17th International Mass Spectrometry Conference :: Prague, 2006
> Go to contents (site navigation)
|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Cathrin Seibert1, Yuqin Wang1, Ahmed Al-Gazzar1, Brian R. Davidson2, Barry J. Fuller2, William J. Griffiths11 School of Pharmacy, University of London, London, United Kingdom
Correspondence address: Cathrin Seibert, School of Pharmacy, University of London, Pharmaceutical and Biological Chemistry, Brunswick Square,29/39, London, WC1N 1AX United Kingdom.
Keywords: Labeling, Isotope; MS/MS, Liquid Chromatography; Proteomic; Quantitative Analysis.
Novel aspect: Absolute quantification of cytochrome P450 2E1 in human liver tissue and liver tumour tissue.
Proteomics can be thought to be the characterisation of proteins found within a cell type, tissue or body fluid. This includes the subsequent determination of their structure, function, modifications and interactions. Today the technology is becoming popularised for the identification of novel markers of physiological and pathological processes and in the determination of disease mechanisms.
Of particular interest are metabolising enzymes which contribute to the metabolism of endogenous as well as xenobiotic substances. Cytochrome P450 proteins (CYPs) represent a class of highly expressed metabolising enzymes in human body. The knowledge of their presence and absence as well their abundance can provide information about the capability of a diseased tissue to metabolise an administered drug. These information could support the development of drug treatments e.g. for novel anti-tumour drugs.
The liver is known as a place of high expression of CYPs. We have chosen this organ to investigate the quantitative differences of CYP-abundance in healthy and cancerous tissue. Therefore a proteomic method was applied to human liver tissue as well as liver tumour sample.
The methodology incorporated one-dimensional polyacrylamide gel electrophoresis (PAGE) combined with nano-scale liquid chromatography – tandem mass spectrometry (LC-MS/MS) and database searching. Microsomal proteins, from human liver samples were reduced, alkylated and separated by SDS-PAGE. Following in-gel digestion of selected bands, the resulting peptides were extracted and subjected to LC-MS/MS analysis on a LCQ-Duo instrument. For identification the obtained data was used to search the detected peptides against a human database of proteins.
Identified proteins were then quantified using a stable isotope dilution mass spectrometry (AQUA, absolute quantification of proteins).1 A known amount of isotope labelled synthetic tryptic peptide was added to the gel band as an internal standard. The band was then treated with trypsin. The native and synthetic peptide show the same physical properties for analysis by LC-MS/MS, such as retention time in the chromatogram and ionisation cross-section. The ratio of the peaks for the native and synthetic peptide was used to determine the quantity of the native peptide.
Using this method it was possible to quantify CYPs in normal and tumour tissue and show differences in their expression levels. With the current study it is also expected to add quantitative information to the already achieved qualitative information.2
1. S. A. Gerber, J. Rush , O. Stemman, M. W. Kirschner , and S. P. Gygi, Proc. Natl. Acad. Sci. USA 100, 6940 (2003).
2. C. S. Lane, S. Nisar, W. J. Griffiths, B. R. Davidson, J. Hewes, K. J. Welham, and L. H. Patterson, Eur. J. Cancer 40, 2127 (2004).