Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Chemistry

Major

Analytical Chemistry

First Advisor

Young-Jin Lee

Abstract

This dissertation presents work that aims to address limitations of and improve current small molecule analysis methods as well as to apply small molecule mass spectrometry (MS) methods to study biological systems. The first chapter consists of a general introduction to MS methods for metabolomics experiments, including chromatography-based methods as well as matrix-assisted laser desorption ionization (MALDI)-MS and mass spectrometry imaging (MSI). The sixth and final chapter summarizes the work presented in this dissertation and also discusses future directions.

The second chapter presents work that utilizes mass spectrometry methods to identify biomarkers in the FERONIA signaling pathway in Arabidopsis thaliana. Wild-type Arabidopsis leaves are compared to tDNA knockouts of FERONIA (fer mutant) leaves, and the Arabidopside family of metabolites is determined to be significantly enriched in the mutant. Related phytohormones are investigated and levels with and without wounding are compared to the changes in Arabidopside compounds after mechanical wounding. Finally the cellular-level localization of Arabidopsides within leaf tissue is determined using MALDI-MSI experiments.

The third chapter illustrates a data acquisition method which allows both MS and MS/MS information to be obtained in-parallel without sacrificing spatial resolution for MSI experiments. This study demonstrates that collecting multiple data sets at the same tissue position, which preserves spatial resolution, is possible, provided enough matrix is present. Utilizing this method allows MS spectra, informative MS/MS, and high-resolution spatial information through MSI to be collected in one instrument run on one tissue sample, thus increasing the information obtained.

The fourth chapter describes the study of six metals applied via sputter coating for their efficiency as MALDI matrices. The noble metal matrices, Au, Ag, and Pt, were highly effective matrices for a broad range of metabolites, and in general out-performed the traditional organic matrices DHB and DAN in positive and negative ion modes, respectively. Additionally as sputter coated metals have little to no background and can be homogenously applied, these are highly useful matrices for small molecule MSI experiments, and so we demonstrate this by applying sputter coated matrices to high spatial resolution imaging of maize root cross-sections.

The fifth chapter discusses the development and optimization of a microarray platform for high-throughput metabolomics experiments using MALDI-MS. Five matrices that provide broad metabolite coverage were selected and used to analyze turkey gut microbiome samples with and without antibiotic treatment. Gut extract samples from turkeys that had received no antibiotics, sub-therapeutic levels of antibiotics, or therapeutic levels of antibiotics across several developmental time-points were studied using this microarray, which allowed distinct metabolite profiles to be discovered.

Copyright Owner

Rebecca Lynn Hansen

Language

en

File Format

application/pdf

File Size

194 pages

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