Separation of N-glycans by HPLC

Most glycoproteins carry a very heterogeneous mixture of oligosaccharides and even a single glycosylation site of a pure glycoprotein is often heterogeneously glycosylated. The structural diversity of oligosaccharides arises from linkage variants, from differences in the size and number of charges of glycans, and from differences in the monosaccharide composition of glycans. Fortunately, the biosynthetic pathway is subject to certain restrictions, so that structural diversity is limited and amenable to laboratory investigation. Different approaches have been developed to the structural characterization of oligosaccharides, including nuclear magnetic resonance (NMR), mass-spectrometry, linkage analysis by gas chromatography-mass spectrometry (GC-MS), sequence analysis using specific exoglycosidases and others, but a crucial part of these strategies is the separation of the glycan mixture into homogeneous glycan fractions. In this chapter some high-performance liquid chromatography (HPLC)-techniques are described for the isolation of oligosaccharides, in particular N-linked glycans.