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First published on June 10, 2005; DOI: 10.1124/dmd.105.004812

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Received for publication March 24, 2005.
Revised May 17, 2005.
Accepted for publication June 8, 2005.

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Michail A. Alterman 1*, Boris Kornilayev 1, Tatyana Duzhak 1, Dmitry Yakovlev 2

1 University of Kansas 2 University of KAnsas

* Address correspondence to: E-mail: malterman{at}ku.edu

Abstract

A novel MALDI-TOF MS method has been developed to quantitate cytochrome P450 isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of CYP isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with ICAT. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized CYP isozyme-specific peptide standards. The absolute quantitation of human CYP isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human CYP isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1 and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.


Key words: analytical pharmacology/toxicology, CYP expression, CYP1A, CYP2B, CYP2C, CYP2E, cytochrome P450 isoforms, human CYP enzymes, mass spectrometry, proteomics


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