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QUANTITATIVE ANALYSIS OF CYTOCHROME P450 ISOZYMES BY MEANS OF UNIQUE ISOZYME-SPECIFIC TRYPTIC PEPTIDES: A PROTEOMIC APPROACH

Michail A. Alterman, Boris Kornilayev, Tatyana Duzhak and Dmitry Yakovlev
Drug Metabolism and Disposition September 2005, 33 (9) 1399-1407; DOI: https://doi.org/10.1124/dmd.105.004812
Michail A. Alterman
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Boris Kornilayev
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Tatyana Duzhak
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Dmitry Yakovlev
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Abstract

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 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 isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 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 P450 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.

Footnotes

  • This work was funded by The University of Kansas Center for Research Grant RDF00254 (M.A.). The Voyager DE STR mass spectrometer of the Biochemical Research Service Laboratory was acquired on funding provided by the National Institutes of Health, Grant 1 S10 RR13020–01A1 (M.A.). B.K. and T.D. were supported by the Research Development Grant from the University of Kansas (KUCR 00254) and National Institutes of Health Center of Biomedical Research Excellence Grant RR017708.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.105.004812.

  • ABBREVIATIONS: P450, cytochrome P450; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; MS, mass spectrometry; PMF, peptide mass fingerprinting; IS, internal standard; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; β-LGA, β-lactoglobulin A; BCA, bicinchoninic acid.

    • Received March 23, 2005.
    • Accepted June 8, 2005.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 33 (9)
Drug Metabolism and Disposition
Vol. 33, Issue 9
1 Sep 2005
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QUANTITATIVE ANALYSIS OF CYTOCHROME P450 ISOZYMES BY MEANS OF UNIQUE ISOZYME-SPECIFIC TRYPTIC PEPTIDES: A PROTEOMIC APPROACH

Michail A. Alterman, Boris Kornilayev, Tatyana Duzhak and Dmitry Yakovlev
Drug Metabolism and Disposition September 1, 2005, 33 (9) 1399-1407; DOI: https://doi.org/10.1124/dmd.105.004812

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QUANTITATIVE ANALYSIS OF CYTOCHROME P450 ISOZYMES BY MEANS OF UNIQUE ISOZYME-SPECIFIC TRYPTIC PEPTIDES: A PROTEOMIC APPROACH

Michail A. Alterman, Boris Kornilayev, Tatyana Duzhak and Dmitry Yakovlev
Drug Metabolism and Disposition September 1, 2005, 33 (9) 1399-1407; DOI: https://doi.org/10.1124/dmd.105.004812
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