Acetaminophen Adducts of Rat Liver Microsomal Proteins using LC-MSMS

The metabolism of xenobiotics in the liver can give rise to reactive metabolites that may covalently bind to tissue macromolecules, such as proteins. Determination of proteins which are targeted by reactive metabolites is of importance in drug discovery and molecular toxicology. However, there are difficulties in the analysis of target proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed for systematic identification of the target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using ultra high-performance liquid chromatography (UHPLC) and high-resolution tandem mass spectrometry. RLM samples were first incubated with and without APAP, digested, and subjected to strong cation exchange (SCX) fractionation prior to the UHPLC-MS/MS analysis. Four data processing strategies were then combined into an efficient label-free workflow, meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in crucial cellular processes, to be covalently modified by APAP.