An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins.

A sensitive, rapid, quantitative strategy has been developed for O-glycan analysis. A structural database has been constructed that currently contains analytical parameters for more than 50 glycans, enabling identification of O-glycans at the subpicomole level. The database contains the structure, molecular weight, and both normal and reversed-phase HPLC elution positions for each glycan. These observed parameters reflect the mass, three-dimensional shape, and hydrophobicity of the glycans and, therefore, provide information relating to linkage and arm specificity as well as monosaccharide composition. Initially the database was constructed by analyzing glycans released by mild hydrazinolysis from bovine serum fetuin, synthetic glycopeptides, human glycophorin A, and serum IgA1. The structures of the fluorescently labeled sugars were determined from a combination of HPLC data, mass spectrometric composition and mass fragmentation data, and exoglycosidase digestions. This approach was then applied to human neutrophil gelatinase B and secretory IgA, where 18 and 25 O-glycans were identified, respectively, and the parameters of these glycans were added to the database. This approach provides a basis for the analysis of subpicomole quantities of O-glycans from normal levels of natural glycoproteins.