Microscale Sequencing of N-Linked Oligosaccharides of Glycoproteins Using Hydrazinolysis, Bio-Gel P-4, and Sequential Exoglycosidase Digestion

Abstract

Hydrazinolysis has been applied to the quantitative liberation of intact N-linked oligosaccharides from small amounts of glycoproteins. The usefulness of this approach was originally described by Mizuochi et al. (1). This method has been extensively utilized for preparation of N-linked oligosaccharides from a number of glycoproteins for structural analysis (2–15). When glycoproteins with N-linked oligosaccharides are heated with anhydrous hydrazine at 100°C for 10 h, almost all peptide bonds in the polypeptide moiety are cleaved and the amino acids are converted to hydrazides, whereas the glycosidic bonds are stable. Simultaneously, there is quantitative cleavage of the GlcNAc-Asn linkage and release of acyl groups linked to the amino groups of amino sugars and sialic acids. Therefore, the procedure for preparing N-linked oligosaccharides from glycoproteins for structural analysis consists of three steps: hydrazinolysis, re-N-acetylation, and reduction with NaB³H₄ for radiolabeling a small amount of liberated oligosaccharides.