Seibert, Cathrin; (2008) Identification and quantification of metabolising enzymes in human tissue: a proteomics approach. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Metabolism is a complex process, taking place in every cell of the organism, comprising a variety of different chemical reactions. Knowledge about enzymes involved in metabolism of endogenous and exogenous substances is crucial for the understanding of detoxifying and toxifying processes in an organism. These in turn can play a role in the generation of a diseased state or contribute to healing. From this knowledge one can also predict e.g. the capability of a diseased tissue to metabolise an administered drug. This information supports the development of drug treatments, as different expression profiles of metalbolising enzymes can be expected in different tissue types. The most prominent group of metabolising enzymes arc presented by the Cytochrome P450 family; in humans there are 57 functioning genes known to dale. Analysis of melabolising enzymes was performed using mass spectrometry, Thereby detection of enzymes is possible directly at the protein level. Microsomal samples were submitted to one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by in-gel tryptic digestion. Extracted peptides were then analysed using nano-liquid chromatography (nano-LC) coupled to nano-electrospray tandem mass spectrometry (ES-MS/MS) and protein database searches were used for protein identification. Emphasis of the initial part of this work was on optimisation of the mass spectrometry method in order to increase the number of identified proteins in human lung microsomes. This was achieved by dividing the mass spectrometry scan range into smaller ranges. Hereby a 1.5-times higher number of proteins was identified hi a quantitative approach, seven human liver tissue samples were analysed and the content of Cytochrome P450 enzymes (CYP2E1 and CYP1A2) determined. This was accomplished using a stable isotope labelled internal standard peptide. The native and synthetic peptides show the same physical properties for analysis by LC-MS/MS, such as retention time in the chromatogram and are only differing in their molecular weight. This method was validated to prove reproducibility and linearity of the quantification technique. Quantification of CYP2E1 resulted in 88 200 pmol per mg of microsomal protein; for CYP1A2 165 240 pmol/mg microsomal protein were detected.

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