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Direct Quantification of Cytochromes P450 and Drug Transporters - A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates

Weiss, F., Hammer, H. S., Klein, K., Planatscher, H. P., Zanger, U. M., Noren, A., Wegler, C., Artursson, P., Joos, T. O., Poetz, O. (2018).

Drug Metab Dispos. 2018 Jan 17. pii: dmd.117.078626. doi: 10.1124/dmd.117.078626.

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample pre-fractionation steps prior to mass spectrometry read-out. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric read-out allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (TXP-antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 CYP enzymes and 9 transporters. We analyzed the cytochrome P450 enzyme and transporter levels in genotyped liver tissue homogenates, microsomes and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. Since for the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.