Date of Award
Doctor of Philosophy
Shawn R. Campagna
Christopher Baker, Michael Best, Elizabeth Fozo
Metabolomics measures all the small, biologically relevant molecules present within a system. An effective way to achieve high-throughput sample analysis and diverse pathway coverage in metabolomics is to use ultra performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS). Depending on the compounds of interest, untargeted or targeted methods can be implemented. Efforts have been focused on analyzing how different forms of stress alter an organism’s metabolite profile.Genetic mutations can inhibit essential metabolic pathways of an organism, but there are often redundant or alternative routes available for continued growth. By systematically deleting single genes in Escherichia coli responsible for the production of individual enzymes in the tricarboxylic acid cycle, it was possible to measure alterations in metabolites and correlate them to specific mutations. With the use of untargeted UPLC-HRMS, 146 known metabolites and over 1,800 significant unknown spectral features were identified. Comparative and multivariate analysis show intense changes were observed in strains lacking Aconitase B and unique global metabolic profiles were enzymatically linked and not strictly based on pathway disruption.Enterococcus faecalis has been shown to develop antibiotic tolerance to daptomycin by incorporation of host-produced fats. The exact biological mechanism by which this bacterium incorporates host lipids into their membranes is currently unknown. The use of a hybrid quadrupole Orbitrap mass spectrometer allowed for the development of a novel method to simultaneously quantify two-tailed phospholipids and four-tailed cardiolipins which pose analytical challenges due to low biological abundances and poor ionization efficiencies. By leveraging HILIC separation and parallel reaction monitoring (PRM), additional information with respect to identities about which specific fatty acids composition was simultaneously measured.A sub-population of Spodoptera frugiperda has been identified as resistant to genetically modified corn and has been causing rampant pest related crop loss. Previous research has shown a link between resistance and a mutation to the ATP binding cassette subfamily C2 gene. Untargeted metabolomic analyses were applied to profile the gut tissue of the larvae and found potential metabolites related to this mutation. From the 126 metabolites, six were found in significantly higher amounts in resistant insects when compared of susceptible insects.
Tague, Eric David, "Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry. " PhD diss., University of Tennessee, 2019.