Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics
Xiaoxiao Luo, Qirui Bi, Dongdong Huang, Yun Li, Changliang Yao, Jianqing Zhang, Wenlong Wei, Jiayuan Li, Zhenwei Li, Jingxian Zhang, Ji Shen, Yurong Wang, De‐an Guo
Abstract
Pheretima, also called “earthworms”, is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in the 2020th edition of China Pharmacopeia. However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), P. vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7451 in PV, and 5896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart Capsules and Xiaohuoluo Pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.