Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.
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.
Kornilayev
Boris A
BA
Biochemical Research Service Laboratory, University of Kansas, Lawrence, KS 66047, United States.
Alterman
Michail A
MA
D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry / Q000276:immunology; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000276:immunology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007120:Immunochemistry; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
D019138:Antibodies, Blocking; D007202:Indicators and Reagents; D007527:Isoenzymes; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D019388:Cytochrome P-450 CYP1A2; D014357:Trypsin