Detection and Quantification of Drug-Protein Adducts in Human Liver.
Publication Title
Journal of proteome research
Document Type
Article
Publication Date
11-1-2024
Keywords
washington; isb; Humans; Microsomes, Liver; Raloxifene Hydrochloride; Tandem Mass Spectrometry; Chromatography, Liquid; Liver; Cytochrome P-450 Enzyme System; Cytochrome P-450 CYP3A
Abstract
Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.
Area of Special Interest
Digestive Health
Specialty/Research Institute
Hepatology
Specialty/Research Institute
Pharmacy
DOI
10.1021/acs.jproteome.4c00663
