Nisar, Sonia;
(2005)
Nano-scale liquid chromatography electrospray tandem mass spectrometric identification of multiple cytochrome P450 isoforms in tissues.
Doctoral thesis (Ph.D.), University College London.
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Abstract
The cytochromes P450 (CYPs) are membrane bound proteins that collectively carry out a wide range of biological oxidations important in the metabolism of steroids, ecosanoids and xenobiotics including anticancer agents. A method of CYP separation and analysis has been developed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nano-scale liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) for the direct identification of multiple CYPs found in rat liver, human tissues and tumours. Endoplasmic reticulum from selected tissues was prepared as microsomes using ultracentrifugation and subjected to SDS-PAGE. The proteins present in the 48-62 kDa gel band region were excised, cut into small pieces and digested with trypsin. The resultant peptides were solvent extracted, separated and analysed by LC-ESI-MS/MS. CYP identifications were made by searching the MS/MS data using Sequest software against a protein database (obtained from National Centre for Biotechnology Information), Twenty-four CYP isoforms from the sub-families lA, 2A, 28, 2C, 2D, 2E, 3A, 4A, 4F, CYP 17 and CYP 19 were positively identified in rat liver, fourteen isoforms of which were shown to be gender biased consistent with previous studies on sexual dimorphism or gender predominance. Fifteen CYP isoforms namely, 1A2, 2A6, 2B6, 2C8, 2C9, 2C17, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 4A11, 4F1 and 4F3 were positively identified to be present in normal human liver. CYPs were also identified from paired samples of human liver: 1A2, 2A6, 2B6, 2C8, 2C9, 2C17, 2C19, 2D6, 2E1, 3A4, 4A11, 4F2 and 4F8 and colon metastasis in the liver: 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, 4A11, 4F2 and 4F8. In normal colon and colon primary tumour only the CYP3A subfamily was identified. These results confirm the use of an MS based proteomic approach to the identification of multiple CYPs that obviates the need to predetermine which CYPs are present. They also suggest that the liver tissue environment is significantly increasing the expression profile of CYPs in colon tumours that metastasise to the liver.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D. |
| Title: | Nano-scale liquid chromatography electrospray tandem mass spectrometric identification of multiple cytochrome P450 isoforms in tissues. |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis Digitised by Proquest. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10122301 |
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