Direct Identification of Cytochrome P450 Isozymes by Matrix-assisted Laser Desorption/Ionization Time of Flight-Based Proteomic Approach

Abstract

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 (Halpert et al., 1994;Kobayashi et al., 2002), antibody-based identification (Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999). However, only a limited number of almost 2000 P450s is characterized in terms of substrate specificity and has available antibodies, not to mention the fact that, to the best of our knowledge, there is no known substrate or inhibitor that is absolutely specific for only one P450 isozyme. As for antibody-based techniques, the main problem is a very high degree of sequence homology characteristic for P450 isozymes. For example, neither polyclonal nor monoclonal antibodies can distinguish C...

A Proteomic Approach to the Identification of Cytochrome P450 Isoforms in Male and Female Rat Liver by Nanoscale Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

“…These isoforms were also found among the 24 P450s identified in male and female rat livers using our LC-ESI-MS/MS methodology. MALDI-TOF-based PMF of clofibrateinduced rat liver microsomes revealed the presence of CYP2B1, 2B2, 4A1, and 4A3 (Galeva et al, 2003). Of these, CYP4A3 was the only isoform detected in our male and female livers, suggesting that noninduced levels of CYP4A1, 2B1, and 2B2 are low.…”

“…Previously, the use of MALDI-TOF-based PMF of onedimensional gel tryptic digests of liver microsomes uncovered four P450s (2A1, 2C11, 2D2, 2D5) in uninduced male Sprague-Dawley rats (Galeva et al, 2003). These isoforms were also found among the 24 P450s identified in male and female rat livers using our LC-ESI-MS/MS methodology.…”

“…In recognition of this, SDS-PAGE (onedimensional gel) separation of microsomal proteins followed by MALDI-TOF and PMF was used to separate and analyze the endoplasmic reticulum proteins from rat and rabbit livers. Using this one-dimensional gel separation, up to eight P450s were positively identified (Galeva et al, 2003). However, PMF is most reliable when samples containing only a few proteins are analyzed because the presence of multiple proteins within an SDS-PAGE band may lead to spurious results.…”

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

“…48 Several efforts to discriminate between CYP isoforms by proteomicsandmassspectrometryaredescribedintheliterature. 15,[47][48][49][50][51][52][53] Most of the studies were performed with rat or mouse liver. But since the CYP pattern of these organisms is mainly different from that in human, studies are hardly comparable.…”

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

“…Consequently, one has to deal with mixtures containing more than one P450, and this superfamily is the largest group of enzymes that share a high degree of similarity in protein sequence. Our laboratory has previously shown that peptide mass fingerprinting (PMF) can be successfully applied to the identification and differential analysis of closely related cytochrome P450 isozymes in liver and brain microsomes from rat, mouse, and rabbit (Galeva and Altermann, 2002;Duzhak et al, 2003;Galeva et al, 2003). In this study, on the examples of rat and human P450s, we demonstrate that members of the P450 superfamily possess unique isozyme-specific tryptic peptide(s) that could be utilized for differential qualitative and quantitative analysis by MALDI-TOF mass spectrometry.…”