A New Approach for Dynamics of Enzyme-Catalyzed Glutathione Conjugation by Electrospray Quadrupole/ Time-of-Flight Mass Spectrometry

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• 作者：Ishigai ; Masaki ; Langridge ; James I. ; Bordoli ; Robert S.
• 刊名：Analytical Biochemistry
• 出版年：2001
• 出版时间：November 1, 2001
• 年：2001
• 卷：298
• 期：1
• 页码：83-92
• 全文大小：151 K

文摘

The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH′ + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH′]. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300–5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity.