Quantitative analysis of cytochrome p450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach.
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.
Alterman
Michail A
MA
Biochemical Research Service Laboratory/Analytical Proteomics Laboratory, University of Kansas, Lawrence, KS 66047-3761, USA. malterman@ku.edu
Kornilayev
Boris
B
Duzhak
Tatyana
T
Yakovlev
Dmitry
D
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010449:Peptide Mapping; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D014357:Trypsin