梁瑞强 孙姗姗 曹进 康帅 林瑞超 邹迪新

摘要 目的：通过蛋白质组学技术手段找到适用于药材全蝎含量测定的目标蛋白质及其特征肽段，用该特征肽段形成定量方法。方法：全蝎粉碎后经PBS缓冲溶液超声提取并过滤后，得到药材的蛋白质提取溶液。上述溶液经胰蛋白酶酶解后通过高分辨质谱进行分析，确定待测的目标蛋白质（β-肌动蛋白）和与之对应的特征肽段（IIAPPER），并进行特征肽段及其同位素内标物（IIA*（13C3，15N）PPER）的合成。用得到特征肽段做对照品，同位素内标物做内标进行含量测定。色谱柱为Waters ACQUITY UPLC HSS T3（2.1 mm×100 mm，1.8 μm），流动相为0.1%甲酸溶液（V/V）-水和乙腈，梯度洗脱。质谱使用电喷雾离子源（ESI）正离子模式，采用多反应监測模式（MRM）进行信号采集。定量方法为内标法。结果：β-肌动蛋白的特征肽段在0.1～40 nmol/L浓度范围内线性良好，相关系数（r2）在0.999以上。方法检出限为0.04 mg/kg，定量限为0.13 mg/kg。加标回收率在87%～93%，重复性的RSD为5.2%，中间精密度的RSD为4.8%，供试品溶液在4 h内稳定。利用该方法对来自不同产地的37批次全蝎药材中的β-肌动蛋白进行含量测定，结果在4.66～100.26 mg/kg之间。结论：该方法灵敏度高，准确度较好和重复性较稳定，可以为全蝎药材药材的质量控制提供一种有效的手段。

关键词 全蝎;超高效液相色谱串联质谱法;蛋白质组学;β-肌动蛋白;含量测定;质量控制

Abstract Objective：To find the target protein and its characteristic peptides suitable for the determination of the whole scorpion of medicinal materials by means of proteomics，and to use this characteristic peptide to form a quantitative method.Methods：After the whole scorpion was crushed，it was ultrasonically extracted with PBS buffer solution and filtered to obtain the protein extract solution of the medicinal material.The above solution was analyzed by high-resolution mass spectrometry after trypsin digestion to determine the target protein to be tested（β-actin）and the corresponding characteristic peptide（IIAPPER），and the characteristic peptide and its isotope internal standard synthesis（IIA*（13C3，15N）PPER）.The obtained characteristic peptide was used as a reference substance，and the isotopic internal standard was used as an internal standard for content determination.The column was Waters ACQUITY UPLC HSS T3（2.1 mm×100 mm，1.8 μm），the mobile phase was 0.1% formic acid solution（V/V）-water and acetonitrile，and the gradient eluted.Mass spectrometry used the electrospray source ion（ESI）positive ion mode and the multi-reaction monitoring mode（MRM）for signal acquisition.The quantitative method was the internal standard method.Results：The characteristic peptide of β-actin was well linear in the concentration range of 0.1-40 nmol/L，and the correlation coefficient（r2）was above 0.999.The detection limit of the method was 0.04 mg/kg，and the limit of quantification was 0.13 mg/kg.The recovery rate of spiked standard was 87%-93%，and the repeatable RSD is 5.2%.The intermediate precision RSD was 4.8%，and the test solution was stable within 4 h.Using this method，the content of β-actin in 37 batches of whole scorpion medicinal materials from different origins was determined，and the result was between 4.66-100.26 mg/kg.Conclusion：The method has high sensitivity，good accuracy and stable repeatability，and can provide an effective method for the quality control of the whole scorpion medicinal materials.

Keywords Whole scorpion; UPLC-MS; Proteomics; β-actin; Content determination; Quality Control

中图分类号：R284.1文献标识码：Adoi：10.3969/j.issn.1673-7202.2020.13.002

全蝎为东亚钳蝎Buthus martensii Karsch的干燥体，生物学分类属于节肢动物门蛛形纲蝎目钳蝎科，作为平肝熄风的常用药材之一，多被用于中医临床治疗痉挛抽搐、疮疡中毒、瘰疬结核、风湿顽痹和顽固性偏正头痛等疾病，同时具有抗凝血、抗肿瘤、抗癫痫和抗菌等药理作用[1-5]。……