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 α-(2→3) and α-(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 α-(2→3) and α-(2→6) sialyl linkages. The method was applied to LNT (LcOse4: β-d-Galp-(1→3)-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-d-Glcp), LSTa (IV3NeuAcLcOse4: α-Neup5Ac-(2→3)-β-d-Galp-(1→3)-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-d-Glcp), LSTc (IV6NeuAcnLcOse4: α-Neup5Ac-(2→6)-β-d-Galp-(1→4)-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-d-Glcp) and a bisialylated biantennary N-glycan in which sialic acid is bound to Gal residues via an α-(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 α-(2→6) versus α-(2→3) sialyl linkages was 31:19.