Article

Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.

Biochemical Research Service Laboratory, University of Kansas, Lawrence, KS 66047, United States.
Toxicology in Vitro (Impact Factor: 3.21). 05/2008; 22(3):779-87. DOI: 10.1016/j.tiv.2007.12.001
Source: PubMed

ABSTRACT P450s are key enzymes responsible for biotransformation of numerous endogenous and exogenous compounds and are located in almost every tissue. This superfamily is the largest group of enzymes (>6000) that share a high degree of similarity in protein sequence. The human genome contains 57 CYP genes and 58 pseudogenes. A major gap exists in our knowledge about differences in CYP expression on a protein level. DNA and mRNA information are not sufficient because transcription and particularly translation events are not necessarily correlated with levels of expressed proteins. The data reported in this study complete the framework of an integrated proteomic method for the simultaneous qualitative and quantitative analysis of CYP isozyme composition using MALDI-TOF-MS and immunochemistry that has been developed in our laboratory over the last several years (Alterman et al., 2005a,b) and is based on the fact that each P450 isozyme possesses unique tryptic peptide(s) (UTP) that could be used for differential analysis of human CYP expression. Here we demonstrate that three different immunochemical techniques (ELISA, Western blot, and peptide affinity enrichment on magnetic beads with attached antibodies) have potential to be incorporated in an integrated proteomic method combining mass spectrometry and immunochemistry. Fundamentally, this approach is based on the measurement of the same chemical entity (isozyme-specific UTP) in the tryptic digest by two orthogonal analytical techniques, mass spectrometry and immunochemistry. The application of this approach is illustrated with two human CYP isozymes--CYP1A2 and CYP2E1.

0 Bookmarks
 · 
76 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The review describes recent approaches used for evaluation of cytochrome P450 concentration and activity. It considers employment of modern methods of proteomic analysis including electrophoresis and chromatography/mass-spectrometry (MS) for investigation of the particular group of proteins. Special attention is paid to targeted quantitative MS analysis of cytochromes P450 in biological samples. Finally, we analyze methods used for assay of cytochromes P450 activities and problems of correlation between content of certain P450 isoforms and their specific enzymatic activities.
    Biochemistry (Moscow) Supplement Series B Biomedical Chemistry 04/2013; 7(2). DOI:10.1134/S1990750813020078
  • [Show abstract] [Hide abstract]
    ABSTRACT: CYP2E1 is an important cytochrome P450 isoform in many endogenous processes and in the metabolism of organic solvents, a number of drugs and pre-carcinogens. Information on the abundance of the enzyme may be valuable in various types of research in the field of toxicology and pharmacology. An indirect ELISA for the quantification of CYP2E1 in human liver microsomes was developed and successfully validated. All samples, including validation samples and calibrators, were diluted to a final concentration of microsomal protein of 10μg/ml. Detection of the antigen was obtained through binding of a polyclonal antibody raised against the full length protein, followed by the addition of horseradish peroxidase conjugated secondary antibodies and enzymatic detection. A five-parameter logistics function with 1/x weighting was used for quantification within the concentration range of 4-256pmol CYP2E1/mg microsomal protein. The method showed acceptable intra- and inter-assay precision, with calculated coefficients of variation of 6.3-15.2% and 11.3-21.0%, respectively. The relative error varied between -2.3 and 8.9%, and the total error between 16.0 and 27.2%. No significant cross reactivity with other abundant CYP isoforms was observed. The method was evaluated through the analysis of samples from a pharmacokinetic study, and the comparison with the CYP2E1 activity in those samples.
    Journal of pharmaceutical and biomedical analysis 10/2013; 88C:536-541. DOI:10.1016/j.jpba.2013.09.008 · 2.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS) - based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of "rare" CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level.
    Toxicology and Applied Pharmacology 12/2012; DOI:10.1016/j.taap.2012.12.008 · 3.98 Impact Factor

Full-text

Download
52 Downloads
Available from
May 23, 2014