Short Communication DIRECT IDENTIFICATION OF CYTOCHROME P450 ISOZYMES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME OF FLIGHT-BASED PROTEOMIC APPROACH
ABSTRACT: The main targets of our investigation were cytochrome P450 isozymes (P450), the key enzymes of the hepatic drug-metabolizing system. Current research approaches to the identification of individual P450 forms include specific P450 inhibitors or substrates, antibody-based identification, and mRNA-based expression profiling. All of these approaches suffer from one common disadvantage-they all are indirect methods. On the other hand, current developments in mass spectrometry provide a direct and reliable approach to protein identification with sensitivity in the femtomole or low picomole range. In this study we have used high-accuracy, matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-based peptide mapping to perform direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes. For the first time, the P450 isozyme composition of clofibrate-induced rat and phenobarbital-induced rabbit liver microsomes was determined by peptide mass fingerprinting (PMF). Application of MALDI TOF-based PMF allows differential identification of such highly homologous P450s as CYP2B1 and CYP2B2. We have found that CYP2A10 previously reported only in rabbit olfactory and respiratory nasal mucosa is present in phenobarbital (PB)-induced rabbit liver microsomes. Two other rabbit P450s, earlier identified only by screening a cDNA library, were found to be present in PB-induced rabbit liver microsomes. In summary, direct identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes. Cytochrome P450 isozymes (P450) are the key enzymes of the hepatic drug-metabolizing system. Eukaryotic P450s are membrane proteins that are expressed in varying amounts, and many forms differ very little in their amino acid sequence and catalytic properties. Currently the number of sequenced and named distinct P450s exceeds 1925 (dnelson. utmem.edu/CytochromeP450.html). Since individual P450 isozymes exhibit a broad, often overlapping substrate specificity, knowledge of the P450 composition in a particular type of microsomes is critical in predicting drug/substrate interactions and formation of reactive intermediates. Current research approaches to the identification of individual P450 forms include: specific P450 inhibitors or substrates On the other hand, tryptic peptide mass fingerprinting (PMF) in conjunction with MALDI TOF mass spectrometry has become the main analytical tool in protein identification due to its ability to analyze picomole quantities of gel-or HPLC-separated proteins in short time In this study we have used MALDI TOF-based peptide mass fingerprinting to perform a detailed direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes and have shown that identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes
