Raymond A. Dwek

HPLC Strategies for Profiling and Sequencing Oligosaccharides

The sugars released from a pure glycoprotein often consist of a heterogeneous population containing both neutral and charged oligosaccharides. For example, the single N-glycosylation site in human erythrocyte CD59 is associated with more than 100 neutral and sialylated complex glycans, each representing a different glycoform (1). The existence of such extensive heterogeneity in biologically important glycoproteins requires refined approaches to the analysis of oligosaccharides. The adaptable technology which is described here represents a significant advance towards faster, more automated and more detailed strategies for the rapid profiling and analysis of sugars. Such technologies may be required for major studies, such as the human genome project, which defines DNA in normal and diseased states, and the proteome project, which sets out to analyse the total amount of protein in a living cell. It is worthy of note that genetic diseases are not caused by the genes themselves, but by the products for which the genes code and their post-translational modifications, which include glycosylation. In this chapter two strategies for rapid oligosaccharide analysis are described: Oligosaccharide profiling and detailed structural analysis.

Glycoproteomics: High-Throughput Sequencing of Oligosaccharide Modifications to Proteins

Genomics establishes the relationship between biological processes and gene activity. Proteomics (James 1997), which relates biological activity to the proteins expressed by genes, is fundamental to our understanding of biology. It is the proteins, rather than the genes that encode them, which engage in biological events (Wilkins et al. 1995). Furthermore, most proteins contain post-translational modifications which are the products of enzyme reactions. Since the enzymes are coded for by different genes, the complete structure of an individual protein cannot be determined by reference to either a single gene or the protein sequence alone. One of the most common ways that a protein is modified is by the process of glycosylation, in which oligosaccharides are attached to specific sites encoded in the primary sequence of the protein (Dwek 1996).