Lane, Catherine Susana; (2004) The analysis of cytochrome P450 proteins by mass spectrometry. Doctoral thesis (Ph.D.), University College London.

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Abstract

The cytochromes P450 (P450s) are a superfamily of mixed function oxidases with a central role in the metabolism of a large number of drugs, xenobiotics and endogenous compounds. Mammalian P450s are membrane bound and hydrophobic in character. The completion of the draft of the human genome revealed the presence of about 106 different P450 genes, of which 57 are thought to be functional. Traditional methods for the analysis of P450s rely on indirect detection techniques such as immunoblotting, activity assays and the detection of P450 mRNA. Mass spectrometry (MS) provides an attractive alternative approach since it offers uniquely the ability to directly detect low levels of multiple proteins simultaneously and without pre-selection. A method for the analysis of cytochrome P450 proteins by MS has been developed. Microsomal proteins are separated by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by in-gel tryptic digestion of the protein bands and peptide analysis by nano-liquid chromatography (nano-LC) coupled to nano-electrospray tandem mass spectrometry (ES-MS/MS), with identification by database searching. Work with biological samples culminated in a study in which the P450 expression profile was determined for six sets of colorectal liver metastases and corresponding liver samples from patients with metastatic colorectal cancer of the liver. Fourteen distinct P450 enzymes were positively identified (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 4A11, 4F2, 4F11, 8B1 and 27A1), thirteen in liver and twelve in tumour tissue. The P450 profiles of the tumours demonstrate that metastatic cancers in liver potentially have extensive drug-metabolising capabilities, which are likely to be important in determining the outcome of treatment. Further studies have been initiated, which aim to identify and quantify functional P450s using mechanism based inactivators (MBIs). These are substrates that form metabolites that covalently adduct to the active site, leaving the enzyme permanently modified. The use of benzyl isothiocyanate (BITC) as a MBI of CYP2E1 led to the identification of a BITC-modified catalytic site peptide, 283LYTMDGITVTVADLFFAGTETTSTTLR309. The site of modification was determined by MS/MS to be one of six C-terminal threonine and serine residues. The mass of the modification was consistent with the adduction of one molecule of benzyl isocyanate and oxygen.

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