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Title: The development and application of Proteomic Complex Detection by Sedimentation (ProCoDeS)
Author: Hartman, Nicholas Todd
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The aim of this project was to develop a novel technique capable of rapidly screening hundreds of proteins, without the use of genetic modification, and identify those that are in stable protein complexes. A range of different technologies was explored, leading to the development of the technique Proteomic Complex Detection by Sedimentation (ProCoDeS). ProCoDeS separates solubilised proteins by their native molecular weight in rate zonal gradient and then uses quantitative proteomics, in this study, isotope-coded tags, to measure the abundance of each protein in successive fractions of the gradient. Using this approach, up to 690 proteins were profiled in a single experiment. This included 30 proteins localized to the Golgi apparatus, and which may be involved in plant cell synthesis. The quality of the quantitative profile was confirmed by demonstrating that proteins known to be in the same complex exhibited very similar distribution profiles. Furthermore, Gaussian fitting was used to model the quantitative profiles and assign a peaking point for each protein in the centrifugation gradient. Through comparison with known complexes, the Gaussian fitting approach was able to model peaking points with an accuracy of much less than a single fraction even though data was collected only at alternate fractions. In conclusion, this project successfully developed and applied the ProCoDeS technique. The results in this thesis represent one of the largest datasets collected to date for the identification of proteins in complexes in Arabidopsis. Furthermore, ProCoDeS is a useful tool for the study of protein complexes in any organism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available