Gray J S, Yang B Y, Montgomery R
Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242, USA.
Carbohydr Res. 1998 Sep;311(1-2):61-9. doi: 10.1016/s0008-6215(98)00209-2.
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)Man3(Fuc)GlcNAc2 (major species), (Xyl)Man2(Fuc)GlcNAc2, (Xyl)Man3GlcNAc2, and Man3(Fuc)GlcNAc2 (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)xManm(Fuc)f GlcNAc2 (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, Eur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogeneity of the glycan pool at each site.
通过甲基化连接分析、外切糖苷酶降解以及基质辅助激光解吸/电离飞行时间质谱法（MALDI-TOF-MS）对辣根过氧化物酶同工酶c（HRPc）的胰蛋白酶消化糖肽进行了研究。在未分级的消化产物中可鉴定出超过90%的HRPc预测胰蛋白酶肽段和糖肽。在所有N-糖基化位点均观察到四种聚糖，即（木糖）Man3（岩藻糖）GlcNAc2（主要种类）、（木糖）Man2（岩藻糖）GlcNAc2、（木糖）Man3GlcNAc2和Man3（岩藻糖）GlcNAc2（次要种类），它们占碳水化合物的比例超过95%。该聚糖家族的其他成员，即（木糖）xManm（岩藻糖）f GlcNAc2（x = 0或1，f = 0或1，m = 4、5、6或7），占其余的聚糖。在HRPc中仅检测到痕量的高甘露糖型聚糖。发现两个位点，即Asn-57和Asn-267处的位点，比Asn-13、Asn-158、Asn-186、198（双糖基化肽段）、Asn-214和Asn-255处的位点具有更高的异质性。观察到两种糖肽是二硫键连接物种的一部分。MALDI-TOF-MS证实了先前报道的N-糖基化位点[K.G.韦林德，《欧洲生物化学杂志》，96（1979）483 - 502]，并用于确定每个位点聚糖库的异质性。
