Novel Fluorescent Methods for Quantitative Determination of Monosaccharides and Sialic Acids in Glycoproteins by Reveresed Phase High Performance Liquid Chromatography

Abstract

Carbohydrate composition analysis of glycoproteins is analogous to the amino acid analysis of proteins and, therefore, is a fundamental part of glycobiology. An accurate composition analysis can give some insights into the type and extent of glycosylation and probable structures of oligosaccharides present in an unknown glycoprotein, based on current knowledge. Obviously, identification and quantitation of the individual monosaccharides present in the purified oligosaccharides is a pre-requisite for structural determination. A review of some useful methods for carbohydrate composition analysis has been published recently (Townsend, 1993). Analysis of monosaccharides and oligosaccharides using high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) has gained some popularity in recent years, since this technique does not involve any derivatization prior to analysis. But, it requires its own hardware components, such as the system for carbohydrate analysis commercially available from Dionex. In addition, PAD is not a truly specific detector for carbohydrates and the glycoprotein hydrolysate containing amino acids, peptides and thiol groups interfere with the monosaccharide analysis.