Analysis of the glycosylation heterogeneity of recombinant human pro-urokinase using UPLC-MS
10.16438/j.0513-4870.2020-1260
- VernacularTitle:液质联用分析重组人尿激酶原的糖基修饰异质性
- Author:
Lei TAO
;
Lei YU
;
You-xue DING
;
Hua BI
;
Chun-ming RAO
- Publication Type:Research Article
- Keywords:
UPLC-MS;
recombinant protein;
pro-urokinase;
glycosylation;
heterogeneity
- From:
Acta Pharmaceutica Sinica
2020;55(11):2713-2718
- CountryChina
- Language:Chinese
-
Abstract:
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 Mr of 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 A2F and A3F accounted for 80% of the total. The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.
