Heterogeneity of glycans at each N-glycosylation site of horseradish peroxidase

The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) were studied by methylation linkage analysis, exoglycosidase degradation, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS). Over 90% of the predicted tryptic peptides and glycopeptides of HRPc could be identified in the unfractionated digest.]Four glycans, namely (Xyl)Man(3)(Fuc)GlcNAc(2) (major species), (Xyl)Man(2)(Fuc)GlcNAc(2), (Xyl)Man(3)GlcNAc(2), and Man(3)(Fuc)GlcNAc(2) (minor species), were observed at all of the N-glycosylation sites and account for greater than 95% of the carbohydrate. Other members of this glycan family, namely (Xyl)(x)Man(m)(Fuc)(f) GlcNAc(2) (x = 0 or 1, f = 0 or 1, m = 4, 5, 6, or 7), account for the rest of the glycans. Only traces of high mannose-type glycans were detected in HRPc. Two sites, namely those at Asn-57 and Asn-267, were found to be more heterogeneous than the sites at Asn-13, Asn-158, Asn-186, 198 (doubly glycosylated peptide), Asn-214, and Asn-255. Two of the glycopeptides were observed as part of disulfide-linked species. MALDITOFMS confirmed the N-glycosylation sites previously reported [K.G. Welinder, fur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogeneity of the glycan pool at each site. (C) 1998 Elsevier Science Ltd. All rights reserved.