A Targeted in Vivo SILAC Approach for Quantification of Drug Metabolism Enzymes: Regulation by the Constitutive Androstane Receptor

作者：MacLeod A Kenneth; Zang Tuo; Riches Zoe; Henderson Colin J; Wolf C Roland; Huang Jeffrey T J^*

来源：Journal of Proteome Research, 2014, 13(2): 866-874.

DOI：10.1021/pr400897t

• drug metabolism
• drug-drug interaction
• constitutive androstane receptor
• protein quantification
• targeted in vivo SILAC

The modulation of drug metabolism enzyme (DME) expression by therapeutic agents is a central mechanism of drug-drug interaction and should be assessed as early as possible in preclinical drug development. Direct measurement of DME levels is typically achieved by Western blotting, qPCR, or microarray, but these techniques have their limitations; antibody cross-reactivity among highly homologous subfamilies creates ambiguity, while discordance between mRNA and protein expression undermines observations. The aim of this study was to design a simple targeted workflow by combining in vivo SILAC and label-flee proteomics approaches for quantification of DMEs in mouse liver, facilitating a rapid and comprehensive evaluation of metabolic potential at the protein level. A total of 197 peptides, representing Si Phase 1 and Phase 11 DMEs, were quantified by LC-MS/MS using targeted high resolution single ion monitoring (tHR/SIM) with a defined mass-to-charge and retention time window for each peptide. In a constitutive androstane receptor (Car) activated mouse model, comparison of tHR/SIM-in vivo SILAC with Western blotting for analysis of the expression of cytochromes P450 was favorable, with agreement in fold-change values between methods. The tHR/SIM-in vivo SILAC approach therefore permits the robust analysis of multiple DME in a single protein sample, with clear utility for the assessment of the drug drug interaction therapeutic compounds.

• 出版日期2014-2