title: Quantitative protein determination for CYP induction via LC-MS/MS.
abstract: The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.
authors.affiliation: AB SCIEX, Framingham, MA 01701, USA. Brian.Williamson@absciex.com
authors.forename: Brian L
authors.initials: BL
authors.lastname: Williamson
authors.forename: Subhasish
authors.initials: S
authors.lastname: Purkayastha
authors.forename: Christie L
authors.initials: CL
authors.lastname: Hunter
authors.forename: Lydia
authors.initials: L
authors.lastname: Nuwaysir
authors.forename: James
authors.initials: J
authors.lastname: Hill
authors.forename: LaHoma
authors.initials: L
authors.lastname: Easterwood
authors.forename: Jeanette
authors.initials: J
authors.lastname: Hill
mesh_terms: D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020033:Protein Isoforms; D003577:Cytochrome P-450 Enzyme System