Journal
JOURNAL OF PHARMACEUTICAL SCIENCES
Volume 100, Issue 1, Pages 341-352Publisher
ELSEVIER SCIENCE INC
DOI: 10.1002/jps.22255
Keywords
CYP enzymes; cytochrome P450; hepatic metabolism; LC-MS/MS; mass spectrometry; metabolism; microsomes; proteomics; quantification
Funding
- Japan Science and Technology Agency (JST)
- New Energy and the Industrial Technology Development Organization (NEDO) of Japan
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Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10 fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100: 341-352, 2011
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