Quantification of Accurate Composition and Total Abundance of Homologous Proteins by Conserved-Plus-Surrogate Peptide Approach: Quantification of UDP Glucuronosyltransferases in Human TissuesS

Characterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucurono-syltransferases (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 surro-gate peptides, which often results in high technical and interlabora-tory variability due to peptide-specific digestion leading to data inconsistencies. To address this problem, we developed a novel conserved-plus-surrogate peptide (CPSP) approach for determin-ing the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kid-ney microsomes (HKM), and human liver S9 (HLS9) fraction. The relative percent composition of UGT1A and UGT2B isoforms in the 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 showed unique compositions of UGT1As and UGT2Bs. The reproduc-ibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2> 0.91). The analysis of the conserved peptides also provided the 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), and 138 and 99 (HKM), respectively. The CPSP approach could be used for applications in the in-vitro-to-in-vivo extrapolation of drug metabolism and PBPK modeling. SIGNIFICANCE STATEMENT We quantified the absolute compositions and total abundance of UDP glucuronosyltransferases (UGTs) in pooled human liver, intestine, and kidney microsomes using a novel conserved-plus-surrogate peptide (CPSP) approach. The CPSP approach addresses the surrogate peptide-specific variability in the determination of the absolute composition of UGTs. The data presented in this manu-script are applicable for the estimation of the fraction metabolized by individual UGTs towards better in vitro-to-in vivo extrapolation of UGT-mediated drug metabolism.

Automated summary

In this study, a novel conserved-plus-surrogate peptide (CPSP) approach was developed to determine the absolute compositions and total abundance of UGTs in human liver, intestine, and kidney. The CPSP approach addresses the issue of data inconsistency caused by surrogate peptide-specific digestion. The results of this study can be applied to the in vitro-to-in vivo extrapolation of UGT-mediated drug metabolism.