RT Journal Article
SR Electronic
T1 Quantification of accurate composition and total abundance of homologous proteins by conserved-plus-surrogate peptide (CPSP) approach: Quantification of UDP glucuronosyltransferases in human tissues
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP DMD-AR-2022-001155
DO 10.1124/dmd.122.001155
A1 Deepak Ahire
A1 Mitesh Patel
A1 Sujal V Deshmukh
A1 Bhagwat Prasad
YR 2022
UL http://dmd.aspetjournals.org/content/early/2022/11/29/dmd.122.001155.abstract
AB Characterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucuronosyltransferases (UGTs), is important for predicting the fractional contribution of individual isoforms involved in the metabolism of a drug for applications in physiologically based pharmacokinetic (PBPK) modeling. Conventional targeted proteomics utilizes surrogate peptides, which often results in high technical and inter-laboratory variability due to peptide-specific digestion efficiency leading to data inconsistencies. To address this problem, we developed a novel universal conserved-plus-surrogate peptide (CPSP) approach for determining the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), and human liver S9 (HLS9) fractions. The relative percent composition of UGT1A and UGT2B isoforms in human liver was 35:5:36:11:13 for UGT1A1:1A3:1A4:1A6:1A9, and 20:32:22:21:5 for UGT2B4:2B7:2B10:2B15:2B17. The human kidney and intestine also show unique compositions of UGT1As and UGT2Bs. The reproducibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2>0.91). The analysis of conserved peptides also provided the absolute abundance for individual UGT isoforms included in this investigation as well as the total abundance (pmol/mg protein) of UGT1As and UGT2Bs across tissues, i.e., 268 and 342 (HLM), 21 and 92 (HIM), 138 and 99 (HKM), respectively. In summary, the CPSP approach could be utilized for applications in the in-vitro to in-vivo extrapolation (IVIVE) of drug metabolism and PBPK modeling. Significance Statement We quantified the absolute compositions and total abundance of UGTs in human liver, intestine, and kidney microsomes using a novel CPSP approach. Unlike the conventional surrogate peptide-based targeted proteomics, the CPSP approach addresses the surrogate peptide-specific variability in the determination of the absolute composition of UGTs. The data presented in this manuscript are applicable for the estimation of the fraction metabolized (fm) by individual UGTs towards better IVIVE of UGT-mediated drug metabolism in the human liver, intestine, and kidney.