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ABSTRACT KALMAR, JACLYN. Development of Innovative ABSTRACT KALMAR, JACLYN. Development of Innovative Strategies for the Analyses of Complex Biological Systems Using Mass Spectrometry. (Under the direction of Dr. David C. Muddiman). Mass spectrometry (MS) is a powerful analytical tool due to its versatility, specificity, and sensitivity. MS has allowed for the proliferation in collection of molecular level –omics data, which, in turn, has provided deep insights into various complex biological systems. This work discusses new strategies for the analysis of biological molecules thought be involved in the pathogenicity of Rice blast disease and Alzheimer’s disease. Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry imaging was used to identify meta-metabolomic features of Magnaporthe oryzae infected barley leaves. Three separate sets of barley were inoculated with Wild type (WT) M. oryzae, an F-box E3 ligase protein knock out (E3 ligase KO) M. oryzae, or a control solution. Over the course of the infection, each treatment was imaged using an advanced polarity switching method, allowing the detection of low and high molecular weight compounds that ionize in positive or negative polarities. Serotonin, a barley defense metabolite, was putatively identified using MS1 data then confirmed with tandem mass spectrometry fragmentation patterns. Metabolites in the melanin pathway, important for infection development of M. oryzae, were also identified using MS1 data but were unable to be confirmed due to their low abundances. Molecules related to the pathogenicity of the fungus were only found in the samples treated with the wild type M. oryzae where those treated with the genetically modified version displayed no metabolic changes related to a fungal infection. A label-free, quantitative proteomic analysis was performed on the mycelia from the same wild type and genetic knock versions of M. oryzae. The post-translational modifications (PTMs) thought to be involved with the knockout protein, phosphorylation and ubiquitination, were also investigated. A total of 4,432 proteins were identified in the WT and E3 ligase KO samples. Eighty-nine proteins were increased and 69 proteins were decreased in the E3 ligase KO strain. Sixty proteins were unique to the WT strain; 13 of which had both phosphorylation and ubiquitination PTMs. Seventy-one proteins were unique to the E3 ligase KO strain; 24 having both phosphorylation and ubiquitination PTMs. Several proteins were associated with key biological processes and greatly assisted in the selection of future genes for functional studies and enabling mechanistic insight related to virulence. The inclusion of systems suitability workflows to ensure optimal and reproducible data collection is critically important. We created mixed system suitability samples by evaluating the addition of either the 6 × 5 Promega Reference mix and the 7 × 5 Pierce™ System Suitability Standard or both with commercially available HeLa cell lysate. These system suitability mixtures provided the capability of monitoring identification (e.g. spectral matches, peptide identifications, etc.), identification free (e.g., mass measurement accuracy, and chromatography), and quantitative (LOD, LOQ, and dynamic range) metrics from a single injection. The analysis of N-linked glycans using MS presents significant challenges, owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to improve ionization and detection sensitivity. The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)TM strategy for labeling glycans, has been utilized in the analysis of N-linked glycans. The protocol using INLIGHT TM derivatization and subsequent analysis were investigated. Optimization of the modified method resulted in 20-100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase including the identification of low abundance glycans, (Fuc)1(Gal)2(GlcNAc)4(Man)3(NeuAc)1 and (Gal)3(GlcNAc)5(Man)3(NeuAc)3. The analysis of glycans has been hindered by the lack of software. GlycoHunter, a user-friendly software created in MATLAB enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pairs are generated for relative quantification, including but not limited to, INLIGHT™. © Copyright 2021 by Jaclyn Kalmar All Rights Reserved Development of Innovative Strategies for the Analyses of Complex Biological Systems Using Mass Spectrometry. by Jaclyn Kalmar A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemistry Raleigh, North Carolina 2021 APPROVED BY: ________________________ ________________________ Dr. David C. Muddiman Dr. Michael S. Bereman Committee Chair ________________________ ________________________ Dr. Erin S. Baker Dr. Edmond F. Bowden ________________________ Dr. Ralph A. Dean DEDICATION To my husband Ben, my love and best friend. Thank you for all of your encouragement. I could not have done this without you. ii BIOGRAPHY Jaclyn (Jackie) Gowen Kalmar was born in Harborcreek, Pennsylvania on the shores of Lake Erie. Jackie loved science and thought her mission in life was to become a surgeon. She began volunteering at a local hospital in high school through a student volunteer program and was placed in the operating room which turned out to be one of the most influential summers of her life. She observed a few procedures in the operating room and when a surgeon realized she was a student he gave her a full tour of the opened thoracic cavity. From that first surgical experience until halfway through college Jackie was riveted by the idea of becoming a surgeon. Her passion lead her to take a job as a patient transporter at the same hospital so she could interact with various clinical personnel and see the interworking’s of healthcare. But, as fate would have it, her fascination for chemistry was also growing and eventually won her heart and mind. During the rest of her college experience Jackie joined biophysical chemist, Dr. Mary Grace Galinato’s research team and worked on analyzing the kinetics of newly reconstituted nitrate reductase enzymes. She also had the opportunity to work with Dr. Bruce Wittmershaus, a physics professor, investigating new fluorescent materials for luminescent solar concentrators. Jackie also ventured into industry as well and completed an internship in the biodiesel industry testing product quality and the developing improvements to make to the processing line starting with their raw material. Jackie received a BS in Chemistry with a minor in Biology from Pennsylvania State University in 2016 and went on to pursue a Ph.D. in Analytical Chemistry at North Carolina State under the direction of Dr. David Muddiman. iii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... ix LIST OF FIGURES...................................................................................................... x LIST OF PUBLICATIONS ........................................................................................... xii Chapter 1: Introduction ............................................................................................. 1 1.1. Systems biology ............................................................................................. 1 1.2. Rice Blast Disease ......................................................................................... 1 1.3. Alzheimer’s Disease ...................................................................................... 3 1.4. Metabolomics ................................................................................................. 5 1.5. Proteomics ..................................................................................................... 6 1.6. Glycomics ...................................................................................................... 7 1.7. Mass Spectrometry ........................................................................................ 10 1.8. Separation and Ionization .............................................................................. 10 1.9. Electrospray Ionization ................................................................................... 11 1.10. Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) .......... 13 1.11. Fourier transform mass spectrometry ............................................................ 15 1.12. Research Synopsis ........................................................................................ 17 1.13. Literature Cited .............................................................................................. 20 Chapter 2: Investigating Host-Pathogen Meta-Metabolic Interactions of Magnaporthe oryzae Infected Barley Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry ....................................... 32 iv 2.1. Introduction ................................................................................................ 32 2.2. Materials and Methods .............................................................................. 34 2.2.1. Fungal Growth ............................................................................. 34 2.2.2. Barley and Inoculation ................................................................. 34 2.2.3. IR-MALDESI
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