Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens

Abstract

Biotransformation by phase I and II metabolizing enzymes represents the major determinant for the oral bioavailability of many drugs. To estimate the pharmacokinetics, data on protein abundance of hepatic and extrahepatic tissues, such as the small intestine, are required. Targeted proteomics assays are nowadays state-of-the-art for absolute protein quantification and several methods for quantification of drug metabolizing enzymes have been published. However, some enzymes remain still uncovered by the analytical spectra of those methods. Therefore, we developed and validated a quantification assay for two carboxylesterases (CES-1, CES-2), 17 cytochrome P450 enzymes (CYP) (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F12, CYP4A11) and five UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT2B7, UGT2B15, UGT2B17). Protein quantification was performed by analyzing proteospecific surrogate peptides after tryptic digestion with stable isotope-labelled standards. Chromatographic separation was performed on a Kinetex® 2.6 µm C18 100 Å core–shell column (100 × 2.1 mm) with a gradient elution using 0.1% formic acid and acetonitrile containing 0.1% formic acid with a flow rate of 200 µl/min. Three mass transitions were simultaneously monitored with a scheduled multiple reaction monitoring (sMRM) method for each analyte and standard. The method was partly validated according to current bioanalytical guidelines and met the criteria regarding linearity (0.1–25 nmol/L), within-day and between-day accuracy and precision as well as multiple stability criteria. Finally, the developed method was successfully applied to determine the abundance of the aforementioned enzymes in human intestinal und liver microsomes. Our work offers a new fit for purpose method for the absolute quantification of CES, CYPs and UGTs in various human tissues and can be used for the acquisition of data for physiologically based pharmacokinetic modelling.