Type of Document Master's Thesis Author Vaghela, Nileshwari URN etd-09222007-013125 Title Proteomic Analysis of the Flavonoid Biosynthetic Machinery in Arabidopsis Thaliana Degree Master of Science Department Biology Advisory Committee
Advisor Name Title Lazar, Iuliana M. Committee Co-Chair Winkel, Brenda S. J. Committee Co-Chair Helm, Richard Frederick Committee Member Walker, Richard A. Committee Member Keywords
- Protein-protein interactions
- Enzyme complexes
- Affinity chromatography
- Flavonoid pathway
- Mass spectrometry
Date of Defense 2007-08-28 Availability unrestricted AbstractWork on a wide variety of metabolic pathways indicates that these systems are often, if not always, organized as multienzyme complexes. Enzyme complexes have the potential to increase catalytic efficiency and provide unique mechanisms for the regulation of cellular metabolism. The flavonoid biosynthetic pathway of Arabidopsis is an excellent model for studying the organization, localization, and regulation of enzyme complexes at the cellular level. Flavonoids are specialized metabolites that perform many important physiological roles in plants. Protein interactions among several key flavonoid enzymes have been described. Moreover, at least two of the flavonoid enzymes have a dual cytoplasmic/nuclear localization. These results indicate that flavonoid enzymes assemble into one or more distinct complexes at different intracellular locations.
The current study integrates a new technology, mass spectrometry, with well-established affinity chromatography methods to further characterize the organization and composition of the Arabidopsis flavonoid enzyme complex. One of the key flavonoid enzymes, chalcone isomerase (CHI), was used in these experiments to detect interacting enzymes. Recombinant thioredoxin (TRX), or TRX-CHI, was produced in E. coli, then purified by metal affinity chromatography, and covalently coupled to an activated resin, Affi-Gel 10. Extracts prepared from 4-day-old wild type or CHI-deficient lines of Arabidopsis were then passed over the column and the bound proteins eluted with sodium dedocyl sulfate (SDS). This eluate was then subjected to a liquid chromatography (LC) - mass spectrometry (MS) protocol developed for the analysis of complex peptide mixtures. An Agilent LC system coupled with an LTQ-MS instrument (Thermo Electron, San Jose, CA) was used for this purpose. Data analysis was performed with the Bioworks software package. Different optimization strategies were performed to improve the affinity chromatography, sample preparation, and the LC separation method. A novel approach has been developed for the MS analysis of biological samples containing contaminants such as salts and detergents.
Protein extracts prepared from wild type Landsburg and mutant tt5(86) were analyzed by LC-MS/MS. A total of 491 proteins were identified for Landsburg and 633 for tt5(86) extracts using a combination of data filters and p-value sorting. All detected proteins had p<0.001 and most were identified by at least 2 unique peptides.
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