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  

Primary Structure Determination of Recombinant Human Erythropoietin by Matrix-assisted Laser Desorption Ionization-Time of Flight-Time of Flight-Mass Spectrometry

作　　者：卢庄[1] 王杉[2] 代景泉[1] 王京兰[1] 张养军[1] 应万涛[1] 徐友宣[2] 钱小红[1] 吴侔天[2] 蔡耘[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]国家体育总局运动医学研究所兴奋剂检测中心,北京100029

出　　处：《分析化学》2006年第5期591-597,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20275044;20275046);国家重点基础研究发展规划(No.2004CB520802;2001CB510201);北京市科技计划重大项目(No.H030230280190);奥运科技专项课题(No.2002BA904B01;2003BA904B05)资助项目

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±23.2Da、27682.5±22.0Da和27914.7±22.5Da;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34·6%。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。A series of method for the primary structure determination of recombinant human erythropoietin （rhEPO）, a recombinant glycoprotein, by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry （MALDI-TOF-TOF-MS） 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 N-glycan 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%, 4. 8%, 5.6% 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.

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 

分 类 号：R914[医药卫生—药物化学]