A strategy for the identification of site-specific glycosylation in glycoproteins using MALDI TOF MS

• Published in: Tetrahedron: asymmetry 2000-01, Vol.11 (1), p.75-93
• Main Authors: Mills, Kevin, Johnson, Andrew W., Diettrich, Ole, Clayton, Peter T., Winchester, Bryan G.
• Format: Article
• Language: English
• ISSN: 0957-4166; 1362-511X
• DOI: 10.1016/S0957-4166(99)00545-5

A strategy for investigation of site-specific glycosylation of glycoproteins has been developed, based on peptide mass fingerprinting using matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF MS). The glycoprotein is subjected to sequential digestion with a protease and glycan-specific endoglycosidases or with the glycan-specific endoglycosidases followed by the protease. Peptides with characteristic masses are detected for sequences containing glycosylated asparagine residues. By using a panel of three proteases, chymotrypsin, protease V8 and trypsin, and endoglycosidases F3 and H and peptide N-glycanase F, it was possible to monitor the state of glycosylation of all putative N-glycosylation sites on three glycoproteins. It was deduced that all potential N-glycosylation sites in human serum transferrin (two) and α1-antitrypsin (three) were occupied by non-fucosylated, biantennary, disialylated, complex glycans. In contrast, only four (asparagines 19, 59, 146 and 270) out of the five potential sites were glycosylated in recombinant human β-glucosylceramidase, with the site nearest the C-terminal (asparagine 462) being unoccupied. The glycans at each site consisted of a mixture of non-fucosylated and core α1–6 fucosylated oligomannose glycans (Man 3 GlcNAc 2), derived from the enzymic truncation of complex glycans.