Date of Award
Doctor of Philosophy
Gregory B. Hurst
Dale A. Pelletier, Robert L. Hettich, Kurt H. Lamour, Frank W. Larimer
The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function.
Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples.
Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively.
Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization.
Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.
Hervey, William Judson, "Targeted Proteomics for the Characterization of Enriched Microbial Protein Isolates and Protein Complexes. " PhD diss., University of Tennessee, 2009.