联合酶解法结合UPLC Q-TOF高分辨质谱检测重组水蛭素蛋白的二硫键结构  

Determination of the Disulfide Bonds in Recombinant Hirudin Protein by Joint Enzymes Digestion with UPLC Q-TOF High Resolution Mass Spectrometry

作　　者：徐金华 赵丽丽 柳常青 张贵民 刘忠 XU Jinhua;ZHAO Lili;LIU Changqing;ZHANG Guimin;LIU Zhong(Shandong New Time Pharmaceutical Co.,Ltd.,Linyi 273400;State Engineering Lab.of High Expression of Mammalian Cells,Linyi 273400;Lunan New Era Biotechnology Co.,Ltd.,Linyi 273400;Lunan Pharmaceutical Group Co.,Ltd.,Linyi 276000)

机构地区：[1]山东新时代药业有限公司,山东临沂273400 [2]哺乳动物细胞高效表达国家工程实验室,山东临沂273400 [3]鲁南新时代生物技术有限公司,山东临沂273400 [4]鲁南制药集团股份有限公司,山东临沂276000

出　　处：《中国医药工业杂志》2023年第11期1636-1645,共10页Chinese Journal of Pharmaceuticals

基　　金：山东省重点研发计划(泰山产业领军人才工程)项目(2018TSCYCX-30、tscy20200329、tscx202306088)。

摘　　要：采用联合酶切法结合UPLC Q-TOF高分辨质谱解析重组水蛭素蛋白的复杂二硫键交联结构。首先从蛋白水平验证重组水蛭素蛋白是否含有游离半胱氨酸,包括高分辨质谱表征相对分子质量和定量检测游离巯基;然后利用单一酶解法标注每个或每组二硫键在蛋白序列中所处的位置;最后根据单酶所测结果,设计混合酶切的方式确定具体的二硫键配对方式,并利用二硫苏糖醇还原法加以验证,最终确证重组水蛭素蛋白的二硫键位置。结果显示,重组水蛭素蛋白存在10个半胱氨酸,共形成5对二硫键,分别为Ṩ1(Cys1-Cys2)、Ṩ2-Ṩ3(Cys3-Cys5/Cys4-Cys6或Cys3-Cys6/Cys4-Cys5)、Ṩ4(Cys7-Cys9)和Ṩ5(Cys8-Cys10)。The complex disulfide crosslinking structure of recombinant hirudin protein was analyzed by joint enzyme digestion with UPLC Q-TOF high resolution mass spectrometry.It was verified whether the recombinant hirudin protein contained free cysteine from the protein level by high resolution mass spectrometry and the free thiol quantitative detection.Then,single enzyme method was used to label the position of each or each group of disulfide bonds in the protein sequence.Finally,according to the results of the single enzyme,the specific disulfide bond pairing method was designed and verified by dithiothreitol reduction method,and the disulfide bond position of recombinant hirudin protein was finally confirmed.It was showed that there were ten cysteine residues included in recombinant hirudin protein sequence,they could establish five pairs of disulfide bonds,includingṨ1(Cys1-Cys2),Ṩ2-Ṩ3(Cys3-Cys5/Cys4-Cys6 or Cys3-Cys6/Cys4-Cys5),Ṩ4(Cys7-Cys9)andṨ5(Cys8-Cys10).

关 键 词：UPLC Q-TOF高分辨质谱 二硫键 蛋白类药物 联合酶解 

分 类 号：O657.63[理学—分析化学] TQ937[理学—化学]