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.The superfamily of cytochrome(s) P450 (P450) plays a key role in hepatic and extrahepatic drug metabolism, and qualitative and quantitative analyses of the P450 isozyme expression in a particular organ are critical in predicting a metabolic fate of a particular drug or in examination of the potential drug-drug interaction. The number of sequenced and named different P450 isozymes surpassed 3100 (dnelson.utmem.edu/CytochromeP450.html), and the degree of sequence homology, particularly among P450s belonging to the same subfamily, is high (Nelson et al., 1996). None of the existing research approaches to the analysis of individual P450 forms, which include specific P450 inhibitors (Halpert et al., 1994;Kobayashi et al., 2003) or substrates (Kobayashi et al., 2002;Stresser et al., 2002), antibodybased identification (Gelboin et al., 1999;Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999;Zhang et al., 1999), is in a position to provide reliable quantitative and qualitative information on the individual P450 composition in a given type of microsomes. First, only a minority of known P450 isozymes is fully characterized by substrate specificity, and since they exhibit a broad, often overlapping substrate specificity, there is no known substrate or inhibitor that is absolutely specific for an individual P450 isozyme. Second, the high degree of sequence homology among members of the P450 superfamily confounds high specificity of antibody-based analysis, particularly among members of the same subfamily. Third, the application of a quantitative mRNA analysis for the evaluation of P450 isozyme expression...
