Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases

Geib, Timon; Lento, Cristina; Wilson, Derek J. et Sleno, Lekha (2019). « Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases ». Frontiers in Chemistry, 7.

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Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S-transferases (GSTs) in vitro. GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro. APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.

Type: Article de revue scientifique
Mots-clés ou Sujets: acetaminophen, reactive metabolite, covalent binding, glutathione S-transferase, bottom-up proteomics, data-dependent acquisition, high-resolution tandemmass spectrometry, multiple reactionmonitoring
Unité d'appartenance: Faculté des sciences > Département de chimie
Déposé par: Lekha Sleno
Date de dépôt: 07 févr. 2020 08:45
Dernière modification: 07 févr. 2020 08:45
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