Use Of Proteomics For The Analysis Of Hard-To-Dissect Biological Samples.

Abstract

The bulk of my research focused on the development and standardization of improved methods to carry out meaningful proteomic analyses of organisms with unannotated or incomplete genome sequences, and the analysis of proteins from hard-to-dissect fractions from these organisms, such as membrane proteins and spore proteins. Within this thesis, I intially provide an introduction to the field of proteomics and it’s methods, with a special attention to proteomic analysis of membrane proteins from microbial systems. I review some of the state-of-the-art techniques used to carry out quantitative proteomic analyses on membranes from microbes, with an emphasis on one of the most popular current methods of choice - iTRAQ. I also discuss options available to investigators wishing to carry out such analyses of microbial membrane proteins. The remaining sections of this thesis describe my work on the proteomic analysis of membrane fractions from the previously unannotated Gram negative bacterium C. crescentus and on the refractile fractions of spore protective structures of the Gram positive bacterium B. subtilis, as well as our preliminary investigations of the spore protective structures of its close relative, B. anthracis. The bioinformatic and computational techniques that were utilized to facilitate and augment these proteomic analyses are also discussed. Additionally, I describe some novel technologies that have wider applications in the field of proteomics beyond improved analysis of hard-to-dissect samples, such as the use of alkaline IPGs for the increased separation of basic proteins and the use of tandem mass spectrometry for more powerful protein identification.