Premium
Quantitative protein determination for CYP induction via LC‐MS/MS
Author(s) -
Williamson Brian L.,
Purkayastha Subhasish,
Hunter Christie L.,
Nuwaysir Lydia,
Hill James,
Easterwood LaHoma,
Hill Jeanette
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000456
Subject(s) - cyp1a2 , cytochrome p450 , cyp3a4 , cyp2b6 , chemistry , gene isoform , microsome , enzyme inducer , enzyme , chromatography , biochemistry , gene
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