Doctor of Philosophy (PhD), Ohio University, 2024, Chemistry and Biochemistry (Arts and Sciences)

We utilized electrospray ionization, collision-induced tandem mass spectrometry, and computational chemistry to investigate the gas phase ion structures and dissociation pathways of model deprotonated petroleum acid anions [M-H]-. The combination of experimental and theoretical approaches provides a mechanistic understanding of the structure-specific dissociation chemistry of targeted petroleum acids. We experimentally and theoretically studied a series of positional isomers and related compounds with variations in aromaticity, size, alkylation, and heteroatoms. Petroleum acids cause significant corrosion during petroleum refining and have been found in weathered petroleum environmental samples as intermediate anaerobic biodegradation products of naphthalene. The differences in structure, size, alkylation, and aromaticity affect the subsequent reactivity of these naphthenic acid species. Understanding neutral losses and fragments from the structural elucidation of various isomers is useful in understanding the availability of transformation products of naphthalene biodegradation, thus enabling the deduction of the extent of biodegradation. Our experiments revealed that the aryl scaffold in each aromatic acid is unsurprisingly very stable, with little or no fragmentation detected. Decarboxylation (CO2 loss) reactions were detected and modeled in all acids. The position of the carboxylate substituent on the aromatic or cyclo-aliphatic ring significantly influenced the ease of4 decarboxylation. Alkyl-substituted acids are far more labile and thus readily cleaved with relative ease, depending on the electron density distribution along the alkyl chain length. We observed that smaller and larger aromatic acids can be differentiated based on their reaction with water. Isomers with cyclo-aliphatic ring systems dissociate through high energy, unique ring-opening reactions such as methane and formaldehyde neutral loss. Conversely, isomers containin (open full item for complete abstract)

Committee: Jixin Chen (Advisor); Benjamin Bythell (Advisor); Peter Harrington (Committee Member); John Schenk (Committee Member) Subjects: Analytical Chemistry; Chemistry

Doctor of Philosophy, University of Akron, 2023, Chemistry

This dissertation discusses the use of various mass spectrometry (MS) techniques to elucidate relevant polymer properties such as chain sequence, primary architecture, degradation mechanisms, and surface segregation. Many of these properties cannot be fully characterized using other analytical techniques such as size exclusion chromatography (SEC), infrared spectroscopy (IR), or nuclear magnetic resonance (NMR), further signifying the importance of MS methods. The studies described in this dissertation focus on application of various ionization sources, mass analyzers, sample preparation techniques, and data processing software to investigate a variety of polymer systems. Degraded poly(ethylene glycol) (PEG) was analyzed using MS and tandem MS (MS/MS) techniques. These methods provided insight into the degradation mechanisms and five main products formed when PEG is exposed to oxidative conditions. Overall, this study provided new information on a polymer frequently used in important biological applications, but whose degradation process had long been debated. Similar methods were used to analyze a newly developed degradable thermoplastic, synthesized from the reaction of butadiene and carbon dioxide (CO2). Many copolymer products can be formed through the reaction of these two starting materials depending on the type of catalyst used. The MS/MS methods used in this work proved the successful production of degradable polymers resulting from simultaneous 1,4 conjugate addition and ring opening polymerization. Collectively, these two studies exhibit the unique capabilities of mass spectrometry for determining vital chain sequence information of polymeric materials. A parallel study applied similar MS/MS techniques to look not only at chain connectivity but also the chain architecture of a branched comb polymer. Thiol-yne comb polymers containing a primary dithiol backbone and PEG side chains were investigated by MS/MS. These experiments provided insight into th (open full item for complete abstract)

Committee: Chrys Wesdemiotis (Advisor); Claire Tessier (Committee Member); James Eagan (Committee Member); Aliaksei Boika (Committee Member); Chunming Liu (Committee Member) Subjects: Analytical Chemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2022, Chemistry

As Analytical Chemists we are constantly demanding more of our methods: greater selectivity, lower detection limits, wider utility, reduced complexity, etc. We are frequently tasked with meeting the ever-changing demands of the many evolving fields that require analytical expertise including the medical, environmental, forensic, and consumer industries. In many cases, new techniques are introduced, or old methods are refined and matured to tackle these challenges. However, there are still times when the development of new and improved instrumentation lags behind demand, and traditional chemical principles must be used to bolster current methods. Chemical derivatization is a well-established approach to overcoming the limitations of available instrumentation and methods. It is commonly used to expand detectability into adjacent areas in the chemical space, to drive signal enhancement for analytes that exhibit low sensitivity, and to improve selectivity in separations. However, conventional protocols can be resource intensive, complex, and time-consuming. The overarching aim of this dissertation is to leverage the virtues of derivatization while mitigating its shortcomings by shifting the procedure from the benchtop to the analytical platform. We do this using a specialized electrospray platform that allows us to carry out chemical reactions rapidly and online during the analysis. Although this approach can be useful for a wide range of compounds, we focus on saccharides due to their broad physiological importance. Recent potential applications in disease diagnosis and monitoring have brought about a need for new analytical tools capable of detecting saccharides at low concentrations and/or for volume-limited samples. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) is the preferred methodology for these compounds due its inherent specificity. However, saccharides generally exhibit limited sensitivity in LC-ESI-MS because they are not (open full item for complete abstract)

Committee: Abraham Badu-Tawiah (Advisor) Subjects: Analytical Chemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2020, Chemistry

The elucidation of protein structure and the interactions of proteins with each other, with ligands, and with other (bio)molecules is a critical step in the understanding of their biological function. Native mass spectrometry (nMS) has emerged as an advantageous technique to study critical attributes of biomolecular complexes. Surface-induced dissociation (SID) is a tandem MS activation method that provides valuable information on biomolecular complex connectivity, relative location of ligand binding interfaces, and relative strength of interaction interfaces between subunits. This dissertation focuses on the implementation and application of SID in high-resolution Orbitrap mass spectrometers and the development of non-denaturing separation techniques coupled to native mass spectrometry. SID has previously been incorporated into time-of-flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometers; however, implementing SID in Orbitrap instruments provides a combination of resolution, speed, and sensitivity that increases the range of samples amenable to nMS-SID analysis. The first part of this work describes the design, fabrication, and implementation of SID into high mass range Orbitrap mass spectrometers. The performance of the modified instruments was benchmarked by analyzing well-studied protein complexes and comparing the results to those previously obtained on well-established SID instrument platforms. Next, the high-resolution capabilities of the SID-Orbitrap instrument were used to probe the relative location of ligand binding within pentameric protein complexes. It was shown that ligands are largely retained on subcomplexes generated by SID and that the stoichiometry of ligand to subcomplex can be used to differentiate between protein complexes that bind ligands at the interface of subunits, and complexes that bind ligands within individual subunits. Transferring samples into MS-compatible solution conditions is time consuming an (open full item for complete abstract)

Committee: Vicki Wysocki (Advisor) Subjects: Analytical Chemistry; Biochemistry; Chemistry

Doctor of Philosophy, University of Akron, 2019, Chemistry

This dissertation focuses on the characterization of polymer architectures and sequences by tandem and multi-stage mass spectrometry (MS2 and MSn) as well as ion mobility mass spectrometry (IM-MS). The experiments described in this dissertation utilized matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) as ionization techniques and collisionally activated dissociation (CAD), electron transfer dissociation (ETD), and laser-induced fragmentation (LIFT) as ion activation and dissociation techniques. Polymers studied include an 8-shaped polystyrene, star-branched polyethers, monodisperse sequence-defined POSS-functionalized polyesters, and poly(t-butyl methacrylate). A well-defined 8‑shaped polystyrene was characterized by MALDI-MS, MALDI-MS2, and IM-MS. 8-shaped polymers could have inter and intra isomers which cannot be distinguished by single stage mass spectrometry. MALDI-MS2 of 8‑shaped polystyrene showed specific fragment species that can only be produced by the inter isomer, thus confirming the existence of the inter isomer. The degree of branching and the length of individual branches of a set of mikto- and homo-arm star polyethers were studied by MALDI-MS2 and ESI-MS2. The number of fragment distributions combined with the mass to charge ratios of the fragment ions gave conclusive evidence for the number of branches in the star polyethers and the corresponding branch lengths. A set of monodisperse, sequence-defined, and POSS-functionalized polyester copolymers were analyzed by MALDI-MS2. Isomeric oligomers with different sequences were readily distinguished by their different fragmentation patterns. Oligomers up to hexamers were successfully sequenced based on the specific backbone fragment species observed. The backbone connectivity of poly(t-butyl methacrylate) (PtBMA) was investigated by tandem and multistage mass spectrometry. The results showed that ETD-MS2 and ETD-CAD-MS3 enable backbone C-C bond cleavages and, thus, (open full item for complete abstract)

Committee: Chrys Wesdemiotis (Advisor); Wiley Youngs (Committee Member); Adam Smith (Committee Member); Aliaksei Boika (Committee Member); Toshikazu Miyoshi (Committee Member) Subjects: Analytical Chemistry; Chemistry; Polymers

Doctor of Philosophy, University of Akron, 2018, Chemistry

Mass spectrometry (MS) has been a powerful technique for the characterization of synthetic polymers especially after the introduction of soft ionization methods such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). One-dimensional MS analysis provides molecular weight information by detection of the mass-to-charge (m/z) ratio of gas-phase analyte ions. However, tandem MS (MS/MS) capabilities, which involve separation and subsequent fragmentation of a selected analyte ion, are needed to obtain more detailed structural information. MS/MS can provide essential information on the backbone connectivity for synthetic polymers. For the sequence analysis of polymers having hydrocarbon backbones, multistage fragmentation (MSn) that combines different ion activation methods may be necessary. In addition to MS or MS/MS analysis, additional dimensions of separation can be achieved by online hyphenation of various dispersion techniques which allows for the investigation of more complex materials and mixtures. While online combination of liquid chromatography (LC) with MS has been the most common hyphenated technique, ion mobility (IM) spectrometry has been emerging as a much more rapid alternative method for the separation of gas-phase analyte ions based on their charge state, size, and architectural differences. These two separation techniques can be combined in a high-throughput 3D LC-IM-MS method or utilized separately depending on the sample complexity. In this dissertation, applications of MSn and hyphenated MS techniques are illustrated for the analysis of synthetic polymers and complex synthetic polymer-protein conjugates. In Chapter IV, various MSn approaches were investigated for the in-depth structural characterization of poly(N-isopropylacrylamide)s (pNIPAM). Different ion activation methods including collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) provided structural information on end gr (open full item for complete abstract)

Committee: Chrys Wesdemiotis (Advisor); Wiley J. Youngs (Committee Member); Adam W. Smith (Committee Member); Leah Shriver (Committee Member); Nita Sahai (Committee Member) Subjects: Analytical Chemistry; Chemistry; Polymer Chemistry

PhD, University of Cincinnati, 2015, Arts and Sciences: Chemistry

This dissertation is focused on developing and implementing liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches for the high-throughput characterization of small RNAs in complex biological matrices. Non-coding ribonucleic acids (ncRNA) have become increasingly recognized as important to a number of biological functions, such as gene expression at both the transcriptional and translational levels. Among the many classes of ncRNAs, transfer ribonucleic acids (tRNA) are noted for their significant role in the center of the central dogma of molecular biology, translating the genetic code into functional proteins. Naturally occurring chemical modifications are applied post-transcriptionally to these polymers giving them added structure and function. Despite biological relevance, the complex nature of mixtures containing a pool of total tRNA typically requires lengthy, impractical sample preparations steps. The “digest and go” methods proposed in this dissertation should be capable of helping advance the field of RNomics, much like the lasting impact LC-MS based strategies have had on the rapidly advancing field of proteomics; lending themselves to applications from basic molecular biology studies to clinical applications previously thought impractical. These advances were accomplished by implementing targeted MS/MS based assays, which take advantage of high-through-put data collection, taking one third the time of previous LC-MS based approaches and capable of identifying more individual tRNA isoacceptors, in conjunction with automated data analysis.

Committee: Patrick Limbach Ph.D. (Committee Chair); Edward Merino Ph.D. (Committee Member); Thomas Ridgway Ph.D. (Committee Member); Apryll Stalcup Ph.D. (Committee Member) Subjects: Analytical Chemistry

Doctor of Philosophy, University of Akron, 2013, Chemistry

This dissertation focuses on coupling mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to thermal degradation, liquid chromatography (LC) and/or ion mobility (IM) spectrometry for the characterization of complex mixtures. In chapter II, an introduction of the history and the principles of MS and LC are discussed. Chapter III illustrates the materials and instrumentation used to complete this dissertation. Polyethers have been characterized utilizing MS/MS, as presented in Chapter IV and Chapter VI. Diblock copolymers of polyethylene oxide and polycaprolactone, PEO-b-PCL, have been characterized by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry (MALDI-Q/ToF) and LC-MS/MS (Chapter V). Thermoplastic elastomers have been characterized by thermal degradation using an atmospheric solids analysis probe (ASAP) and ion mobility mass spectrometry (IM-MS), as discussed in Chapter VII. Interfacing separation techniques with mass spectrometry permitted the detection of species present with low concentration in complex materials and improved the sensitivity of MS. In chapter IV, the fragmentation mechanisms in MS/MS experiments of cyclic and linear poly(ethylene oxide) macroinitiators are discussed. This study aimed at determining the influence of end groups on the fragmentation pathways. In the study reported in Chapter V, ultra high performance liquid chromatography (UHPLC) was interfaced with MS and MS/MS to achieve the separation and in-depth characterization and separation of amphiphilic diblock copolymers (PEO-b-PCL) in which the architecture of the PEO block is linear or cyclic. Applying UPLC-MS and UPLC-MS/MS provides fast accurate information about the number and type of the blocks in the copolymers. Chapter VI reports MS/MS and IM-MS analyses which were performed to elucidate the influence of molecular size and collision energy on the fragmentation pathways of polyethers subjected to collisionally activated dissocia (open full item for complete abstract)

Committee: Chrys Wesdemiotis Dr. (Advisor); Michael J. Taschner Dr. (Committee Member); Peter L. Rinaldi Dr. (Committee Member); Matthew Espe Dr. (Committee Member); Yu Zhu Dr. (Committee Member) Subjects: Analytical Chemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2013, Chemistry

There is a growing interest in application of mass spectrometry as a high throughput technique for quaternary structure studies of protein complexes. One way to study protein complexes by mass spectrometry is to specifically label peptides segments that carry critical structural information, and after protein digestion subsequently identify the labeled peptides using liquid chromatography - mass spectrometry. A chemical crosslinker forms covalent bonds at specific amino acid sidechains that are in proximity in the protein structure. This approach is used to probe the binding interface of LexA/RecA proteins in Escherichia coli (Chapter 3). In contrast, intact noncovalent protein complexes can be directly transferred into the gas phase, while retaining memory of their solution structures. Accurate molecular weight measurement by mass spectrometry can be used for stoichiometry determination of protein-protein and protein-ligand systems, as manifested by the two examples of stoichiometry determination of differently treated adiponectin oligomers (Chapter 4), and the silver binding properties of the N-terminal region of a bacterial protein CusB (Chapter 5). Finally, tandem mass spectrometry (MS/MS) can be used to dissociate protein complexes into smaller subcomplexes for more structural information. It is necessary to develop alternative MS/MS methods for large protein complexes, because the commonly used and widely available method, collision induced dissociation (CID), often does not provide sufficient and informative dissociation for quaternary structure studies of large protein complexes. The dissertation is mainly focused on the modification of a commercial ion mobility (IM) mass spectrometer with the addition of a custom surface induced dissociation (SID) device as an alternative MS/MS method for characterization of large protein complexes (Chapter 6). This work is a continuation of a previous project from the research group, in which SID has produced dissociati (open full item for complete abstract)

Committee: Vicki Wysocki (Advisor); Mark Foster (Committee Member); Terry Gustafson (Committee Member) Subjects: Analytical Chemistry; Biophysics

Doctor of Philosophy, The Ohio State University, 2003, Chemistry

The posttranslational modifications of core histones, such as the acetylation and methylation on lysine residues, play a critical role in a variety of gene activities such as transcription regulation, Deoxyribose Nucleic Acid (DNA) replication and damage repair, gene silencing and the regulation of cell developmental processes such as proliferation and differentiation. These modifications, as well as the responsible enzymes, can be related to the occurrence and development of many diseases such as leukemia, lupus and Huntington's disease. A thorough investigation of these post-translational modification sites will shed light on the mechanism and function of histone modifications in these disorders. Traditional methods for the investigation of histone post-translational modifications are primarily immunoassay techniques. These techniques are very sensitive, but they rely greatly on the availability of the site specific antibodies. The lack of a comprehensive set of antibodies has limited the studies of the novel modification sites. An additional complication with immunoassays arises when multiple modifications occur on the same histone. The simultaneous presence of other modifications may impact the specificity of antibodies directed at a specific individual modification. In this study, mass spectrometry and its related techniques were applied for the investigation of histone post-translational modification. A model was first established to demonstrate that mass spectrometry and peptide mapping can be successfully applied to study the detailed localization of acetylation at specific lysine residues. Core histones were extracted from bovine thymus and identified by LC/MS. After the identification, these proteins were separated and digested using different enzymes. Direct peptide mapping was applied for these proteolytic digestion mixtures on a 7T FT ICR MS. The results showed more than 20 novel modification sites in addition to previously reported modification sites (open full item for complete abstract)

Committee: Michael Freitas (Advisor) Subjects: Chemistry, Analytical

Doctor of Philosophy, University of Akron, 2009, Chemistry

The utilization of MS and MS/MS techniques have resulted in the complete characterization of chain-end, in-chain and cyclic polystyrene and polybutadiene as well as an analytical method which allows the rapid determination of the location of functionality as well as determining if the material is linear or cyclic.The characterization of in-chain polystyrene is described and compared to chain-end polymers comprised with similar functionality. This allowed the differentiation of fragment ions resulting from the CAD fragmentation between the two types of functionalization. Additionally, the MS/MS spectra of in-chain functional polymers allow the determination of average chain length on either side of the functional group. Further expansion of polystyrene understanding was accomplished by characterizing macrocycle polystyrene which also contained a functional group. The CAD spectrum provided conclusive proof that the material was in fact cyclic due to the observed monomer losses as a result of CAD induced ring opening. After ring opening the macrocycle behaves similarly to an in-chain functional polymer and produces a mid- mass range Poisson distribution corresponding to the chain length on each side of the functional group, however, in this case it does not directly correlate to average chain length. This cyclic fragmentation pattern was confirmed when cyclic non-functionalized polybutadiene was characterized. Here the mid-range Poisson distribution was absent due to no functionality being present. However, the same monomer loss was observed which further confirmed that the monomer loss was indeed a function of the ring opening rather than a spectral feature induced by the ToF/ToF mechanism being akin to PSD. This was further confirmed by using a Q/ToF to verify the monomer loss after ring opening. Characterization of polybutadiene without pyrolysis was also conducted. While the adherence to free radical degradation was maintained it was discovered that unlike poly (open full item for complete abstract)

Committee: Chrys Wesdemiotis PhD (Advisor) Subjects: Analytical Chemistry

Doctor of Philosophy, University of Akron, 2013, Chemistry

This dissertation focuses on utilizing mass spectrometry (MS) and tandem mass spectrometry (MS/MS), combined with the coupling of liquid chromatography (LC) and/or ion mobility (IM) spectrometry, to characterize polymeric materials. Chapter II will introduce history and basic principles of mass spectrometry and liquid chromatography including background on instrumentation and theory. Chapter III will discuss the materials and instrumentation used in the various projects. Chapters IV-VII are research project chapters as described below. Lastly, Chapter VIII concludes this dissertation with a summary. Following the last chapter are appendices with supplemental data and copyright permissions for this dissertation. Chapter IV details the characterization of two nonionic surfactants consisting of a methoxylated glucose core (glucam) that was either chain-extended with ethylene oxide and then esterified with stearic acid to generate poly(ethylene oxide) glucam stearate(s) or only esterified with stearic acid which made glucam stearate(s). The use of LC-MS, LC-MS/MS, IM-MS, and MS/MS made it possible to separate and determine the compositions for each component in the mixtures resulting from the synthesis of these amphiphilic polymers. Components of (PEO)n-glucam stearate(s) were readily separated by their hydrophobicity and degrees of esterification by LC-MS and IM-MS. Each method offers complementary information, with LC-MS excelling in the separation and characterization of highly hydrophobic amphiphilles and IM-MS provides a quicker analysis of minor trace components. Electron transfer dissociation (ETD) was also applied as a novel MS/MS application to poly(ethylene oxide) glucam stearate(s). However, when compared to traditional MS/MS activation techniques (collisionally activated dissociation (CAD)), ETD unveiled no significant structural information when compared to CAD activation of the same oligomer. Specifically, ETD caused no unique cleavages acro (open full item for complete abstract)

Committee: Chrys Wesdemiotis Dr. (Advisor); Claire Tessier Dr. (Committee Member); David Perry Dr. (Committee Member); Leah Shriver Dr. (Committee Member); Kevin Cavicchi Dr. (Committee Member) Subjects: Analytical Chemistry; Chemistry; Polymer Chemistry; Polymers

Master of Science, The Ohio State University, 2010, Chemistry

Mass spectrometry-based protein quantification approaches are powerful tools for biomarker discovery. In this thesis, a spectral counting-based label-free analysis platform and tandem mass tag isobaric labeling quantification platform are described and evaluated for detecting differential protein abundances of an in vivo system in response to UV radiation. Sample preparation and separation of global protein digests were optimized for both label-free and labeling methods. Statistical evaluations such as principal component analysis and Venn diagram were used to evaluate the reproducibility of the analytical system. For spectral counting analysis, a few different normalization and modification methods such as ratioing of normalized spectral counting, normalized spectral abundance factors, and spectral index were applied and the results were compared. Correlations of the results from spectral counting and tandem mass tags labeling were also carried out. Potential protein biomarkers related to DNA damage repair in the in vivo system are proposed based on both labeling and label-free approaches.

Committee: Michael Freitas (Advisor); Susan Olesik (Advisor) Subjects: Chemistry

Doctor of Philosophy, University of Akron, 2008, Chemistry

Matrix Assisted Laser Desorption Ionization (MALDI) time of flight (ToF) mass spectrometry is a technique that has been used widely to characterize synthetic polymers. Mass spectrometric characterization is an essential tool in the determination of the comonomer composition and functionality distribution of synthetic polymers. Tandem mass spectrometry (MS/MS) is a two-dimensional technique that characterizes synthetic polymers in more detail. In this dissertation, poly (methyl methacrylate) (PMMA) and poly (methyl acrylate) (PMA) were examined by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Particular emphasis was devoted to the elucidation of MS/MS fragmentation mechanisms. Such knowledge is a prerequisite for interpreting MS/MS spectra correctly, so that structural information can be deduced from them with confidence. Within the MS/MS studies, a nomenclature was developed to classify MS/MS fragments in a meaningful manner. Briefly, terminal fragments are labeled by one of the letter codes given in the chart, while internal fragments are labeled by J, K, L, etc. A radical site is indicated by • and the number of repeat units by the subscript. Both, PMMA and PMA chain ionized by alkali metal addition dissociate in the mass spectrometer by homolytic cleavages along the backbone which produce the incipient radical ions an•, bn•, yn• and zn•. These radical ions undergo consecutive, radical-induced reactions which lead to the ultimately observed MS/MS fragments. Radical ions from PMMA as well as PMA dissociate by β-scissions; those from PMA also dissociate by backbiting reactions which produce abundant internal fragments. Hyperbranched acrylate polymers were analyzed by MALDI mass spectrometry and MS/MS. The MALDI-MS data reveal the composition of the polymer, while the MALDI-MS/MS data provide information about the branching architecture. Upon MS/MS, linear as well as hyperbranched polymers dissociate through 1,5-hydrogen rearrangement over the ester (open full item for complete abstract)

Committee: Chrys Wesdemiotis (Advisor) Subjects: Chemistry

Doctor of Philosophy (PhD), Ohio University, 2024, Chemistry and Biochemistry (Arts and Sciences)

In this dissertation, the gas-phase fragmentation chemistries of deprotonated carbohydrates were characterized. Tandem mass spectrometry and density functional theory (DFT) reaction pathway calculations were applied to rationalize the gas-phase fragmentation mechanisms. The fragmentation behavior of singly charged deprotonated glucose-α-glucose positional isomers: b,b-trehalose and isomaltose was investigated. The gas-phase chemistry of two other glucose-α-glucose positional isomers was studied: kojibiose and nigerose. The fragmentation mechanisms of singly deprotonated glucose- 1,4-glucose stereochemistry isomers α/β for maltose and cellobiose, respectively were examined. Finally, the fragmentation chemistry of acidic glycan galacturonic acid is studied, both singly and doubly deprotonated precursor ions were individually fragmented, and theoretical modeling was used to compare the charge-state effect on the fragmentation behavior of digalacturonic acid.

Committee: Benjamin Bythell Mr (Advisor); Howard Dewald Mr (Committee Member) Subjects: Chemistry

Bachelor of Science (BS), Ohio University, 2023, Chemistry

We investigate deprotonated and regiospecifically labeled glucosamine phosphate derivatives using tandem mass spectrometry. The spectral results were supplemented with computational modeling of each analyte's key gas-phase dissociation reactions to calculate specific reaction energetics. The dissociation chemistries underpinning dihydrogen phosphate anion (H2PO4-, m/z 97) loss, phosphenate anion loss (PO3-, m/z 79), and formation of the structurally informative cross-ring anion (0,2A1, C4H8PO7-, m/z 199 and 0,4A1, C2H4PO5-, m/z 139) products were examined. Deprotonated α-1-phosphate glucosamine anion analytes fragment primarily through the loss of phosphenate anions and dihydrogen phosphate. In contrast, deprotonated 6-phosphate glucosamine anion analytes exhibit multiple active fragmentation patterns. The phosphate group at the 6-position enables cross-ring cleavage to occur, producing 0,2A1 and to a lesser extent 0,4A1 ion products. Furthermore, analytes with a 6-phosphate also exhibited dissociation via ejection of H2PO4- and PO3-.

Committee: Lauren McMills (Other); Benjamin Bythell (Advisor) Subjects: Chemistry

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2020, College of Sciences and Health Professions

Nonalcoholic fatty liver disease (NAFLD) is a progressive liver condition of increasing significance worldwide. NAFLD ranges from simple steatosis to aggressive nonalcoholic steatohepatitis (NASH), which can evolve to progressive fibrosis and in the long-term to cirrhosis. Although the mechanism of steatosis transition to NASH the clinical demarcation boundary between the reversible and irreversible progression of the disease is not fully elucidated, early detection of this transition is crucial for intervention before fibrosis appearance. This study aims to define the circulating profile across the entire spectrum of NAFLD in a biopsy-proven NAFLD cohort using targeted metabolomics analysis with robust stable isotope dilution liquid chromatography-tandem mass spectrometry and to find potential non-invasive biomarkers related to disease progression. Metabolomics analysis revealed significant changes in plasma bile acids, aromatic amino acids related metabolites, branched amino acids, and carnitine metabolites. Plasma bile acid alterations are associated with the presence and severity of fibrosis more than NASH. Subjects with fibrosis had higher plasma levels of 7-Keto-DCA, GCDCA, GCA, and GUDCA, while patients with NASH had higher levels of 7-Keto-DCA and 7-Keto-LCA. Plasma levels of 7-Keto-DCA and 7-Keto-LCA bile acids are associated with increases in NAFLD Activity Score and were significantly higher in NASH compared to steatosis. Higher plasma levels of aromatic amino acids-derived metabolite (4-OH-phenyllactic acid, 2-OH-benzoic acid, phenylacetic acid) are associated with a higher NASH risk. Plasma levels of 4-OH-phenyllactic acid and phenylacetic acid showed a stepwise increase through steatosis transition to NASH. Higher plasma levels of hippuric acid aromatic amino acids-derived metabolite and branched amino acids track with a lower risk of fibrosis in NAFLD. Branched amino acid plasma levels exhibited a gradual decrease with steatosis transition to ballooni (open full item for complete abstract)

Committee: Valentin Gogonea PhD (Advisor); Stanley Hazen MD, PhD (Advisor); David J Anderson PhD (Committee Member); Jonathan Mark Brown PhD (Committee Member); Petru S Fodor PhD (Committee Member) Subjects: Analytical Chemistry

Doctor of Philosophy, University of Akron, 2019, Polymer Science

This dissertation describes the development of novel mass spectrometry (MS) methodologies and techniques to improve, simplify and allow for specific characterizations of synthetic materials. Successful MS analyses are governed by the mass spectrometry principles surrounding specific ionization sources, mass analyzers and ion detecters. The methods described within this dissertation provide information about polymer materials that is historically difficult to determine using other available analytical techniques. Self-assembled terpyridine (tpy)-based metallomacrocycles that are coordinatively-bound using various transition metals were investigated through the analysis of their collision cross-section (CCS) values obtained by travelling wave ion mobility (TWIM)-MS separation, and through comparison to modeled/simulated structures and theoretical CCS values. This work demonstrated the uniquely powerful ability of TWIM-MS separation for architectural verification in samples containing stoichiometrically-defined isobaric ion species ranging from dimeric macrocycles to hexameric macrocycles. Furthermore, TWIM-MS was utilized in a separate project which allowed for the development of a method to monitor concentration dependent supramolecular structural equilibria and conformational trends. Using the methodology in this work, equilibrium constants were derived from these data; this was illustrated for two equilibria concerning the self-assembly of a tpy-based ligand containing a flexible crown ether moiety with Zn2+ ions, and a tetrakisterpyridinyl ligand with Cd2+ ions, which have previously been shown to reversibly interconvert. Additionally, experimental CCS values were also derived from drift time measurements and compared with theoretical predictions for these complex self-assembled terpyridine-based supramolecules to provide insight into their size, stoichiometry and architecture. Parallel studies have focused on the development of methods that can obtain conne (open full item for complete abstract)

Committee: Chrys Wesdemiotis (Advisor); Mark Foster (Committee Chair); George Newkome (Committee Member); Matthew Becker (Committee Member); Kevin Cavicchi (Committee Member) Subjects: Analytical Chemistry; Chemistry; Materials Science; Polymer Chemistry; Polymers

Master of Science, The Ohio State University, 2015, Human Ecology: Human Nutrition

Biofortification is defined as the enrichment of staple crops with essential micronutrients. At present, it is one of the strategies used to alleviate vitamin A deficiency (VAD) by breeding staple crops with β-carotene. Staple crops that have been successfully biofortified with β-carotene under the HarvestPlus program are cassava, maize (corn) and sweet potato. Recently, β-apocarotenoids have been identified and quantified in cantaloupe melons and orange-fleshed honeydew. These cleavage products of β-carotene are formed by chemical and enzymatic oxidations. However, there are no detailed analyses of these compounds in biofortified foods and little is known about their bioavailability and intestinal absorption. Hence, this research focused on the analysis of β-apocarotenoids in biofortified cassava and kinetics of cell uptake and metabolism of β-apocarotenoids. In the first study, we analyzed the β-carotene and β-apocarotenoids content of conventionally bred cassava biofortified with β-carotene. Using a previously described high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, we identified and quantified β-apo-13-carotenone, β-apo-14'-carotenal, β-apo-12'-carotenal, β-apo-10'-carotenal, and β-apo-8'-carotenal in hexane/acetone extracts of raw, boiled, and fried roots of the two biofortified cassava varieties investigated. The levels of β-apocarotenoids in roots of non-biofortified cassava varieties were lower than those of biofortified varieties and some of these values approached the limit of detection (LOD) or limit of quantification (LOQ). The purpose of the second study was to determine kinetics of uptake and metabolism of β-apocarotenoids using Caco-2 intestinal cells as a model. We hypothesized that these compounds are directly absorbed from the diet similarly to β-carotene. Pure β-apocarotenoids were delivered to fully differentiated monolayers of Caco-2 cells using tween-40 micelles. Carotenoids were extracted from media (open full item for complete abstract)

Committee: Earl Harrison (Advisor); Steven Schwartz (Committee Member); Joshua Bomser (Committee Member) Subjects: Biochemistry; Nutrition

Doctor of Philosophy, The Ohio State University, 2005, Pharmacy

Overexpression of the anti-apoptotic protein Bcl-2 has been found in about half of human cancers. G3139 is an 18-mer phosphorothioate antisense oligonucleotide designed to bind to the first six codons of the open reading frame of the human Bcl-2 mRNA. In this project, the preclinical and clinical pharmacokinetics, pharmacodynamics and metabolism of this novel therapeutics were investigated. A novel, ultrasensitive, nonradioactive hybridization ELISA method has been developed and validated for quantification of G3139. Plasma pharmacokinetics of G3139 in acute myeloid leukemia (AML) patients was characterized and found to fit a two-compartment open infusion model. There was no major pharmacokinetic interaction between G3139 and concomitant chemotherapeutic agents. Robust intracellular concentrations of G3139 in bone marrow (BM) and peripheral blood mononuclear cells obtained from treated AML patients were achieved and a correlation between the Bcl-2 mRNA/protein down-regulation and disease response was found. A higher median intracellular concentration of G3139 was detected in the complete responders as compared with non-responders. Cellular uptake and distribution of G3139 was studied in K562 cells. When exposed to free G3139, only low intracellular concentrations of G3139 were found in the cells with no significant suppression of Bcl-2 mRNA. In contrast, a 10 to 25-fold increase of the intracellular G3139 was observed when G3139 was delivered with cationic lipids. Dose-response curve shows that G3139 concentration that produces 50% down-regulation was 0.29 iM. Two in vitro PK/PD models were developed for AML cells NB4, which describe the relationship between drug exposure and target down-regulation reasonably well. A novel ESI LC/MS/MS method has been developed. Using this method, several chain-shortened G3139 metabolites were identified in mice, rats and humans, implicating the involvement of 3'-exonuclease in G3139 metabolism. In the mouse, tissue distribution is (open full item for complete abstract)

Committee: Kenneth Chan (Advisor) Subjects: Health Sciences, Pharmacy