Quantitative cleavage of the N-glycosidic bond under the normal conditions of methanolysis used for the analysis of glycoprotein monosaccharides

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.