Identificationof Acetaminophen Adducts of Rat LiverMicrosomal Proteins using 2D-LC-MS/MS.

Xenobiotic metabolism in the livercan give rise to reactive metabolitesthat covalently bind to proteins, and determining which proteins aretargeted is important in drug discovery and molecular toxicology.However, there are difficulties in the analysis of these modifiedproteins in complex biological matrices due to their low abundance.In this study, an analytical approach was developed to systematicallyidentify target proteins of acetaminophen (APAP) in rat liver microsomes(RLM) using two-dimensional chromatography and high-resolution tandemmass spectrometry. In vitromicrosomal incubations,with and without APAP, were digested and subjected to strong cationexchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Fourdata processing strategies were combined into an efficient label-freeworkflow meant to eliminate potential false positives, using peptidespectral matching, statistical differential analysis, product ionscreening, and a custom-built delta-mass filtering tool to pinpointpotential modified peptides. This study revealed four proteins, involvedin important cellular processes, to be covalently modified by APAP.Data are available via ProteomeXchange with identifier PXD002590. [ABSTRACT FROM AUTHOR]