Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with In silico target peptide selection

Hirotaka Kawakami, Sumio Ohtsuki, Junichi Kamiie, Takashi Suzuki, Takaaki Abe, Tetsuya Terasaki

Research output: Contribution to journalArticle

117 Citations (Scopus)

Abstract

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 10fmol, 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.

Original languageEnglish
Pages (from-to)341-352
Number of pages12
JournalJournal of Pharmaceutical Sciences
Volume100
Issue number1
DOIs
Publication statusPublished - 2011 Jan

Keywords

  • CYP enzymes
  • Cytochrome P450
  • Hepatic metabolism
  • LC-MS/MS
  • Mass spectrometry
  • Metabolism
  • Microsomes
  • Proteomics
  • Quantification

ASJC Scopus subject areas

  • Pharmaceutical Science

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