Abstract

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.