摘要
目的:应用刀豆素A(ConA)亲合层析结合液相色谱串联质谱法(HPLC-MS)发现结核分枝杆菌H37Rv的新的糖蛋白。方法:收集生长2周的结核菌培养液,经过滤浓缩得到培养滤液蛋白(culture filtrate protein,CFP)。应用ConA亲合层析法纯化CFP中的糖蛋白,经SDS-PAGE电泳结合考马斯亮蓝染色后,对蛋白条带进行胰蛋白酶消化。应用HPLC-MS检测消化后的糖肽结构并与结核菌H37Rv蛋白质数据库进行比对,寻找新的糖蛋白。结果:CFP经ConA亲合层析纯化后的电泳结果显示有数条蛋白条带出现,经筛选相对分子质量26×103蛋白最可能为糖蛋白。酶切消化该蛋白得到的多肽在HPLC-MS上保留时间26.24min处显示有多肽色谱峰,其对应的一级MS图提示该多肽总质量为1467原子质量单位(AMU),二级MS图显示为多个质荷比(m/z)逐级减少为54(一个己糖的分子质量单位为162AMU)的多肽,提示有己糖丢失。同时,MS分析肽链氨基酸序列并证实蛋白的糖基化存在。与结核菌H37Rv蛋白质数据库比对结果提示,结核菌分泌的26×103蛋白为一新的糖蛋白,即铜-锌超氧化物歧化酶。结论:应用ConA亲合层析结合HPLC-MS发现了一个新的结核分枝杆菌糖蛋白。
Objective： To find novel glycoproteins from Mycobacterium tuberculosis H37Rv by ConA affinity chromatography and HPLC-MS. nethods：M, tuberculosis H37Rv was cultivated at 37℃ for 2 weeks, and the culture supematant was collected by centrifugation. The supematant was filtrated and concentrated to obtain culture filtrate protein （CFP）. The glycoprotein in the CFP was purified by ConA affinity chromatography and visualized by SDS-PAGE and Coommassie brilliant blue staining. The protein bands on gel were cut and digested by trypsin for identification by HPLC- MS. The structure of glycoprotein was compared with M. Tuberculosis H37Rv protein database. Results： After screening, a 26×10^3 protein was chosen from SDS-PAGE for further analysis. Then, the peptides from 26×10^3 protein were separated by HPLC. MS analysis revealed that a peptide peak with retention time of 26.24 min was glycopeptide. Its average mass was 1 467 atomic mass units （AMU）, and MS/MS results exhibited the neutral loss of 54 （m/z）, which suggested that this peptide be hexose-glycosylated. Meanwhile, the amino acid sequence for this peptide was found. Compared with M. tuberculosis H37Rv protein database, this 26 ×10^3 protein was identified as a glycosylated Cu-Zn superoxide dismutase. Conclusion： A new glycoprotein from M. tuberculosis H37Rv is identified by ConA affinity chromatography and HPLC-MS.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第4期326-329,共4页
Bulletin of the Academy of Military Medical Sciences
