Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics

Publication date: Available online 4 May 2019Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Alexia Grangeon, Valerie Clermont, Azemi Barama, Fleur Gaudette, Jacques Turgeon, Veronique MichaudAbstractThe cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450 s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450 s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoenzymes (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450 s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 μm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 μL/min). Calibration curves were linear over a wide range of concentrations (0.1-50 nM) and the assay met all requirements of...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research