液相色谱-质谱联用方法用于严重急性呼吸系统综合症冠状病毒结构蛋白质的研究  

Studies on Structural Proteins of Severe Acute Respiratory Syndrome-Coronavirus by Liquid Chromatography Coupled with Electrospray Ionization-Quadrupole Time-of-Flight Tandem Mass Spectrometry

作　　者：张养军[1] 王京兰[1] 蔡耘[1] 应万涛[1] 郝运伟[1] 李蕾[1] 李晓海[1] 戴舒佳[1] 代景泉[1] 贺福初[1] 钱小红[1] 王杰[2] 魏开华[2] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]军事医学科学院仪器测试分析中心,北京100850

出　　处：《分析化学》2004年第4期415-420,共6页Chinese Journal of Analytical Chemistry

基　　金：国家重点基础研究发展规划项目 (No .2 0 0 1CB5 10 2 0 1);国家自然科学基金 (No .2 0 2 75 0 46);国家高技术项目 (No .2 0 0 1AA2 3 3 0 3 1;No .2 2 0 2 BA711A11)资助课题

摘　　要：通过高效液相反相色谱、毛细管反相色谱 -串联质谱联用方法对SARS病毒攻击细胞的研究 ,鉴定出了N、S和M 3种SARS结构蛋白质 ,并准确测定了N蛋白质的分子量。由分子量结果和生物信息学推断的N蛋白质的理论分子量比较 ,可以确定N蛋白质不存在常见的磷酸化修饰和糖基化修饰或含量很低。进一步的研究表明 :N蛋白质可能存在降解现象 ,其降解机理尚需探索。另外 ,通过对样品直接酶切后进行两维毛细管分离和串联质谱鉴定与对样品先进行分离 ,然后再进行毛细管反相分离 串联质谱鉴定方法比较 ,表明两种方法在蛋白质组学研究中各具优缺点 。Various structural proteins including nucleocaspid protein, spike protein and membrane glycoprotein of severe acute respiratory syndrome-coronavirus (SARS-CoV) in the culture supernatant of virus infected-Vero E6 were identified by two dimensional (SCX and RPLC) capillary liquid chromatography (2D-CapLC) coupled with electropray ionization-quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS) and analytical reversed-phase high-performance liquid chromatogram by-hybrid capillary liquid chromatography (CapLC) with ESI-QTOF MS/MS. Matrix-assisted laser desorption/ionization-mass spectrometric (MALDI-MS) determination shows that relative molecular weight of intact N protein is 45929 Da, which is very much close to its theoretically calculated molecular weight (45935. 38 Da) based on the amino acid sequence with the first amino acid methionine in N-terminus depleted and second serine acetylated, indicating that phosphorylation and glycosylation do not happen or the amount of phosphorylated and glycosylated protein is very low in the predicted phosphorylation and glycosylation sites within infected cells. Further study shows that possible degradation of N protein occurs. Comparing 2D-CapLC coupled with ESI-QTOF MS/MS and analytical reversed-phase high-performance liquid chromatography-hybrid Capl,C with E SI-QTOF MS/MS indicates that there are advantages and disadvantages to these two methods in proteome study and choice may be made in terms of different experimental aims.

关 键 词：严重急性呼吸系统综合症 冠状病毒 结构蛋白质 液相色谱-质谱联用 SARS 非典型肺炎 

分 类 号：R373[医药卫生—病原生物学]