液质联用分析重组人尿激酶原的糖基修饰异质性

Analysis of the glycosylation heterogeneity of recombinant human pro-urokinase using UPLC-MS

应用超高效液相色谱-质谱联用技术,对重组人尿激酶原的糖基修饰异质性进行了分析鉴定。首先通过测定切除N-糖基前、后完整蛋白的相对分子质量,确定异质性的来源;然后在肽段水平鉴定糖基化位点并确定不同O-糖肽的比例;最后通过绘制N-糖图谱,对N-糖基进行定性定量分析。结果显示,该蛋白的异质性主要由糖基化修饰造成。T18全部发生岩藻糖化;6.4%的S138/139发生O-糖基化,糖型主要有2种,比例分别为6.0%和0.4%;N302全部发生N-糖基化,糖型有十多种,以A2F和A3F最多,二者约占总比例的80%。本文对该制品糖基修饰异质性的分析可为其质量标准的提高提供数据参考。

The glycosylation heterogeneity of recombinant human pro-urokinase(pro-UK)was assessed using ultra-performance liquid chromatography-mass spectrometry(UPLC-MS).Firstly,the source of heterogeneity was determined by measuring the Mrof intact protein before and after N-deglycosylation.Glycosylation sites and the proportion of O-glycopeptides then were determined at the peptide level.Finally,the N-glycans were confirmed and quantified using the N-glycan profile.Results show that the structural heterogeneity of pro-UK is mainly caused by glycosylation.All T18 were fucosylated,and 6.4%of S138/139 was O-glycosylated with two kinds of oligosaccharides with a ratio of 6.0%and 0.4%respectively.All N302 positions were N-glycosylated by more than ten types of glycans,among which A2 F and A3 F accounted for 80%of the total.The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.