Discovery of in-vitro generated metabolitesof Thymosin beta4 by UHPLC--Exactive Orbitrap MS

Abstract

Thymosin β4 is a highly active protein that exerts biological activities such as actin-binding, tissue repair, regeneration, anti-inflammation, and maturation in cells. Few athletes in sports world reportedly used thymosin β4 for doping purposes. Based on its biological activities, thymosin β4 undoubtedly has the high potential to be abused by athletes. In this experiment, we found metabolites of thymosin β4 in 6 different enzyme-buffer systems. We used porcine carboxypeptidase B, porcine leucine aminopeptidase N, trypsin, recombinant human carboxypeptidase B, recombinant human aminopeptidase, and recombinant human carboxypeptidase M with each specific buffer for the metabolism of thymosin β4. Thymosin β4 was incubated for 22 hrs with addition of an appropriate buffer system. The samples were collected and analyzed for metabolites by UHPLC-Q-Exactive Orbitrap MS. The metabolites were calculated by mass prospector (http://prospector.ucsf.edu/). The standards for detected metabolites were ordered synthetically and will be used to confirm the discovered metabolites in our study. In this study, we detected 13 new metabolites (y13, y8, a3-NH3, y42, c11, c23, y27, y41, y20-NH3, y24, c14, c15, c17). Detected were 7 metabolites in porcine carboxypeptidase B, 8 metabolites in porcine leucine aminopeptidase N, 5 metabolites in trypsin, 5 metabolites in recombinant human carboxypeptidase B1, 4 metabolites in recombinant human aminopeptidase N, and 2 metabolites in recombinant human carboxypeptidase M. There are common metabolites observed from various enzymes, but their peak abundance was different. We aim to characterize all the detected metabolites according to their respective mass spectra by in-silico and to match them to the synthesized standards ordered. Biological activities of these novel 13 metabolites detected in this study could be screened and applied for in-vivo models. Furthermore, these metabolites are potential key biomarkers for detecting thymosin β4 doping in sports.