The primary goal of this research project is to develop procedures to structurally characterize the carbohydrate side chains of naturally occurring mammalian glycoproteins, despite their limited sample quantities. We are developing a series of glycosidase digestions coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to elucidate the complete primary structures of both N- and O-linked carbohydrate side chains. Preliminary studies have shown that this approach is capable of fully elucidating the primary structures of each carbohydrate side chain attached to a glycoprotein using only 30 picomoles of sample. Similar quantities of released oligosaccharide side chain mixtures and heterogeneous glycopeptides have also been analyzed by the glycosidase/MALDI-MS procedure. These sample requirements are approximately 1,000 times lower than those needed for current analytical procedures used to elucidate the complete primary structures of thes e carbohydrate side chains. The proposed research will refine the glycosidase/MALDI-MS procedure, develop new techniques to simplify these analyses, and test these procedures by analyzing several glycoproteins whose carbohydrate side chains have not been previously characterized. This work offers potential for advancing the field of glycobiology by providing a method to characterize the carbohydrate side chains of the numerous naturally occurring glycoproteins that cannot be obtained in sufficient quantities for analysis by current procedures.
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