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-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 (LcOse4: beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)-beta-D- Galp-(1-->4)-D-Glcp), LSTa (IV3NeuAcLcOse4: alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D- GlcpNac-(1-->3-beta-D-Galp-(1-->4)-D-Glcp), LSTc (IV6NeuAcn LcOse4: 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.
Cointe
D
D
Laboratoire de Chimie Biologique, CNRS No 111, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Leroy
Y
Y
Chirat
F
F
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D008401:Gas Chromatography-Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D000509:alpha-Fetoproteins / Q000737:chemistry*
D011134:Polysaccharides; D000509:alpha-Fetoproteins; D019158:N-Acetylneuraminic Acid