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Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics

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Abstract

Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model. A strategy integrating proteogenomics and novel pseudotargeted peptidomics was developed to characterize the natural peptide phenotype and screen for signature peptides of four leech species. First, natural peptides were sequenced against an in-house annotated protein database of closely related species constructed from RNA-seq data from the Sequence Read Archive (SRA) website, which is an open-sourced public archive resource. Second, a novel pseudotargeted peptidomics integrating peptide ion pair extraction and retention time transfer was established to achieve high coverage and quantitative accuracy of the natural peptides and to screen for signature peptides for species authentication. In all, 2323 natural peptides were identified from four leech species whose databases were poorly annotated. The strategy was shown to significantly improve peptide identification. In addition, 36 of 167 differential peptides screened by pseudotargeted proteomics were identified, and about one-third of them came from the leucine-rich repeat domain (LRR) proteins, which are widely distributed in organisms. Furthermore, six signature peptides were screened with good specificity and stability, and four of them were validated by synthetic standards. Finally, a dynamic multiple reaction monitoring (dMRM) method based on these signature peptides was established and revealed that one-half of the commercial samples and all of the Tongxinluo capsules were derived from WP. All in all, the strategy developed in this study was effective for natural peptide characterization and signature peptide screening, which could also be applied to other animal-derived drugs, especially for modelless species that are less studied in protein database annotation.

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Funding

This work was financially supported by the Key Program of the National Natural Science Foundation of China (NO.82130111), National Natural Science Foundation of China (NO.81803716), and Chief Scientist of Qi-Huang Project of National Traditional Chinese Medicine Inheritance.

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  1. Jingmei Liao and Min Gao contributed equally to this work.

Authors and Affiliations

  1. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China

    Jingmei Liao, Yelin Ding & De-an Guo

  2. Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Research Center for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    Jingmei Liao, Min Gao, Yelin Ding, Qirui Bi, Dongdong Huang, Xiaoxiao Luo, Peilei Yang, Yun Li, Yong Huang, Changliang Yao, Jianqing Zhang, Wenlong Wei, Zhenwei Li & De-an Guo

  3. University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, China

    Min Gao, Dongdong Huang & De-an Guo

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  1. Jingmei Liao
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Contributions

Jingmei Liao: methodology, formal analysis, writing—original draft, visualization. Min Gao: methodology, formal analysis, writing—original draft, visualization. Qirui Bi: conceptualization, writing—review and editing, project administration. Yelin Ding: resources, writing—original draft. Dongdong Huang: software, formal analysis, visualization. Xiaoxiao Luo: formal analysis, visualization. Peilei Yang: resources. Yun Li: data curation. Yong, Huang, Changliang Yao: formal analysis. Jianqing Zhang: formal analysis. Wenlong Wei: formal analysis. Zhenwei Li: formal analysis.

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Correspondence to De-an Guo.

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Liao, J., Gao, M., Ding, Y. et al. Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics. Anal Bioanal Chem 415, 2795–2807 (2023). https://doi.org/10.1007/s00216-023-04692-w

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