液质联用法分析重组抗肿瘤抗病毒蛋白的一级结构  

Analysis of Primary Structure of Recombinant Antitumor-Antivirus Protein by Liquid Chromatography-Mass Spectrometry

作　　者：李萌[1,2] 裴德宁[2] 陶磊[2] 李永红[2] 王兰[2] 高凯[2] 饶春明[2] 王军志[1,2] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2011年第12期1473-1476,共4页Chinese Journal of Biologicals

基　　金：国家科技重大专项课题(2009ZX09307-001)资助

摘　　要：目的通过液质联用法分析重组抗肿瘤抗病毒蛋白(乐复能)的一级结构。方法采用高效液相-电喷雾四级杆飞行时间质谱技术(High performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry,HPLC-ESI-Q-TOF-MS)绘制乐复能经胰蛋白酶酶切后的液质肽图,并定位二硫键;采用电喷雾四级杆飞行时间质谱(ESI-Q-TOF-MS)测定乐复能的精确相对分子质量。结果乐复能液质肽图的22个理论酶切肽段中,有6个肽段(T2、T5、T12、T15、T19、T22)共7个氨基酸未被检测到,氨基酸覆盖率为96%;通过分析胰蛋白酶酶切液质肽图,发现1种二硫键连接方式:Cys1-Cys29、Cys99-Cys139;质谱测定相对分子质量为19 311.63,与理论相对分子质量(19 311.27)的相对误差为0.000 02%。结论建立了乐复能的液质肽图分析方法,为该类制品的质量控制及其参考品的研制提供了参考。Objective To analyze the primary structure of recombinant antitumor-antivirus protein（Novaferon） by liquid chromatography-mass spectrometry（LC-MS）.Methods The peptide map of Novaferon digested with trypsin was measure by high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry（HPLC-ESI-Q-TOF-MS） which could identify the sites of disulfide bonds.The exact relative molecular mass of Novaferon was measured by ESI-Q-TOF-MS.Results Of the 22 theoretical trypsin-digested fragments of Novaferon,6（i.e.T2,T5,T12,T15,T19 and T22,7 amino acids in total） were undetected,with a amino acid coverage of 96%.Peptide mapping showed that the disulfide bonds in Novaferon were Cys1-Cys29 and Cys99-Cys139.The relative molecular mass of Novaferon was 19 311.63,with a relative error of 0.000 02% to the theoretical value（19 311.27）.Conclusion The method for peptide mapping of Novaferon was developed by LC-MS,which provided a reference for quality control of products of the same kind and preparation of Novaferon reference.

关 键 词：液质联用 重组蛋白质类 氨基酸序列 二硫键定位 分子量 

分 类 号：Q657.63[生物学—生物物理学] Q516