期刊
PROTEOMICS
卷 8, 期 20, 页码 4186-4196出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/pmic.200800144
关键词
Absolute quantification; CYP3A; Cytochrome P450; LC-MS/MS; Pharmacoproteomics
资金
- Bill and Melinda Gates Foundation
The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in M S-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HIM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was similar to 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV <= 27%). The MS-based method strongly correlated with the immunoquantification method (r(2)>= 0.87) and marker activities (r(2)>= 0.88), including testosterone 6 beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.
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