Quantitative protein determination for CYP induction via LC-MS/MS

Proteomics. 2011 Jan;11(1):33-41. doi: 10.1002/pmic.201000456. Epub 2010 Dec 6.


The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.

MeSH terms

  • Cells, Cultured
  • Chromatography, Liquid / methods*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Microsomes, Liver / metabolism
  • Protein Isoforms / metabolism
  • Proteomics / methods*
  • Tandem Mass Spectrometry / methods*


  • Protein Isoforms
  • Cytochrome P-450 Enzyme System