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Quantitative analysis of γ-glutamylpeptides by liquid chromatography-mass spectrometry and application for γ-glutamyltransferase assays.

Analytical biochemistry (2019-05-07)
Sho Kobayashi, Yoshinori Tokairin, Takeru Miyakoshi, Takuya Saito, Keita Nagaoka, Yoshitaka Ikeda, Junichi Fujii, Hiroyuki Konno
ABSTRACT

γ-Glutamylpeptides are largely produced via the action of γ-glutamylcysteine synthetase or γ-glutamyltransferase (GGT). GGT transfers the γ-glutamyl moiety from glutathione (GSH) and other γ-glutamyl compounds to amino acids, peptides, or water. A conventional GGT assay employs a synthetic donor substrate, which facilitates monitoring cleavage activity by means of colorimetric analyses but provides no information on the resulting γ-glutamylpeptides. In this study, we report on the use of liquid chromatography-mass spectrometry (LC-MS) to quantitatively measure the levels of 21 γ-glutamylpeptides including GSH and 45 amino acids, including Cys. Authentic compounds consisting of 17 chemically synthesized and commercially available 4 γ-glutamylpeptides were adopted as references. We applied this method to the characterization of γ-glutamylpeptides in blood plasma and livers of mice that had been treated with an overdose of acetaminophen. The established LC-MS-based assay was found to be useful for characterizing the γ-glutamylation reaction under in vivo and in vitro conditions and was clearly helpful for understanding the physiological significance of the production of γ-glutamylpeptides.