducts. Capillary HPLC-MS and CID will be utilized to understand the diverse and isomeric mixtures anticipated following depolymerization. Chromatographic protocols will be established for the methylated products under investigation, (e.g., modified and methylated glycans, oligosaccharides, and glycosphingolipids), using a new silica C-18 with improved alkane packing densities. Classical techniques of composition and linkage analysis (methanolysis, methylated alditol acetates) by GC-MS will support methods development projects. The overall goal of this research is to obtain sufficient sequence information to completely define high mannose, complex, and hybrid glycotypes with the trace amounts of sample frequently available.
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