Detection and Quantification of Drug-Protein Adducts in Human Liver

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.

Keywords: DDA; DIA; adduct; cytochrome P450; mass spectrometry; raloxifene; reactive metabolite.

Figure 1

Raloxifene adducts identified in HLM incubations from 3 individual donors. (a) Molecular function Gene Ontology (GO) terms for proteins containing raloxifene modifications are presented as a hierarchical graph. Each term is labeled with its accession number, name, and the number of PSMs and distinct proteins containing raloxifene modifications. Terms with a PSM count under 125 were removed and the graph was limited to 4 levels. More intense shading represents more PSMs. (b) Number of peptides containing raloxifene modification mass detections in untreated (green) and raloxifene treated samples (purple) classified by the amino acid modified. One PSM in untreated sample had 2 modifications. Thus, 5 PSMs result in 6 locations for untreated modifications. (c) Raloxifene modified peptides found in CYPs are listed along with the number of PSMs in untreated versus raloxifene treated samples. Residues adducted by raloxifene are represented by [*]. Alkylation and oxidation are indicated by [57] and [16], respectively. Results represent comet searches allowing a variable modification of 471.15 Da (d₀-raloxifene) or 475.18 Da (d₄-raloxifene) postprocessed with TPP and filtered at 1% FDR. Raw data are available on Limelight at: https://limelight.yeastrc.org/limelight/go/LzWY1QCWYR.

Figure 2

Retention time and charge state distributions for PSMs of raloxifene adducted peptides relative to PSMs of their unadducted peptide counterparts from HLMs. (a) The mean retention time of the PSMs for each unadducted peptide was defined as 0 and all individual PSM retention times for corresponding unadducted and adducted peptides were calculated as a delta in minutes relative to that value. (b) The lowest observed charge state in the PSMs for each unadducted peptide counterpart was defined as 0 and all individual PSM charge states were calculated as a delta to that value. Raw data are the same as for Figure 1.

Figure 3

Quantification of peptides in untreated and raloxifene treated human liver microsome (HLM) preparations from 3 individual donors. Representative extracted ion chromatograms (XICs) and resulting peak areas are shown for fragment ion transitions from a selection of peptides. Yellow (top) panels: Control peptides. Yeast enolase (ENO1) was spiked into all samples as a process control. GP3 (GAPDH) is an endogenous housekeeping protein. Quantification of representative unadducted peptides from proteins found to be modified by raloxifene are also shown. Green (left) panel: Quantification of unadducted peptides corresponding to the raloxifene-adducted peptides shown in the purple (right) panel. Protein name, peptide sequence and precursor transition charge state are shown for each peptide. Example XICs are shown for treated and untreated HLM donor 1 samples. Peak areas are shown for all three donors. Horizontal green bars indicate untreated samples, and horizontal purple bars indicate raloxifene treated samples. Raw Skyline quantification data are available at https://panoramaweb.org/raloxifene-adducts.url.

Figure 4

CYP2C9 active site C216 is modified by raloxifene. Annotated spectrum of raloxifene-d₀ adducted C216 peptide (ILSSPWIQIC*NNFSPIIDYFPGTHNK) plus extracted ion chromatograms (XICs) and integrated peak areas for this peptide from untreated, raloxifene-d₀ and raloxifene-d₄ treated recombinant CYP2C9 supersome samples. Precursor signal is specific to raloxifene-d₀ sample. Quantified product ions are shared between raloxifene-d₀ and d₄ samples, are in the same DIA isolation window, and therefore show up in both treated samples. Expanded figure is presented in SI Figure S9.