Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes

Anal Biochem. 2015 Jun 1;478:8-13. doi: 10.1016/j.ab.2015.03.001. Epub 2015 Mar 10.


The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography-tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

Keywords: Cytochrome P450 enzymes; Microsomal epoxide hydrolase; Microsomal glutathione-S-transferases; Microsomes; Nano liquid chromatography–tandem multiple reaction monitoring.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Liquid / methods
  • Cytochrome P-450 Enzyme System / analysis*
  • Enzyme Assays / methods
  • Epoxide Hydrolases / analysis*
  • Glutathione Transferase / analysis*
  • Humans
  • Intestines / enzymology
  • Kidney / enzymology
  • Lung / enzymology
  • Microsomes, Liver / enzymology*
  • Molecular Sequence Data
  • Tandem Mass Spectrometry / methods


  • Cytochrome P-450 Enzyme System
  • Glutathione Transferase
  • Epoxide Hydrolases