A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

• Published in: Proteomics (Weinheim) Vol. 8; no. 20; pp. 4186 - 4196
• Main Authors: Wang, Michael Zhuo, Wu, Judy Qiju, Dennison, Jennifer B., Bridges, Arlene S., Hall, Stephen D., Kornbluth, Sally, Tidwell, Richard R., Smith, Philip C., Voyksner, Robert D., Paine, Mary F., Hall, James Edwin
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.10.2008; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: Absolute quantification; Adolescent; Adult; Aged; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Child; CYP3A; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - analysis; Cytochrome P450; Female; Fundamental and applied biological sciences. Psychology; Humans; LC-MS/MS; Male; Mass Spectrometry - methods; Microsomes, Liver - chemistry; Middle Aged; Miscellaneous; Pharmacoproteomics; Proteins; Proteomics - methods; Sequence Homology, Amino Acid; Uncertainty; Human; CYP3A; LC-MS/MS; Cytochrome P450; Liver; Proteomics; Absolute quantification; Metabolic activation; Pharmacoproteomics; Microsome; Protein; Quantitative analysis
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.200800144

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 MS‐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 (HLM). 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 ˜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 (r2≥0.87) and marker activities (r2≥0.88), including testosterone 6β‐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.