Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples |
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Authors: | Ahmad Al Ali David Touboul Jean-Pierre Le Caër Isabelle Schmitz-Afonso Jean-Pierre Flinois Catherine Marchetti Isabelle De Waziers Alain Brunelle Olivier Laprévote Philippe Beaune |
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Institution: | 1. Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette Cedex, France 2. Médecine Personnalisée, Pharmacogénomique et Optimisation Thérapeutique, INSERM UMR-S1147, Centre Universitaire des Saints-Pères, Université Paris Descartes, Sorbonne Paris-Cité, 45 rue des Saint Pères, 75270, Paris Cedex 06, France 3. Chimie-Toxicologie Analytique et Cellulaire, UMR CNRS 8638, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l’Observatoire, 75006, Paris, France 4. Service de Toxicologie Biologique, H?pital Lariboisière, 2 rue Ambroise Paré, 75010, Paris, France
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Abstract: | Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost. Figure Calibration curve in complex matrix for CYPs quantification |
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