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Relative and absolute quantitative expression profiling of cytochromes P450 using isotope‐coded affinity tags
Author(s) -
Jenkins Rosalind E.,
Kitteringham Neil R.,
Hunter Christie L.,
Webb Sally,
Hunt Tony J.,
Elsby Robert,
Watson Rod B.,
Williams Dominic,
Penningtonand Stephen R.,
Park B. Kevin
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500432
Subject(s) - cytochrome p450 , computational biology , chemistry , drug development , profiling (computer programming) , drug , chromatography , biochemistry , biology , enzyme , computer science , pharmacology , operating system
The development of a novel method for absolute quantification of proteins based on isotope‐coded affinity tagging using ICAT® reagents is described. The method exploits synthetic peptide standards to determine protein content at the femtomole level in biological samples. The approach is generally applicable to any subset of proteins, but is particularly appropriate for quantitative analysis of multiple, closely related isoforms, and for hydrophobic proteins that are poorly represented in 2‐D gels. Relative and absolute quantification techniques are applied to an important group of microsomal metabolic enzymes, the cytochromes P450 (P450), which are critical in determining the disposition, safety and efficacy of drugs in man. Measurement of the P450 induction profile in response to chemicals is a fundamental aspect of drug safety evaluation and is currently achieved by low‐throughput methods employing poorly discriminatory antibodies or substrates. Tagging technology is shown to supersede conventional methods for P450 profiling in terms of discriminatory power and throughput, exemplified by the simultaneous detection of distinct induction profiles for cyp2c subfamily members in response to phenobarbitone: cyp2c29 expression, but not cyp2c40 or cyp2c50, was induced threefold by treatment. This technology should abbreviate the drug development pathway, and provide a widely applicable, rapid means of quantifying proteins.