21] Glycoform analysis of glycoproteins

Abstract

Publisher Summary This chapter discusses the glycoform analysis of glycoproteins. Analytical strategies to perform glycoform analysis are now well defined and involve the general steps. First, a determination of the primary amino acid sequence of the protein; this is a prerequisite for subsequent assignment of oligosaccharides to specific glycosylated sites along the polypeptide. Second, some characterization of the total pool of oligosaccharides associated with the intact glycoprotein; this is optional, but useful to check the experimental accuracy of site-specific analysis. Third, identification of those amino acid residues that carry N - or O -linked oligosaccharides. Fourth, a separation of glycosylated sites into glycopeptides carrying just one of each glycosylated site and retaining a sufficient length of peptide to allow an assignment of that glycopeptide along the amino acid sequence of the protein. Fifth, a determination of the degree of occupancy of each glycosylated site. Sixth, analysis of the oligosaccharide structures associated with each glycopeptide. Other steps include a reconstruction of the glycoforms constituting the glycoprotein and an experimental check of the accuracy of the analysis. Primary amino acid sequence analysis of the intact glycoprotein can give approximate information on the degree of occupancy of partially glycosylated amino acid residues, but these data are essentially semiquantitative. Where partial site occupancy is indicated, a more accurate method is to perform amino acid sequence analysis on the peptides and glycopeptides obtained after proteolytic or chemical cleavage of the glycoprotein.