摘要
蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer^(2).2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI+模式进行了多反应监测(MRM)定性定量分析。结果显示,特征肽在2.5~30 ng/mL范围内线性关系良好,相关系数(r)大于0.9996,多水平加标回收率范围为95.5%~101.9%,各水平平行测定结果的相对标准偏差(RSD)为1.1%~3.2%,完全能够满足实际样品检测需求。方法简便快捷,灵敏度高,专属性强,可用于矛头蝮蛇蛇毒种属鉴别及svTLE含量测定,从源头保证血凝酶类产品的质量,并可为其他蛇毒类产品的质量控制提供参考。
Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom,and the main active ingredients are snake venom thrombin-like enzymes(svTLEs). The svTLEs derived from different snake species differ in their structures,hemostatic mechanisms,and pharmacological effects. Therefore,accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology,the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study,and an ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox,enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatographyquadrupole/electrostatic field orbitrap high resolution mass spectrometry(Nano LC-Q-ExactiveMS). The mass spectrum data were analyzed by Proteome Discoverer 2. 2 software. The maker peptide “EAYNGLPAK ”,which characterized the svTLE from Bothrops atrox,was finally screened and validated by comparison of the basic local alignment search tool(BLAST)with the NCBI and UniProt databases. For the marker peptide,the enzymolysis temperature,enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows:enzymolysis temperature,37 ℃;enzymolysis time,4h;and amount of enzyme,10 μL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly,20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then,25 mmol/L ammonium bicarbonate solution was added to dissolve the sample,and the solution was diluted to the scale. Precisely 1. 00 mL of the solu
作者
咸瑞卿
杭宝建
巩丽萍
王聪聪
张迅杰
彭丽
石峰
XIAN Ruiqing;HANG Baojian;GONG Liping;WANG Congcong;ZHANG Xunjie;PENG Li;SHI Feng(National Medical Products Administration(NMPA)Key Laboratory for Research and Evaluation of Genetic Drugs,Shandong Institute for Food and Drug Control,Jinan 250101,China;School of Pharmaceutical Sciences,Shandong University,Jinan 250012,China;Department of Quality Control,China Resources Double-Crane Limin Pharmaceutical(Jinan)Co.,Ltd.,Jinan 250200,China)
出处
《色谱》
CAS
CSCD
北大核心
2022年第9期810-816,共7页
Chinese Journal of Chromatography
基金
国家药典委员会药品标准制修订研究课题(2019H04).
关键词
超高效液相色谱-串联质谱
特征肽
蛇毒类凝血酶
矛头蝮蛇
蛇毒
ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)
marker peptide
snake venom thrombin-like enzymes(svTLEs)
Bothrops atrox
snake venom
