High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates

This chapter discusses the basic high-pH anion-exchange chromatography (HPAEC) methods for monosaccharide and oligosaccharide analysis of glycoproteins and a sample preparation approach that extends these methods to the analysis of glycoproteins requiring complicated sample matrices for their isolation. Analogous to amino acid analysis of proteins, hydrolysis with heat and acid is used to release monosaccharides from glycoproteins. The susceptibility of different types of monosaccharides to these conditions varies considerably. Because monosaccharides are hygroscopic, a significant portion of their weight may be from retained water. To prepare a standard monosaccharide solution, the sample must be dried to a constant weight under vacuum. Whenever glycoprotein sample is not limiting, duplicate HCl and trifluoroacetic acid (TFA) hydrolysis should be performed. Because HPAEC gives sufficient chromatographic resolution for all classes of oligosaccharides found on glycoproteins there is a high probability that each peak represents a single oligosaccharide structure; thus, HPAEC is a useful “oligosaccharide mapping” method. Retention times have been shown to vary by less than 1% when internal standards are employed.