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INVESTIGATION OF SELENOPROTEOME AND METALLOPROTEOME OF SEEDS OF BERTHOLLETIA EXCELSA AND METALLOPROTEOME OF SEEDS OF GLYCINE MAX BY LC AND MS TECHNIQUES

by JAYASINGHE, SARATH B

Abstract (Summary)
Research directed to identify compounds containing selenium and their chemical and biochemical pathways in the environment and in organisms has gained momentum in recent years, in part owing to the ability of selenium compounds to serve as cancer-preventive agents. Even though an extensive knowledge of small molecules containing selenium has been obtained and analytical methodologies to analyze small molecules containing selenium are well established, the wealth of knowledge regarding selenoproteins is limited except in a few selenoproteins such as glutathione peroxidase. Even in studies in which selenoproteins were analyzed in detail, the method of analysis has been based on the classical biochemical approach-one protein at a time. This dissertation describes the analysis of selenium containing proteins based on the proteomics approach. Bertholletia excelsa (Brazil nut) which has been identified to be rich in selenoproteins is used as the source of selenoproteins. In chapter one, chromatographic methods of analysis and three critical mass spectrometric techniques employed in selenoproteomics research, namely ICPMS, MALDI MS and ESI-MS are briefly described. Chapter two describes the factors affecting the selenium isotope pattern-the most important observation in identifying peptides containing selenium. The manner in which the isotope pattern changes with the molecular weight of the peptide, the number of selenium atoms in the peptide, the effect of post-translational modifications and the instrumental parameters such as the resolving power of the mass spectrometer and chromatographic separation conditions, is presented in detail. In chapter three, analysis of selenium-containing proteins in Brazil nuts is described, demonstrating the way the different experimental methodologies affect the outcome of the analysis, and highlighting the advantages, disadvantages as well as limitations of the analytical methodologies employed. Chapter four presents the usefulness of fractional precipitation by ammonium sulfate as a front-end sample fractionation and enrichment technique in metalloproteomics research. Metalloproteome of soybean seeds and metalloproteome of Brazil nuts are used as examples to prove the proposed methodology.
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Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

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ISBN:

Date of Publication:01/01/2007

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