Determination of the sialylation level and of the ratio α-(2→3)/α-(2→6) sialyl linkages of N-glycans by methylation and GC/MS analysis

A methodology for the determination of the sialylation pattern of N-glycans, extent of sialylation and the ratio between alpha-(2-->3) and alpha-(2-->6) sialyl linkages, is presented based on the labelling of the C-3 and C-6 hydroxyl groups of Gal residues obtained after permethylation, saponification, selective desialylation of sialylated oligosaccharides and methanolysis. Deuteromethylation and GC/MS analysis of Gal derivatives allow to determine the sialylation level of glycans. O-Ethyl ether labelling followed by GC analysis of the resulting Gal derivatives 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 -->3)-beta-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--> 3)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp), LSTc (IV(6)NeuAcn LcOse(4): alpha-Neup5Ac-(2-->6)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp) and a bisialylated biantennary N-glycan in which sialic acid is bound to Gal residues via an alpha-(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.