基质辅助激光解吸附电离飞行时间串联质谱研究重组人促红细胞生成素一级结构
基质辅助激光解吸附电离,,基质辅助激光解吸附电离飞行时间串联质谱，重组人促红细胞生成素，肽质量指纹谱，二硫键，N连接糖基化位点，糖型结构,基质辅助激光解吸附电离飞行时间串联质谱研究重组人促红
摘 要建立了生物质谱研究rhEPO 一级结构的系列方法，包括分子量、唾液酸含量以及N糖含量等糖基化性质的测定方法，并用于4种国产rhEPO 样品的分析；在此基础上优化酶切条件，使测定的肽质量指纹谱(PMF)的覆盖率均达到98%，成功鉴定了4个样品中的全部N糖基化位点以及对分子中两对二硫键进行了结构确证，测定4个样品分子量分别为：27754.1±27.4Da、27941.4±23.2 Da、27682.5±22.0 Da和27914.7±22.5 Da；唾液酸含量分别为：5.0%、4.8%、5.6%和5.4%；糖含量分别为：34.3%、34.7%、34.1%和346%。此外还对4种产品在O连接糖型及N连接糖型上的区别做了初步探讨。
关键词 基质辅助激光解吸附电离飞行时间串联质谱，重组人促红细胞生成素，肽质量指纹谱，二硫键，N连接糖基化位点，糖型结构
Primary Structure Determination of Recombinant Human Erythropoietin by Matrixassisted Laser Desorption IonizationTime of FlightTime of FlightMass Spectrometry
Lu Zhuang, Wang Shan, Dai Jingquan, Wang Jinglan, Zhang Yangjun,Ying Wantao, Xu Youxuan， Qian Xiaohong, Wu Moutian, Cai Yun
(Beijing Institute of Radiation Medicine, Beijing 100850)
(China Doping Control Center, National Research Institute of Sports Medicine, Beijing 100029)
Abstract A series of method for the primary structure determination of recombinant human erythropoietin (rhEPO), a recombinant glycoprotein, by matrixassisted laser desorption ionizationtime of flighttime of flightmass spectrometry (MALDITOFTOFMS) was developed , which includes the methods of the measurement of molecular weight, content of sialic acid and carbohydrate in rhEPO etc. Based on the developed methods, four kinds of rhEPO coming from different manufactories were determined. The coverage of PMF of all rhEPOs reached 98% by optimization of digestion condition, and Nglycan site and two disulphide bridges in the rhEPOs were determined successfully. Molecular weight of four samples are (27754.1±27.4)Da, (27941.4±23.2)Da, (27682.5±22.0)Da and (27914.7±22.5)Da; content of sialic acid are 5.0%, 48%, 56% and 5.4%; and content of carbohydrate are 34.3%, 34.7%, 34.1% and 34.6%. Moreover, the difference of the glycan structure type on among these four rhEPOs was probed and discussed. ......
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