Mass Spectrometric Analysis of O-Linked Glycans Released Directly from Glycoproteins in Gels Using β-Elimination

Abstract

Extensive posttranslational modification of proteins through the glycosidic attachment of carbohydrate moieties to the backbone of polypeptides makes glycosylation an important area of study. The function of these glycan chains may be to affect the conformation and stability of a protein (1), or to perform essential roles in cell surface or intracellular recognition and cell adhesion and therefore play important roles in mediating biological activity (2). Analysis of glycoproteins is made difficult by the sheer structural diversity of glycans attached to proteins, and because each glycosylated polypeptide is generally associated with a population of different glycan structures frequently attached at more than one site, possibly via more than one different type of chemical linkage. Consequently, detailed structural analysis of one or more glycans is impractical while the glycans are still attached to the polypep-tide; release of the glycan is therefore required. Traditional methods used for glycan release employ both chemical and enzymatic approaches. The most commonly used chemical methods for the release of O-linked glycans are based on β-elimination in solution. Once the glycans have been released from the protein, methods for the analysis of glycoprotein glycans generally require a combination of techniques that include mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, monosaccharide compositional analysis and linkage analysis.