Protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, toxicity and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods capable of discovering the proteins and peptides adducted by reactive metabolites in complex matrices. The current study presents a proteomics workflow that achieves this task. This workflow combines data-dependent, and data independent acquisition (DDA and DIA) based LC-MS/MS and is sensitive enough to detect very low abundance adducts resulting from CYP mediated drug metabolism in human livers. Human liver microsomes (HLMs) or recombinant CYPs were incubated with raloxifene as a model compound. The resulting adducts were identified using this workflow. In HLMs, raloxifene adducts were detected in 78 proteins, including multiple adducts in 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 inactivation (TDI) by raloxifene. These data suggest many adducts formed by reactive intermediates are benign. DIA analysis showed variable abundance of raloxifene adducts in many proteins between livers, but no concomitant decrease in abundance of unadducted peptides. The current study sets a new standard for adduct detection in complex samples, offering valuable insights into the human adductome resulting from exposure to reactive metabolites. The developed methodology forms a foundation for mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and drug-drug interactions and adducts that do not.

HLM and supersome incubations were diluted with 50 mM ammonium bicarbonate or 50 mM tris pH 8.5 to a final protein concentration of 0.45 to 0.66 µg/µL. Yeast enolase was spiked in as a process control at 16 ng enolase/µg total sample protein. Samples were reduced, alkylated and digested for 6 hours at 37oC at an enzyme to substrate ratio of 1:15. Digestion was stopped by acidification. Pierce Peptide Retention Time Calibration Mixture (PRTC) was added as a system suitability standard. Samples were spun at max speed in a benchtop microfuge for 10 min and supernatants were transferred to an autosampler vial and stored at -80oC until analysis. Full details can be found in the associated manuscript.

For human liver microsome (HLM) incubations HLMs were obtained from the tissue bank of University of Washington School of Pharmacy. HLMs were prepared from 3 donors, HLM131, HLM135 and HLM157, as previously described. The donor characteristics for the human liver samples were as follows: HL131 was from a 62 year old white female who died of stroke after 6 days at the ICU, HL135 was from a 45 year old female who died of cerebrovascular accident after 6 days in ICU. This liver was mildly fatty. HL157 was from a 41 year old female who died of stroke after 5 days in ICU. All of the donors had CYP3A5*3/*3 genotype. The microsomes were stored in 50 mM potassium phosphate (KPi) containing 250 mM sucrose and 50 mM KCl, pH 7.4. Insect cell microsomes (supersomes) expressing a single human CYP enzyme (CYP3A4, CYP3A5, CYP2C8, CYP2C9 or CYP2C19) along with human cytochrome P450 reductase (reductase) and cytochrome b5 were purchased from BD Gentest/Corning. All samples were treated with raloxifene-d0 or raloxifene-d4 dissolved in DMSO, or pure DMSO as a control. The reactions were initiated by addition of NADPH. Samples were digested using trypsin or trypsin/LysC. Full details can be found in the associated manuscript.