Li Zehua, Li Kexin, Xu Bin, Chen Jia, Zhang Ying, Guo Lei, Xie Jianwei
State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, P. R. China.
Forensic Science Service of Beijing Public Security Bureau, Key Laboratory of Forensic Toxicology, Ministry of Public Security, Beijing, P. R. China.
Electrophoresis. 2023 Jan;44(1-2):337-348. doi: 10.1002/elps.202200051. Epub 2022 Aug 27.
Snake venom is a complex mixture of proteins and peptides secreted by venomous snakes from their poison glands. Although proteomics for snake venom composition, interspecific differences, and developmental evolution has been developed for a decade, current diagnosis or identification techniques of snake venom in clinical intoxication and forensic science applications are mainly dependent on morphological and immunoassay. It could be expected that the proteomics techniques directly offer great help. This work applied a bottom-up proteomics method to identify proteins' types and species attribution in suspected snake venom samples using ultrahigh-performance liquid chromatography-quadrupole-electrostatic field Orbitrap tandem mass spectrometric technique, and cytotoxicity assay was amended to provide a direct evidence of toxicity. Toward the suspicious samples seized in the security control, sample pretreatment (in-sol and in-gel digestion) and data acquisition (nontargeted and targeted screening) modes complemented and validated each other. We have implemented two consequent approaches in identifying the species source of proteins in the samples via the points of venom proteomics and strict forensic identification. First, we completed a workflow consisting of a proteomics database match toward an entire SWISS-PROT (date 2018-11-22) database and a result-directed specific taxonomy database. The latter was a helpful hint to compare master protein kinds and reveal the insufficiency of specific venom proteomics characterization rules. Second, we suggested strict rules for protein identification to meet the requirements of forensic science on improved identification correctness, that is, (1) peptide spectrum matches confidence, peptide confidence, and protein confidence were both high (with the false-discovery ratio less than 1%); (2) the number of unique peptides was more than or equal to two in one protein, and (3) within unique peptides, which at least 75% of the ∆m/z of the matched y and b ions were less than 5 ppm. We identified these samples as cobra venom containing 10 highly abundant proteins (P00597, P82463, P60770, Q9YGI4, P62375, P49123, P80245, P60302, P01442, and P60304) from two snake venom protein families (acid phospholipase A2 and three-finger toxins), and the most abundant proteins were cytotoxins.
蛇毒是毒蛇从其毒腺分泌的蛋白质和肽的复杂混合物。尽管针对蛇毒成分、种间差异和进化发育的蛋白质组学已发展了十年，但目前在临床中毒和法医学应用中蛇毒的诊断或鉴定技术主要依赖于形态学和免疫分析。可以预期蛋白质组学技术能直接提供很大帮助。这项工作应用自下而上的蛋白质组学方法，使用超高效液相色谱 - 四极杆 - 静电场轨道阱串联质谱技术来鉴定疑似蛇毒样品中蛋白质的类型和物种归属，并改进细胞毒性测定以提供毒性的直接证据。对于安全检查中查获的可疑样品，样品预处理（溶液内和凝胶内消化）和数据采集（非靶向和靶向筛选）模式相互补充和验证。我们通过蛇毒蛋白质组学和严格的法医学鉴定要点，实施了两种连续的方法来鉴定样品中蛋白质的物种来源。首先，我们完成了一个工作流程，包括对整个SWISS - PROT（2018 - 11 - 22日期）数据库进行蛋白质组学数据库匹配以及针对结果的特定分类数据库。后者有助于比较主要蛋白质种类并揭示特定蛇毒蛋白质组学表征规则的不足。其次，我们提出了严格的蛋白质鉴定规则，以满足法医学对提高鉴定准确性的要求，即：（1）肽谱匹配置信度、肽置信度和蛋白质置信度都很高（错误发现率小于1%）；（2）一种蛋白质中独特肽段的数量大于或等于两个；（3）在独特肽段中，匹配的y和b离子的∆m/z至少75%小于5 ppm。我们将这些样品鉴定为眼镜蛇毒液，其中含有来自两个蛇毒蛋白质家族（酸性磷脂酶A2和三指毒素）的10种高丰度蛋白质（P00597、P82463、P60770、Q9YGI4、P62375、P49123、P80245、P60302、P01442和P60304），最丰富的蛋白质是细胞毒素。
