Metabolite detection of Thymosin β4 and its peptide drug TB-500 generated in in-vitro by High resolution LC/MS-MS

Abstract

PT-005 Metabolite detection of Thymosin β4 and its peptide drug TB-500 generated in in-vitro by High resolution LC/MS-MS Khandoker Asiqur Rahaman1,2, Anca Raluca Muresan1,2, Farzana Binte Rafique1,2, Hophil Min1, and Oh-Seung Kwon1,2,; 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 43 amino acid protein that found abundantly in the nature. It plays an essential role in actin binding, tissue repair, regeneration, anti-inflammation, and cell maturation. TB-500 (Ac-LKKTETQ) is a 7 amino acid peptide prepared from the active site of thymosin β4. Based on its biological activities, thymosin β4 and TB-500 would undoubtedly have the potential to be abused by athletes. Likewise, thymosin β4, TB-500, and their metabolites should be investigated for detection purposes as an anti-doping tool. In this study, we detected metabolites of thymosin β4 and TB-500 in six different enzyme-buffer systems. At first, thymosin β4 was metabolized with an appropriate buffer system for 22 hours. TB-500 metabolism was conducted in various in-vitro enzyme systems such as human kidney microsomes, rat liver microsomes, rat liver cytosol, rat liver S9, human serum, and human skin S9 after prolonged incubation (22 hr) with various enzymes. The metabolites were analyzed in a full-scan mode by liquid chromatography coupled with MS/MS (Q-Exactive). Total 13 new metabolites of thymosin β4 were detected in this study (y13, y8, a3-NH3, y42, c11, c23, y27, y41, y20-NH3, y24, c14, c15, c17) in 6 in-vitro enzyme systems. TB-500 metabolism produced N-acetylleucine (Acetyl-Leu-OH; m/z 174) as a new metabolite. The new metabolite, N-acetylleucine, was also found in all enzyme systems used. The other metabolites, such as acetyl-Leu-