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An approach for identifying in silico peptides against authentic metabolites: in vitro characterization of thymosin β4 metabolites

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Abstract

Purpose

Thymosin β4 is a highly active protein that exerts multiple biological activities such as tissue repair, anti-inflammation, and cell maturation. Thymosin β4 has also been listed as a prohibited drug by the World Anti-doping Agency (WADA). Based on its biological activities, thymosin β4 has a high potential of abuse for the performance enhancement among athletes. This study aimed to investigate and characterize the metabolism of thymosin β4 in vitro system.

Methods

TB4 protein was metabolized in six different enzyme–buffer systems in vitro. After TB4 was metabolized with an appropriate buffer system, the resulting metabolites were detected by high resolution LC–MS/MS. The mass spectrum data of the observed metabolites were characterized in silico, and confirmed the structures based on synthesized authentic standards.

Results

Total 13 new metabolites, some of which were detected in more than one enzyme system, were found. This study characterized all of the detected metabolites according to their in silico m/z ions and compared our findings with synthesized standards. Finally, metabolites M1, M5, M7, M11, M12, and M13 were confirmed based on their synthesized authentic standards.

Conclusion

By using an approach for metabolizing a protein to detect, characterize and identify new peptide metabolites, 6 metabolites are identified among 13 expected potential metabolites. Newly detected metabolites may have the potential for biological activities after further screening compared to their parent protein.

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Acknowledgements

This research was supported, in part, by Project 2V06980 of the Korea Institute of Science and Technology (KIST).

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Authors and Affiliations

  1. Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, 02792, Korea

    Khandoker Asiqur Rahaman, Anca Raluca Muresan, Min-Jung Kang & Oh-Seung Kwon

  2. Doping Control Center, Korea Institute of Science and Technology, Hwarangno 14 gil-5, Seongbuk-gu, Seoul, 02792, Republic of Korea

    Hophil Min, Junghyun Son & Oh-Seung Kwon

  3. Center for Advanced Biomolecular Recognition, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea

    Khandoker Asiqur Rahaman, Anca Raluca Muresan & Min-Jung Kang

Authors
  1. Khandoker Asiqur Rahaman
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  2. Anca Raluca Muresan
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  3. Hophil Min
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  4. Junghyun Son
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  5. Min-Jung Kang
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  6. Oh-Seung Kwon
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Contributions

KAR conceived the study, performed experiments, analyzed data, and prepared the manuscript. ARM contributed to method development and interpreted MS/MS data. HM interpreted MS/MS data, edited and reviewed the manuscript. JS provided support, resources, and funding as a project leader and reviewed the manuscript. M-JK interpreted MS/MS data, edited and reviewed the manuscript. O-SK conceived the study, analyzed mass spectra, interpreted data, and provided support, resources, and funding as a project leader.

Corresponding author

Correspondence to Oh-Seung Kwon.

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Conflict of interest

All authors (K.A. Rahaman, A.R. Muresan, H. Min, J. Son, M.‑J. Kang, and O.-S. Kwon) declare that they have no conflicts of interest or personal relationships that could influence this work.

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Rahaman, K.A., Muresan, A.R., Min, H. et al. An approach for identifying in silico peptides against authentic metabolites: in vitro characterization of thymosin β4 metabolites. J. Pharm. Investig. 52, 611–621 (2022). https://doi.org/10.1007/s40005-022-00581-z

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  • DOI: https://doi.org/10.1007/s40005-022-00581-z

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