Development of Proteomics Methods for Profiling Cytochrome P450s
Chris Sutton, Head of the ICT Proteomics and Mass Spectrometry Facility, Bradford University
Date Posted: Friday, October 16, 2009
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Launch presentation
About the speaker
Chris Sutton graduated with a PhD in Biochemistry from University of Liverpool, with combined studies in the University of California, Berkeley. He did a Postdoc. at the London School of Hygiene and Tropical Medicine and then joined the UK biotechnology company, Celltech as a Senior Scientist in Protein Biochemistry. Chris then went to ThermoFinnigan to develop mass spectrometers (MS) applications and later joined Kratos Analytical as Product Manager with responsibility for the marketing of MS instruments. In August 2005, He joined the Institute of Cancer Therapeutics in the University of Bradford as Senior Lecturer in Proteomics Chemistry and Mass Spectrometry.
Abstract
Cytochrome P450s are a superfamily of mixed-function oxidases, with 103 isoforms identified in mouse and 60 in humans. They are characterised into families and sub-families by their sequence similarities. Although most cytochrome P450s are found in the liver, some isoforms are over-expressed in the major clinically derived solid tumours. They are of great interest within pharmacology because they modify a diverse range of drugs, activating some whilst inactivating others before reaching their target. A strategy has been developed for the isolation and identification of cytochrome P450s using proteomics techniques.
Tissues are homogenised and subject to subcellular fractionation by ultra-centrifugation to isolate microsomes. Microsomal proteins are separated by SDS PAGE, bands excised and proteins digested with trypsin. Peptides are extracted, separated by nano-HPLC and fractions collected onto a MALDI mass spectrometry plate. MALDI TOF-TOF mass spectrometry is used to screen fractions in MS mode with subsequent analysis of identified peptides using MS/MS analysis and the resulting data searched against protein sequence databases for protein identification.
Cytochrome P450s were identified in liver, xenografts and cell lines using this approach. The potential of this strategy for characterising cancer biopsies and the wider pharmaceutical field will be discussed.
Launch presentation
Launch Presentation