Detection of metabolites of thymosin b4 in-vitro by liquid chromatography coupled with high-resolution orbitrap mass spectrometry

Abstract

Detection of Metabolites of Thymosin β4 in-vitro by liquid chromatography coupled with high-resolution orbitrap mass spectrometry Khandoker Asiqur Rahaman1, 2, Anca Raluca Muresan1, 2, Farzana Binte Rafique1, 2, Hophil Min1 and Oh-Seung Kwon1,2 2, *1Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Korea, 2Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea. * Corresponding author: oskwon@kist.re.kr Thymosin β4 is a highly active protein (SDKPDMAEIEKFDKSKLKKTETQEKNPLP SKETIEQEKQAGES) found in platelets, red blood cells, spleen, and thymus gland of human. Biological activities exerted by thymosin β4 are actin-binding, tissue repair, regeneration, anti-inflammation, and maturation of cells. Unfortunately, few athletes in the international sports world reportedly used thymosin β4, and the investigation was carried out by anti-doping organizations. Based on its biological activities, thymosin β4 would undoubtedly have the potential to be abused by athletes. Likewise, thymosin β4, its active sites, and metabolites should be investigated for detection purposes as an anti-doping tool. In this study, we detected 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 a different buffer for the metabolism of thymosin β4. At first, thymosin β4 was metabolized with an appropriate buffer system for 22 hours. Then samples were collected and analyzed for metabolites by liquid chromatographic separation coupled with full scan mass spectrometry. Total 13 new metabolites were detected in this study (y13, y8, a3-NH3, y42, c11, c23, y27, y41, y20-NH3, y24, c14, c15, c17). We have detected 7 metabolites in porcine carbo