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Rahaman Khandoker Asiqur,Muresan Anca Raluca,Min Hophil,손정현,강민정,권오승 한국약제학회 2022 Journal of Pharmaceutical Investigation Vol.52 No.5
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.
Identification of thymosin β4 in-vitro metabolites with synthesized standards
Khandoker Asiqur Rahaman,Anca Raluca Muresan,Hophil Min,Junghyun Son,Min-Jung Kang,Oh-Seung Kwon 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Thymosin β4 is a highly active protein that exerts biological activities such as actin-binding, tissue repair, regeneration, anti-inflammation, and cell maturation. Thymosin β4 has also been listed in WADA prohibited drugs. Based on its biological activities, thymosin β4 undoubtedly has a high potential to be abused by athletes. This study aims to identify metabolites of thymosin β4 in different enzyme-buffer systems, characterize each m/z ion of the detected metabolite in-silico and confirm its structure with the synthesized authentic standards. At first, thymosin β4 was metabolized with an appropriate buffer system for 22 hours. Then, the samples were collected and analyzed for metabolites by UHPLC-Q-Exactive Orbitrap MS. The metabolites were calculated by in-silico software. Finally, the standards for detected metabolites were custom synthesized and validated through respective TIC, MS, and MS/MS. In this study, we detected 13 new metabolites. Moreover, more than one enzyme system shared many metabolites, but the abundance was different. We characterized all the detected metabolites according to respective m/z ion in-silico and matched them to the synthesized standards. Metabolite M1, M5, M7, M11, M12, M13 were confirmed to their synthesized authentic standards, respectively. This study established a method of detection and identification of new peptide metabolites. Remaining further studies by screening these peptides could discover new drug development with the potential of numerous biological activities.