Effect and limitation of excess ammonium on the release of O-glycans in reducing forms from glycoproteins under mild alkaline conditions for glycomic and functional analysis.

Ammonium-based alkali-catalyzed β-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with β1,3-linked cores. In contrast to the previously studied β1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the β1,3-linkage is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages (α1,2-, β1,2-, α1,3-, β1,3-, α1,4-, β1,4-, α1,6-, and β1,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O- and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O- and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glycan release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies.