Separation and identification of O-linked oligosaccharides derived from glycoproteins by high-pH anion-exchange...

A high-pH anion-exchange chromatography (HPAEC) system with a pulsed amperometric detector was developed for analysis of O-linked oligosaccharides in glycoproteins. Since O-linked oligosaccharides are usually released from glycoproteins as alditols by beta-elimination with reduction and they are retained less on an anion-exchange resin than their reducing counterparts, a special set of HPAEC conditions optimized for separation of sialyl O-linked oligosaccharides different from those for N-linked oligosaccharide separation had to be devised. HPAEC separation of desialylated O-linked oligosaccharides is even more difficult because of the lack of carboxylic acids or anomeric hydroxyls. Therefore, a precolumn derivatization technique was developed for these oligosaccharides. Reduced desialylated oligosaccharides were de-N-acetylated followed by N-succinylation, which endowed a negative charge at each amino sugar residue in the oligosaccharides. The desialylated oligosaccharides are derivatized in the same vial and can be easily separated on HPAEC. The new HPAEC technique presented here promises to be a valuable tool for analysis of O-linked oligosaccharides in glycoproteins.