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.
Molecules. 2022 Apr 20;27(9):2651. doi: 10.3390/molecules27092651.
Quality control of animal-derived traditional Chinese medicines has improved dramatically as proteomics research advanced in the past few decades. However, it remains challenging to identify quality attributes with routine proteomics approaches since protein with fibrinolytic activity is rarely reported in pheretima, a typical animal-derived traditional medicine. A novel strategy based on bioinformatics combined with parallel reaction monitoring (PRM) was developed here to rapidly discover the marker peptides associated with a fibrinolytic effect. Potential marker peptides were found by lumbrokinase sequences' alignment and in silico digestion. The fibrinogen zymography was used to visually identify fibrinolytic proteins in pheretima. As a result, it was found that the fibrinolytic activity varied among different portions of pheretima. Fibrinolytic proteins were distributed regionally in the anterior and anterior-mid portion and there was no significant fibrinogenolytic activity observed in the mid-posterior and posterior portion. Finally, PRM experiments were deployed to validate and quantify selected marker peptides and a total of 11 peptides were identified as marker peptides, which could be potentially used in quality control of pheretima. This strategy provides a robust workflow to benefit the quality control of other animal-derived traditional medicines.
在过去几十年中，随着蛋白质组学研究的进展，动物源中药材的质量控制有了显著提高。然而，由于常规蛋白质组学方法很少报道蚯蚓中具有纤溶活性的蛋白质，因此仍然难以鉴定质量属性。在这里，我们开发了一种基于生物信息学结合平行反应监测（PRM）的新策略，以快速发现与纤溶作用相关的标记肽。通过蚓激酶序列比对和计算机消化发现潜在的标记肽。纤维蛋白原凝胶电泳用于直观地鉴定蚯蚓中的纤溶蛋白。结果表明，不同部位的蚯蚓纤溶活性存在差异。纤溶蛋白在前段和前中段呈区域性分布，中后段和后段没有明显的纤维蛋白溶活性。最后，进行 PRM 实验来验证和定量选择的标记肽，共鉴定出 11 种肽作为标记肽，它们可能被用于蚯蚓的质量控制。该策略为其他动物源中药材的质量控制提供了一个稳健的工作流程。
