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Browsing Department of Chemistry by Author "Allison, Christopher L., author"

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    Development of LC-MS and degradation techniques for the analysis of fungal-derived chitin
    (Colorado State University. Libraries, 2020) Allison, Christopher L., author; Reynolds, Melissa M., advisor; Farmer, Delphine, committee member; Bailey, Travis, committee member; Popat, Ketul, committee member
    The research contained within this dissertation began with the following question: Can liquid chromatography-mass spectrometry (LC-MS) be used as a screening method for fungal infections? The ensuing projects investigated various aspects of that question, taking a ground-up approach that started with the analysis of the simplest constituents of the biomarkers used, chitin and chitosan. The complexity of the systems investigated was gradually increased, culminating in the extraction and detection of these biomarkers from pertinent fungal cells. Chitin constitutes 10-30% of the mass of filamentous fungi. While not found endogenously, it is the second most abundant naturally-occurring polysaccharide, next to cellulose. Chitin is composed of >50% N-acetylglucosamine (GlcNAc) and D-glucosamine (GlcN). In nature and in numerous applications, chitin can be deacetylated to produce chitosan. Chitosan is the deacetylated (>50% GlcN) counterpart to chitin and is also found in some species of fungi. This dissertation began with the development of electrospray ionization mass spectrometry (ESI-MS) methods to analyze GlcN and GlcNAc, as well as oligomers composed of both residues. The optimization of methods to analyze the components of chitin served as the foundation on which to advance the applicability of these methods. Following method optimization, the ability of mass spectrometry to analyze polymeric chitosan was explored. Detecting polymeric chitosan was determined to be infeasible using ESI-MS; hence, the focus of subsequent studies was turned to the use of degradation studies to generate low molecular weight chemical fingerprints that could be correlated to the presence of chitin and chitosan. The first experiments performed to study the degradation of chitosan evaluated the impact nitrosating conditions have on the structure of chitosan. Both mass spectrometry and spectroscopic methods were used to track the formation of a degradation product, 2,5-anhydro-D-mannose (2,5-AM), to demonstrate that nitrous acid-based conditions induce degradation in polymeric chitosan. Following these experiments, degradation studies were expanded to include a wider range of starting materials. Chitosan polymer was used again, in addition to two chitin polymers with varied degrees of deacetylation. In addition to examining the effect of nitrosating conditions on these polymers, degradation methods were expanded to include hydrochloric acid (HCl), hydrogen peroxide (H2O2), and enzymatic degradation agents (lysozyme, lipase, and hemicellulase). The susceptibility of each polymer to degradation protocols was assessed by ESI-MS or LC-MS analysis of degradation products generated. Results from these studies indicated that HCl, H2O2, HNO2, and lysozyme generate distinct products from chitin and chitosan polymers. Identification of unique chemical fingerprints produced from chitin and its derivatives provided the necessary information to apply these studies to pertinent fungal cells. The final experiments in this dissertation apply cleanup, cell lysis, degradation methods, and LC-MS to identify GlcN produced from Aspergillus niger fungi. Cumulatively, the following research contains a thorough overview of degradation methods for chitin and its derivates, along with the characterization of low molecular weight fingerprints that each protocol generates. ESI-MS or LC-MS methods were used to identify low molecular weight products formed during degradation. Finally, both degradation and LC-MS methods were applied to Aspergillus niger to validate that representative fungal species can be detected using the proposed techniques.