TY - JOUR T1 - Enrichment-free high throughput LC-MRM quantification of cytochrome P450 proteins in plated human hepatocytes direct from 96-well plates enables routine protein induction measurements JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.090480 SP - dmd.120.090480 AU - John Savaryn AU - Ning Liu AU - Jun Sun AU - Junli Ma AU - David M Stresser AU - Gary Jenkins Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/04/29/dmd.120.090480.abstract N2 - Despite the availability of LC-MS methods for quantifying cytochrome P450 (CYP) proteins, incorporation of CYP protein quantification into induction study workflows has not been widely adopted. To more readily enable CYP protein quantification in induction study workflows, DMPK research groups need a simple, robust, cost-effective, high-throughput method compatible with 96-well plated human hepatocyte formats. Here, we provide such a methodology. Our method bypasses both microsomal enrichment and antibody-based enrichment to go directly from the plate to LC-MS/MS analysis. We use this "plate-to-peaks" approach for quantifying CYP3A4, CYP2B6, and CYP1A2 - the major inducible hepatic CYPs representative of PXR, CAR and AhR mediated induction, respectively. We leveraged our induction study-aligned assay format to assess induction across mRNA, protein, and enzyme activity using known induction control compounds. As expected, results from the 3 methods using model inducers were broadly concordant, but the magnitude of the induction response differed. Induction of CYP3A4 using 10 μM rifampicin was 12-fold for RNA, 8-fold for protein, and 3-fold for activity; for CYP1A2 with 50 μM omeprazole induction was 30-fold for RNA, 13-fold for protein, and 17-fold for activity; for CYP2B6 with 50 μM phenytoin induction was 23-fold for RNA, 2-fold for protein, and 5-fold for activity. Most importantly, we anticipate the relative ease of this method will enable researchers to routinely adopt CYP protein quantification as part of non-clinical evaluation of CYP induction.SIGNIFICANCE STATEMENT Current methodologies for quantifying CYP proteins by LC-MS/MS are either cumbersome, too costly, or both, to be widely adopted into induction study workflows by the ADME research community. We present a simplified LC-MS/MS methodology for quantifying CYP proteins directly from human hepatocytes, without any form of enrichment, in 96-well induction assay plate format that should be readily adoptable by any ADME lab with LC-MRM capabilities. ER -