Cytochrome P450s (CYPs) constitute a large superfamily of membrane-bound proteins that participate in metabolism of
environmental contaminants, drugs, and endogenous compounds. Advances in mass spectrometry (MS)-based proteomics
have provided a potential for the identification and expression profiling of CYP proteins. However, the application of MS-based
proteomics is still limited due to the high amino acid sequence homology and insolubility of CYP proteins. Only a few studies have
successfully identified CYPs by combination of 1-dimensional electrophoresis (DE) and liquid chromatography tandem mass
spectrometry (LC-MS/MS). In previous studies, CYP proteins have been poorly separated in 2-DE. The objective of this study
is thus to establish a method using 2-DE and a high-performance matrix-assisted laser desorption/ionization- (MALDI) time of
flight/time of flight (TOF/TOF) tandem mass spectrometry for the comprehensive analysis of CYP isozymes expressed in animal
tissues. Microsomal fractions were isolated from the liver of MRL/lpr female mice treated with phenobarbital (PB) (80μg/g body
weight x 3 times). Total CYP content in the liver microsomes of PB-treated mice was significantly induced 5.4-fold, compared
with those of vehicle-treated mice. Moreover, O-dealkylation activities of alkoxyresorufins including methoxyresorufin (MROD),
ethoxyresorufin (EROD), pentoxyresorufin (PROD), and benzyloxyresorufin (BROD) were determined by fluorescence assays.
MROD, EROD, PROD, and BROD activities were significantly induced at 3.8, 6.4, 17, and 97-fold, respectively. 2-DE showed
high resolution of microsomal proteins in the range of the predicted gel migration of CYPs (40-60 kDa, pH 7-10). Peptide mass
fingerprinting and MS/MS ion searchusing MALDI TOF/TOF allowed successful identification of some CYP2 isozymes.
Maria Claret Lauan is currently a first year PhD student at Ehime University, Matsuyama, Japan under the supervision of Prof. Hisato Iwata
at Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University. Her research interests include wildlife
toxicology, proteomics, and bioinformatics. One of the goals of Prof. Iwata?s laboratory is to develop novel bioassay methods to explore bioactive/
toxic contaminants and its effects in a variety of wild and model animals.
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