DETERMINATION OF THE SIALYLATION LEVEL AND OF THE RATIO ALPHA-(2-]3) ALPHA-(2-]6) SIALYL LINKAGES OF N-GLYCANS BY METHYLATION AND GC/MS ANALYSIS/

A methodology for the determination of the sialylation pattern of N-gl ycans, extent of sialylation and the ratio between alpha-(2-->3) and a lpha-(2-->6) sialyl linkages, is presented based on the labelling of t he C-3 and C-6 hydroxyl groups of Gal residues obtained after permethy lation, saponification, selective desialylation of sialylated oligosac charides and methanolysis. Deuteromethylation and GC/MS analysis of Ga l derivatives allow to determine the sialylation level of glycans. O-E thyl ether labelling followed by GC analysis of the resulting Gal deri vatives allows to obtain the ratio between alpha-(2-->3) and alpha-(2- ->6) sialyl linkages. The method was applied to LNT (LcOse(4): beta-D- Galp-(1 eta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp), LSTa (IV(3) NeuAcLcOse(4): alpha-Neu-p5Ac-(2-->3)-beta-D-Galp-(1--> eta-D-GlcpNAc- (1-->3)-beta-D-Galp-(1-->4)-D-Glcp), LSTc (IV(6)NeuAcn LcOse(4): beta- D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp) and a bisialylated biant ennary N-glycan in which sialic acid is bound to Gal residues via an a lpha-(2-->6) linkage. Using this method, it was found that 92.8% of N- glycans in bovine fetuin is sialylated and that the ratio of alpha-(2- ->6) versus alpha-(2-->3) sialyl linkages was 31:19. (C) 1998 Elsevier Science Ltd. All rights reserved.