The analysis of oligosaccharide heterogeneity will be addressed by the development of several complementary analytical methods based on Fourier transform mass spectrometry. Oligosaccharides are involved in a host of biological functions including cell-cell and cell-matrix recognition, hormonal actions, inter- and intracellular trafficking, and protection. However, unlike DNA and proteins where sequence provides nearly all the primary structure, oligosaccharides are characterized by their sequence, linkage, and stereochemistry. Additionally, the large diversity in the monosaccharides due to chemical modification and isomerism, the labile nature of the glycosidic bonds, and the poor intrinsic basicity all combine to make the structural elucidation of oligosaccharides significantly more difficult than other biopolymers. There is currently no analogous method for oligosaccharides with the sensitivity, reliability and accuracy of the Edman degradation for proteins. We propose to develop analytical methods to rapidly elucidate structures in oligosaccharide libraries. The methods will be developed in the study of the jelly coat of the South African toad, Xenopus laevis. X. laevis is an important model for the study of the early stages of fertilization. The jelly coat plays a role in several important processes including the block to polyspermy, prevention of cross fertilization and protection. Collision induced dissociation will be employed to determine structures of the released oligosaccharide alditols. A catalog of structural motifs with their corresponding CID fragmentation pattern (an oligosaccharide fingerprint) will be produced using the structures of known oligosaccharides. From this catalog, the structure of the minor components will be determined. Alkaline degradation (AD) will be used to obtain linkage and sequence information of unknown oligosaccharides. AD coupled with matrix-assisted laser desorption/ionization and Fourier transform mass spectrometry has recently been demonstrated in this laboratory to provide sequence and linkage information on a host of model compounds. The method will be further refined for greater sensitivity and shorter analysis time. It will also be coupled with electrospray ionization. This method will be tested with the oligosaccharides of X. laevis. However, its application is aimed towards libraries where little structural information exists. Finally, a method will be developed to eliminate the tedious process of separation prior to analysis. Strong biotin-avidin interaction will be the basis for the development of an analyte specific MALDI probe. A probe with immobilized avidin will be used to extract biotinylated oligosaccharides directly from solution for immediate MALDI-FTMS analysis. This method bypasses the long separation process necessary for the isolation of released oligosaccharides.
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