药学学报, 2013, 48(6): 896-900
引用本文:
陶 磊, 丁有学, 郭 莹, 饶春明, 王军志. 液质联用分析rhTNK-tPA的一级结构[J]. 药学学报, 2013, 48(6): 896-900.
TAO Lei, DING You-xue, GUO Ying, RAO Chun-ming, WANG Jun-zhi. Characterization of the primary structure of TNK-tissue plasminogen activator using LC-MS[J]. Acta Pharmaceutica Sinica, 2013, 48(6): 896-900.

液质联用分析rhTNK-tPA的一级结构
陶 磊, 丁有学, 郭 莹, 饶春明*, 王军志*
(中国食品药品检定研究院, 卫生部生物技术产品检定方法及其标准化重点实验室, 北京 100050)
摘要:

液质联用分析重组人组织型纤溶酶原激活物变构体 (TNK-tPA) 的一级结构。用糖苷酶切除其糖基, 测定质谱分子量; 对其进行还原、烷基化、胰蛋白酶酶解, 测定液质肽图及串联质谱图, 通过质谱图的解析对氨基酸序列进行验证, 并对翻译后修饰进行鉴定。该蛋白氨基酸序列与理论一致, 其中约5% M207发生氧化; 80%T61发生岩藻糖化修饰; N103N448N184 (15%) 存在N-糖基化修饰, 糖型均为复杂型N-糖。液质联用结合适当的样品前处理方式, 可快速、准确地对该蛋白的一级结构进行鉴定

关键词:   
Characterization of the primary structure of TNK-tissue plasminogen activator using LC-MS
Abstract:

The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS).  Firstly, the molecular mass of deglycosylated protein was measured.  Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications.  Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with ~80% occupancy; N103, N448 and N184 (~15% occupancy) were glycosylated with complex-type oligosaccharides.  LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.

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