Sugar Chain Analysis by Enzymatic Digestion and 2D Mapping by HPLC

Abstract

Structural analysis is crucial to elucidating the physiological functions of oligosaccharides. The rapid development of both hardware and software for NMR spectrometry and mass spectrometry (ESI-MS and MALDI-MS) has enabled significantly faster and easier structural analysis. When analyzing a small amount of oligosaccharide, the combined use of endo- and exo-glycosidases having clear-cut substrate specificities provides large and reliable advantages for structural analysis. For structural analysis using glycosidases, which are used for high-sensitivity detection, oligosaccharides are usually labeled with tritium or fluorescence reagents: 2-aminopyridine (Hase et al. 1978, 1987) or 2-aminobenzamide (Rudd et al. 1997) in advance. When fluorescent reagents are used for labeling oligosaccharides, reverse-phase HPLC can be used for structural analysis because of the increase in the hydrophobicity of sugar chains. Therefore, two-dimensional (2D)-HPLC using both reverse-phase and normal-phase columns can be used for identifying oligosaccharide structures. 2D-HPLC can distinguish the structural difference (anomeric or branching structures) between isomeric oligosaccharides of the same molecular size or sugar composition using a small amount of sample. Here, as a typical example, we describe the structural analysis of a novel N-glycan obtained from royal jelly glycoproteins using fluorescence labeling and 2D-HPLC developed by Hase et al. (1978, 1987).