STRUCTURE DETERMINATION OF N- AND O-GLYCANS USING SOFT IONIZATION MASS SPECTROMETRY

Abstract

Glycosylation is a crucial event in the post-translational modification of proteins. Numerous investigations have shown clearly that both the primary structure and the conformation of glycoproteins are intimately connected with their biological function and metabolic fate. Many laboratories are currently directing their efforts towards the development of more specific and sensitive methods for the determination of the primary and secondary structure of glycopeptides as a first step, towards the analysis of their biological function. In view of the complexity of the structures involved, it is evident that only a combination of different methods will yield the desired results. In this instance soft ionization mass spectrometry and high resolution NMR have proved to be expecially useful in determining such different structural parameters as, for example, the type and number of sugar components, the sequence of sugar residues, the sites of the glycosidic linkages and their anomeric confuguration, the sites of glycosylation on the peptide chain, and the 3D-conformation of these structures. Glycans linked N-glycosidically through a chitobiosyl-Asn linkage are usually released enzymatically from the peptide chain prior to analysis ‚whereas O-glycans, e.g., of the mucin type with a GalNAc (al-3)-Ser/Thr linkage, can be released by alkaline borohydride treatment. In this contribution special emphasis will be given to the potential of mass spectrometric methods in analysing the complex mixtures that are obtained after enzymatic release of N-glycans from a single glycosylation site, or after alkaline borohydride treatment of mucin O-glycans.