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Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry

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Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2019 Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry Eric David Tague University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Tague, Eric David, "Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry. " PhD diss., University of Tennessee, 2019. https://trace.tennessee.edu/utk_graddiss/5348 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Eric David Tague entitled "Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Chemistry. Shawn R. Campagna, Major Professor We have read this dissertation and recommend its acceptance: Christopher Baker, Michael Best, Elizabeth Fozo Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Evaluating Metabolic Stress Response Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Eric David Tague May 2019 Copyright © 2019 by Eric David Tague All rights reserved. ii DEDICATION To all those in graduate school, just remember science is bigger than you. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Shawn R. Campagna for the opportunity to research in his lab and continue to grow as a chemist. Without his motivation and constant support, I would have never reached beyond what I thought was my limit. I also want to thank my committee members, Drs. Michael Best, Christopher Baker, and Elizabeth Fozo for giving up some of their valuable time to sit on my committee. I want to thank all my wonderful collaborators who have come into the lab with desires to answers biological questions that I did not even know existed. They have given me the opportunity to diversify my knowledge base and apply new mass spectrometry techniques. Specifically, I would like to thank Dr. Fozo and Dr. John Harp for introducing me to the world of microbiology and putting up my simple questions about how to pour plates and grow bacteria. Over the past several years, I have become great friends with all the lab members, and I could not have survived/thrived without everyone. Stephen Dearth, my graduate student mentor, showed me how to do my first metabolomics extraction and how to clean and calibrate an Orbitrap. Allen Bourdon made all those long hours in cold room much more bearable. Josh Powers was always around and willing to listen to my crazy rants. You know what they say about 3 guys in a room? Caleb Gibson was always there to motivate me to keep working on the CRP and ORP. Brittni Woodall was always willing to pick another set of lipids for me, so I could focus on writing. Brandon Kennedy for always ready to help me with some crazy synthesis scheme. Dr. Hector Castro was an invaluable asset providing me projects and allowing me to network with new collaborators as well as being a great friend. And finally, thanks to rest of the Campagna lab (former and present) for making the laboratory a great environment to learn. I would like to thank my parents, Vince and Kathy, for the constant support to keep doing my best and never give up. My Aunt Linda and Uncle Cliff for not letting me take a job at Gateway Packing and encouraging me to keep continuing my education. iv The completion of this degree would have never even begun without my amazing wife, Jennifer. Even through her own graduate school struggles, she was there to comfort me during the dark times and celebrate with me during the successful times. I cannot thank her enough for being that beacon of motivation and inspiration that I needed. v ABSTRACT 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. vi 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. vii TABLE OF CONTENTS INTRODUCTION .................................................................................................. 1 Metabolomics .................................................................................................... 2 Analytical Technique for Metabolite Analysis .................................................... 3 Types of Metabolomics Analyses ...................................................................... 5 Data Analysis for Metabolomics ........................................................................ 6 The Future of Metabolomics .............................................................................. 7 Outline of Dissertation ....................................................................................... 8 Chapter 1 GENETIC MUTATIONS TO THE TRICARBOXYLIC ACID CYCLE INFLUENCE METABOLOMIC AND LIPIDOMIC PROFILES ............................ 10 Preface ............................................................................................................ 11 1.1 Abstract ..................................................................................................... 14 1.2 Introduction ................................................................................................ 15 1.3 Experimental ............................................................................................. 17 1.3.1 Strain generation ................................................................................ 17 1.3.2 Bacterial growth conditions ................................................................. 17 1.3.3 Microscopy .......................................................................................... 19 1.3.4 Metabolomics ...................................................................................... 19 1.3.5 Lipidomics ........................................................................................... 21 1.3.6 Flux analysis ....................................................................................... 22 1.3.7 Data processing .................................................................................. 23 1.3.8 Unidentified spectral features ............................................................. 24 1.4 Results and Discussion ............................................................................. 24 1.4.1 Growth changes .................................................................................. 24 1.4.2 Global
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