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

A strategy for investigation of site-specific glycosylation of glycoprotein s has been developed, based on peptide mass fingerprinting using matrix ass isted laser desorption ionisation time of flight mass spectrometry (MALDI T OF MS). The glycoprotein is subjected to sequential digestion with a protea se and glycan-specific endoglycosidases or with the glycan-specific endogly cosidases followed by the protease. Peptides with characteristic masses are detected for sequences containing glycosylated asparagine residues. By usi ng a panel of three proteases, chymotrypsin, protease V8 and trypsin, and e ndoglycosidases F3 and H and peptide N-glycanase F, it was possible to moni tor the state of glycosylation of all putative N-glycosylation sites on thr ee glycoproteins. It was deduced that all potential N-glycosylation sites i n human serum transferrin (two) and alpha 1-antitrypsin (three) were occupi ed by non-fucosylated, biantennary, disialylated, complex glycans. In contr ast, only four (asparagines 19, 59, 146 and 270) out of the five potential sites were glycosylated in recombinant human beta-glucosylceramidase, with the site nearest the C-terminal (asparagine 462) being unoccupied. The glyc ans at each site consisted of a mixture of non-fucosylated and core alpha 1 -6 fucosylated oligomannose glycans (Man(3) GlcNAc(2)), derived from the en zymic truncation of complex glycans. (C) 2000 Elsevier Science Ltd. All rig hts reserved.