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

Abstract

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 (LcOse 4: β- d-Gal p-(1→3)- β- d-Glc pNAc-(1→3)- β- d-Gal p-(1→4)- d-Glc p), LSTa (IV 3NeuAcLcOse 4: α-Neu p5Ac-(2→3)- β- d-Gal p-(1→3)- β- d-Glc pNAc-(1→3)- β- d-Gal p-(1→4)- d-Glc p), LSTc (IV 6NeuAcnLcOse 4: α-Neu p5Ac-(2→6)- β- d-Gal p-(1→4)- β- d-Glc pNAc-(1→3)- β- d-Gal p-(1→4)- d-Glc p) 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.