摘要
针对5个疑似蛇毒毒液及其沾染样品,基于纳升级超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(Nano LC-MS/HRMS)技术,结合尺寸排阻色谱分离,建立了一种蛋白质种类及物种归属的严格鉴定方法。5个样品经尺寸排阻色谱分离后均得到3个洗脱峰,分别冻干后以胰蛋白酶进行溶液内酶解处理并进行液相色谱-高分辨质谱分析鉴定。首先采用全扫描-数据依赖型MS/MS(Full MS/dd MS^(2))采集模式对样品中的肽段信息进行非靶向采集,依次与Swiss-Prot、蛇亚目(Serpentes)、游蛇科(Colubroidea)、眼镜蛇科(Elapidae)、眼镜蛇亚科(Elapinae)、眼镜蛇属(Naja)蛋白质数据库逐级收缩比对;再筛选符合肽谱匹配度、肽段错误发现率小于1%和特征肽段数目大于等于2的蛋白质,共鉴定到32种蛋白质均来自中华眼镜蛇(Naja atra),可归属于Naja atra的10个家族,主要为三指毒素、金属蛋白酶、磷脂酶A2等。最后,采用平行反应监测模式选取每种蛋白质的两条特征肽段进行靶向验证,当两条特征肽段均满足“至少75%的y^(+)和b^(+)离子的Δm/z小于5 ppm”时,方认为鉴定到了样品中的某一蛋白质。最终鉴定出5个样品均含有Naja atra蛇毒。此鉴定方法研究系统、严格,可为蛇毒中毒司法鉴定以及蛇毒药物研究等提供有效的技术支持。
Snake venom is a complex mixture secreted from the glands of poisonous snakes,which contains proteins,peptides,lipids,nucleosides,sugars,amino acids,amines,metal ions,and other components.According to the toxicological classification,snake venoms can be classified as neurotoxins,anticoagulants and procoagulant toxins,cardiac toxins,other toxin proteins,and enzymes.Proteins and peptides are the key components of snake venom.The establishment of rapid,accurate analysis and identification methods for proteins in snake venom is a prerequisite for snake venom-related forensic identification,intoxication events,and pharmaceutical development.Until now,the classical analysis and identification methods have mainly been biochemical or immunoassays for DNA or proteins,such as polymerase chain reaction,agglutination test,enzyme-linked immunosorbent assay,fluorescent immunoassay,and various biosensing approaches.These methods have some limitations such as a high false-positive ratio,low sensitivity,poor anti-interference ability,and limited species discrimination capability.In recent years,with the rapid development of mass spectrometry(MS)techniques,the proteomics of snake venom has also attracted much attention and has contributed to the identification of snake species,in which non-targeted and targeted proteomics represent two main divisions.However,species identification via proteomics is in its infancy in forensic science.First,the tandem MS spectra of peptide sequences are highly complex,which poses a great challenge for the strict and accurate matching of peptides based on the rational speculation of MS fragmentation rules and theoretical calculations in non-targeted proteomics.Second,for the confirmation and identification of unknown substances,reference substances are commonly needed,but those for snake venom are lacking.Proteomics in snake venom identification is still in progress to improve the identification confidence and clarify the identification rules.In this work,a method based on nano-ultra-high performance
作者
李泽华
王闯
徐斌
陈佳
张瑛
郭磊
谢剑炜
LI Zehua;WANG Chuang;XU Bin;CHEN Jia;ZHANG Ying;GUO Lei;XIE Jianwei(State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China;Key Laboratory of Ethnic Medicine,Ministry of Education,College of Pharmacy,Minzu University of China,Beijing 100081,China;Forensic Science Service of Beijing Public Security Bureau,Key Laboratory of Forensic Toxicology,Ministry of Public Security,Beijing 100192,China)
出处
《色谱》
CAS
CSCD
北大核心
2023年第2期122-130,共9页
Chinese Journal of Chromatography
关键词
纳升级超高效液相色谱
高分辨质谱
蛇毒
蛋白质组学
平行反应监测
nano-ultra-high performance liquid chromatography(Nano LC)
high resolution mass spectrometry(HRMS)
snake venom
proteomics
parallel reaction monitoring
