Chapter 5 Analysis of Glycoconjugates Using High-pH Anion-Exchange Chromatography

This chapter discusses the analysis of monosaccharide and oligosaccharide of glycoproteins, glycolipids, proteoglycans, and glycosylphosphoinositol (GPI) anchors using high-pH anion-exchange chromatography (HPAEC). HPAEC with pulsed amperometric detection (PAD) does not require the derivatization and associated sample preparation steps that are needed for gas chromatography and other high-performance liquid chromatographic (HPLC) approaches. This relative simplicity of HPAEC with PAD for the composition analysis of glycoconjugates has made this fundamental analysis more generally accessible. The analysis of monosaccharides commonly found in the acid hydrolyzates of glycoproteins using isocratic elution (16 mM NaOH) on a Dionex AS-6 ion exchange column is reported in this chapter. Because PAD detects compounds other than carbohydrates, interfering and extraneous peaks can be observed. Quantitative monosaccharide composition is often the initial analysis toward the structural elucidation of glycoprotein glycans. An accurate molar ratio of covalently linked sugars relative to protein (1) provides direct proof that the polypeptide is glycosylated, (2) provides the global basis for strategies for identifying key structural features, (3) suggests classes of oligosaccharide chains, (4) can indicate changes in biosynthetic pathways, and (5) can be used as a quality control measure for therapeutic glycoproteins and glycoconjugates.