Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.

The distribution of neutral and sialyl N-linked oligosaccharides from human serum glycoproteins has been studied using three kinds of HPLC columns as described below. N-linked oligosaccharides were released from chymotrypsin- and trypsin-digested glycopeptides of human serum by means of glycoamidase (from almond) digestion. The reducing ends of the oligosaccharides were derivatized with the fluorescent reagent 2-aminopyridine (PA). The mixture of PA-oligosaccharides was separated by high-performance liquid chromatography on a diethylaminoethyl column according to the sialic acid content. Each fraction separated was then applied on an octadecylsilyl (ODS) column, and the elution volume of each peak was recorded as a glucose unit on the X-axis. Then, each of the separated oligosaccharides was applied to the amide column. Each peak's elution volume was recorded as a glucose unit on the Y-axis. The elution volumes from these two columns (ODS and amide) provide a unique set of coordinates. The structure of each sialyl oligosaccharide fraction was analyzed by the same two-dimensional sugar-mapping technique as previously developed for neutral oligosaccharides. This was combined with exoglycosidase digestion and high-resolution proton NMR measurement. Fourteen neutral, eight mon-sialyl, four di-sialyl, and five tri-sialyl oligosaccharides were isolated from human serum and their structures were characterized.