NMPA Key Laboratory for Quality Control of Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control, 1500 Zhangheng Road, Shanghai, 201203, China.
China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Shanghai, 201203, China.
Anal Bioanal Chem. 2021 May;413(12):3167-3176. doi: 10.1007/s00216-021-03254-2. Epub 2021 Mar 9.
Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.
地龙是一种常见且有价值的动物源性药物，在中国传统医学中被广泛应用。《中国药典》（2015 年版）规定了四种地龙，即参环毛蚓、通俗环毛蚓、威廉环毛蚓和栉盲环毛蚓。最近的一份报告显示，市面上约有 55%的地龙可能与药典标准不符，被其他物种所掺杂。地龙的安全性、功效和真实性是一个重要问题。目前，各种物种的特异性质量标志物和有效的鉴定方法仍然有限。在本研究中，我们使用纳升液相色谱-串联质谱（nano LC-MS/MS）对来自环毛蚓属和颤蚓属的物种进行了无标记定量蛋白质组学分析，并基于其离子强度使用多元数据分析（主成分分析和有监督偏最小二乘判别分析）来鉴定标记肽。从所有样品中鉴定出了 48476 个具有高可信度的肽段，对应于 13397 种蛋白质。利用 LC-MS/MS 多反应监测模式，通过与合成肽参考标准的比较对标记肽进行了验证。提出了一种用于鉴定地龙的多肽鉴定策略，并随后将其应用于从中国不同地区零售店获得的样本中。结果表明，在测试的 15 个样本中，有 8 个被认为是真正的地龙。
