Detection\nand\nQuantification of Drug–Protein\nAdducts in Human Liver

Covalent protein\nadducts formed by drugs or their reactive metabolites\nare risk factors for adverse reactions, and inactivation of cytochrome\nP450 (CYP) enzymes. Characterization of drug–protein adducts\nis limited due to lack of methods identifying and quantifying covalent\nadducts in complex matrices. This study presents a workflow that combines\ndata-dependent and data-independent acquisition (DDA and DIA) based\nliquid chromatography with tandem mass spectrometry (LC-MS/MS) to\ndetect very low abundance adducts resulting from CYP mediated drug\nmetabolism in human liver microsomes (HLMs). HLMs were incubated with\nraloxifene as a model compound and adducts were detected in 78 proteins,\nincluding CYP3A and CYP2C family enzymes. Experiments with recombinant\nCYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested,\nincluding CYPs not subject to time-dependent inhibition by raloxifene.\nThese data suggest adducts can be benign. DIA analysis showed variable\nadduct abundance in many peptides between livers, but no concomitant\ndecrease of unadducted peptides. This study sets a new standard for\nadduct detection in complex samples, offering insights into the human\nadductome resulting from reactive metabolite exposure. The methodology\npresented will aid mechanistic studies to identify, quantify and differentiate\nbetween adducts that result in adverse drug reactions and those that\nare benign.