title: Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples.
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.
authors: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette Cedex, France.
authors: Ahmad
authors: A
authors: Al Ali
authors: David
authors: D
authors: Touboul
authors: Jean-Pierre
authors: JP
authors: Le Caër
authors: Isabelle
authors: I
authors: Schmitz-Afonso
authors: Jean-Pierre
authors: JP
authors: Flinois
authors: Catherine
authors: C
authors: Marchetti
authors: Isabelle
authors: I
authors: De Waziers
authors: Alain
authors: A
authors: Brunelle
authors: Olivier
authors: O
authors: Laprévote
authors: Philippe
authors: P
authors: Beaune
mesh_terms: D003545:Cysteine / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007527:Isoenzymes; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D003545:Cysteine