<p> Bovine heparin is characteristically different than porcine intestinal heparin. These differences include sulfation, molecular weight properties, activity, structure, and shape. Bovine lung heparin has a higher amount of GlcNY6S (where Y can represent Ac or S) while the amount of GlcNY6S is much lower in bovine intestinal heparin. All heparins have high amounts of trisulfated TriS disaccharide but the level of TriS is in lower in bovine intestinal heparin. The amount of NS2S is much higher in bovine intestinal heparin than in bovine lung and porcine intestinal heparins. The average molecular weight of bovine intestinal heparin is similar to porcine intestinal heparin but the molecular weight of bovine lung heparin is much lower. The activities of bovine tissue heparins were comparable to, but lower than, the activity of porcine intestinal heparin. </p><p> The differences in heparins from different sources become much more subtle as they are depolymerized into low molecular weight heparin (LMWH). These differences are often sufficiently small so that they require principle component analysis (PCA) to determine. Differences in the reducing end and the non-reducing end structures in heparin determined by mass spectrometry (MS) as well as differences in the glucosamine and uronic acid residues determined by nuclear magnetic resonance spectroscopy (NMR) were selected for PCA. Using PCA it was possible to link parent heparin starting material to its daughter LMWH. This analysis demonstrated the lower variation between LMW bovine and LMW porcine heparins than between bovine and porcine heparins. This lower variation afforded the LMW bovine heparin similar anti-Xa and anti-IIa activity comparable to commercial heparin. </p><p> The harsh purification process used to prepare heparin leaves the heparin product largely unaffected except for its reducing end tetrasaccharide linkage region. GlyserineAc is present in porcine and bovine heparin but is absent in LMW heparin. We hypothesized that the peracetic acid bleaching adds an O-acetyl group that is selectively lost during &beta;-elimination in the LMWH production process. The tetrasaccharide composition of bovine and porcine heparin is similar as are different batches from the same supplier. This emphasizes how similar processing results in similar heparin regardless of whether bovine or porcine is used. </p><p> Small differences in counterfeit heparin, i.e. blended porcine and bovine heparins were next examined. Porcine heparin and bovine heparin of similar molecular weights to obtain a bovine heparin counterfeit drug of enhanced activity. Such counterfeit blends are undetectable by current methods of analysis. Diffusion ordered spectroscopy (DOSY) method for analysis was developed. DOSY exploits differences in the molecular shape of bovine heparin and porcine heparin and achieved partial separation in the diffusion dimension. Additional spectra for component resolution (SCORE) analysis were used to demonstrate detection and identification of blend mixture.</p><p>

<p> The challenges to forensic body fluid analysis have placed limitations on the type of information that investigators can acquire and how that information can be collected. In recent years, Raman spectroscopy has proven itself useful for characterizing body fluids. In 2008, a large-scale investigation was undertaken to explore the use of Raman spectroscopy as a means of identifying body fluids. This work resulted in multidimensional Raman spectroscopic signatures for the five main body fluids: semen, peripheral blood, saliva, vaginal fluid, and sweat. These studies were incredibly successful and created the foundation for years of continued research. Accordingly, the studies included in this thesis have been specifically chosen to frame the previous research projects. They include a suite of projects aimed to advance and validate the developed method. </p><p> First, a statistical model was developed to automatically identify and differentiate body fluids based on their Raman spectra. The multidimensional spectroscopic signatures mentioned above are very effective at identification, but they are body fluid-specific. In other words, they individually evaluate whether or not an unknown spectrum is from a particular body fluid, such as blood. Additionally, each signature was built on spectra from a limited number of donors. To improve on this capability, a single classification model was built on the Raman spectra from 60 donors (12 for each body fluid). This model was externally validated with an additional 15 donors in order to objectively assess the model&rsquo;s performance. All of the external validation donors were correctly identified, illustrating how accurate and robust the model is. </p><p> Second, the limit of detection (LOD) for the classification model was explored as a form of validation. It is vitally important that a method&rsquo;s limits be established before deploying it into use. The LOD of peripheral blood was investigated. Peripheral blood is unique from other body fluids because its Raman spectrum has been attributed almost entirely to one molecule- hemoglobin. Because hemoglobin is only found in red blood cells (RBCs), the Raman spectrum of peripheral blood essentially results purely from RBCs. Given this, we chose to start with a single RBC, and then increase the volume until identification was successful. We found that we were able to conclusively and confidently identify peripheral blood using a single red blood cell. This limit is 5000X smaller than the amount of blood required for DNA analysis, demonstrating the sensitivity of the developed method. </p><p> Finally, the method was further advanced by incorporating donor characterization into the process. Besides identifying body fluids, the method can now extract &ldquo;phenotypic&rdquo; information about the donor. Raman spectroscopy and multivariate data analysis were used to determine the biological sex of saliva donors, and the race of semen donors. These studies will help forensic investigators extract incredibly useful information about a potential suspect or victim, and can be performed directly at a crime scene for instant results. </p><p> Altogether, these studies combine to strengthen the method previously developed by our research group. More importantly, they help to bridge the gap between research and application. Creating a universal method to differentiate and identify body fluids, investigating the method&rsquo;s LOD, and developing additional techniques to characterize body fluids represents a significant contribution to the field of forensic chemistry. The universal method created within this thesis will be adapted to perform on-site analysis of physical evidence at crime scenes. The methods&rsquo; incredible sensitivity has been demonstrated by determining that it can identify peripheral blood based on a single RBC. Finally, by developing models to characterize body fluid donors, investigators will be able to extract useful information about individuals that may have been present at a crime scene. Additional studies are already being conducted to make further improvements, and our method is poised to make a significant contribution to the field of crime scene investigation. </p><p>

<p> Metabolomics is a rapidly growing field in &ldquo;omics&rdquo; research where metabolites are analyzed in biological systems. Over the past decade, mass spectrometry (MS) based metabolomics has been used for its superior analytical performance to reveal how these biological systems respond to genetic and environmental changes. MS is both sensitive and selective and is capable for providing comprehensive information for metabolic profiling by combining separation methods such as liquid chromatography (LC-MS) or gas chromatography (GC-MS). However, in untargeted metabolomics identification of small molecules is the bottleneck. In the research described here, I have combined both <i> in silico</i> and experimental methods for compound dereplication of natural products using MS-based metabolomics. </p><p> <b>Chapter 1</b> addresses the advancement of fragmentation and mass spectral trees used for unknown metabolite identification. Tools used for metabolite identification from the past 10 years are discussed, including algorithms, software, mass spectral libraries, and databases that implement fragmentation and mass spectral trees. Due to the inherent complexity of natural products in plants and microbes, unknown compound identification is increasingly difficult and limiting. Resolving this problem requires better computational tools and informative data such as those acquired by multi-stage mass spectrometry (MSⁿ). MSⁿ yields more fragmentation data and allows for more complex structural elucidation as needed for compounds with positional isomers. The limitation with using tandem mass spectrometry (MS/MS) only is that many ions are shared between positional isomers and full structural information is not available to elucidate an unknown metabolite. Fragmentation and mass spectral trees both describe the fragmentation processes of a metabolite and aid in fragmentation rule generation and substructure identification. The major difference between fragmentation and mass spectral trees is that fragmentation trees use elemental compositions to describe the fragmentation process and mass spectral trees or ion trees use precursor and product ion spectra from MSⁿ mass spectral acquisition. As a result, there has been a large increase in efforts to develop MS^{n > 2} data and tools for both structure elucidation and spectral annotations with the use of fragmentation and mass spectral trees in recent years. </p><p> <b>Chapter 2</b> describes research and development of iTree, a MSⁿ mass spectral tree library of plant natural products and its aid in compound identification of natural products. In metabolomics, mass spectral library searching is a standard method for compound identification, correctly known as compound dereplication. Mass spectral libraries are either freely or commercially available and can contain both experimental and <i> in silico</i> MS/MS reference spectra. The coverage of MS^{n > 2} reference spectra is much smaller in many of these MS/MS libraries and databases. To date the largest MS^{n > 2} libraries are HighChem and mzCloud, which also support mass spectral trees. The chemical coverage of such libraries and databases are very low in comparison to the number of known compounds. iTree was developed to expand the coverage of fragmentation spectra for natural products. iTree contains more than 2,000 natural products and more than 9,000 ion tree spectra annotated with <i>in silico</i> generated substructures from both Mass Frontier 7.0 and CFM-ID. iTree is freely available through MassBank of North America (MoNA), an open-access mass spectral database. As a result of the high number of natural products, and specifically flavonoid aglycones, previously published fragmentation rules were studied and validated. A new rule for flavanonols was proposed as a loss of &ndash;CCO to occur specifically for this class. In addition, iTree was used to profile secondary metabolites in the roots and nodules of the host plant <i> Datisca glomerata</i>. More than 100 natural products were identified by combining LC-MSⁿ, high resolution LC-MS/MS, and ion tree analysis using iTree. Overall, iTree has shown to provide a method to facilitate metabolite identification for plant natural products. </p><p> Although MS^{n > 2} data is more useful for complex structural elucidation, the predominant data used in untargeted metabolomics is MS/MS. For this reason, <i>in silico</i> tools that focus on the interpretation of MS and MS/MS spectra alone must be evaluated. In Chapters 3 through 5, I discuss how the Critical Assessment of Small Molecule Identification (CASMI) has allowed for such an evaluation by presenting unknown challenge data sets to the metabolomics community to evaluate the tools and methods they currently use for unknown compound identification. The results submitted by each user are compared and discussed to provide greater insight into how <i>in silico</i> tools can be further improved to aid in the advancement and accuracy of unknown compound identification methods. </p><p> <b>Chapter 3</b> focuses specifically on the performance of MS-FINDER, a software that uses MS and MS/MS spectra for structural elucidation of unknown compounds, presented in the CASMI 2016 Category 1. (Abstract shortened by ProQuest.) </p><p>

Solvated transition metal solvent cluster ions were generated by electrospray ionization and their spectroscopy and photo dissociation dynamics have been studied by laser photofragment spectroscopy. Solvation of Co 2+ by water and methanol has been examined by studying Co2+ (H2O)n and Co2+(CH3OH) n (n = 4–7). An electrospray ionization reflectron time-of-flight mass spectrometer (ESI-RTOFMS) was developed to produce gas phase transition metal solvent cluster ions. This system couples with a Nd:YAG pumped dye laser to produce a unique mass spectrometry-laser photodissociation system. This instrument was compared to ESI-mass spectrometers in the literature. It shows a mass resolution (m/Δm) of 250 and a detection limit of 480 pmol. The instrument compares favorably to existing analytical instrumentation and several improvements are suggested. Hydrated cluster ions, Co2+(H2O)n (with n = 4–7), have been generated by electrospray ionization and studied by laser photofragment spectroscopy. The similarity between the spectrum of gas-phase Co2+(H2O)6 and the absorption spectrum of aqueous cobalt (II) suggests that Co2+(H2 O)6 (aq) is responsible for the room-temperature solution absorption spectrum. The observed photodissociation spectrum of Co 2+(H2O)4 is similar to new bands that appear in aqueous cobalt(II) at high temperatures and have been assigned to Co 2+(H2O)4 (aq) in the literature. The hexahydrate dissociates by loss of one or two water molecules, whereas the heptahydrate dissociates by loss of two or three water molecules. In both cases, loss of two water molecules is the preferred dissociation pathway. The tetrahydrate dissociates either by simple loss of water or by charge separation to form CoOH+(H2O)2 and H3O +, with charge separation being the preferred dissociation channel. Methanolic cluster ions, Co2+(CH3OH)n (with n = 4–7), show behavior similar to that of the hydrated clusters. The gas phase spectra display similar shifts from the solution absorption spectra, but absorb more strongly than the corresponding hydrated clusters. The hexamethanol cluster dissociates via loss of one or two methanol molecules; the heptamethanol cluster dissociates via loss of one, two or three methanols. The tetramethanol cluster primarily dissociates by a charge separation mechanism similar to that observed in the hydrated clusters, forming Co(OCH3) +(CH3OH)2 and H+(CH3OH); dissociation by simple loss of methanol is a minor channel.

Arsenic contamination in groundwater is a worldwide problem. The existing portable field test kits can not provide accurate results when the arsenic concentration is around 10 µg L-1 or lower. This research first was focused on the development and validation of methods in which portable instrumentation, such as electrochemistry instruments or quartz crystal microbalances, could be used to accurately determine arsenic concentrations in water even when the concentration is below 10 µg L-1. A modified anodic stripping voltammetry (ASV) and cathodic stripping voltammetry (CSV) method with measurement at a microarray electrode manufactured by TraceDetect Inc. was developed. When the ASV method with a gold electrode was applied for real water analysis, the detection limit of arsenite was 2.2 µg L-1, and for arsenate was 0.13 µg L-1 . In the CSV method the more commonly used hanging mercury drop electrode was replaced with a mercury film array electrode. Under the optimum condition, this method had a detection limit for arsenite of 0.58 µg L-1 and for arsenate of 2.7 µg L-1. A method for the determination of arsenic using a quartz crystal microbalance was developed in which the crystal surface was modified in situ by dithiolthreitol, an arsenite-selective ingand. The method was applied to real water sample analysis with a limit of 0.6 µg L-1. The second was concerned with an investigation of the kinetics of the reactions that are the basis of several currently available field test kits (as exemplified by the Hach Kit) using inductively coupled plasma mass spectrometry (ICP-MS) with the goal of improving the performance of the test kit. The time for arsine gas reaches to the maximum concentration in the headspace of the vessel was about 60 min without continuous stirring and only 20% of arsenic was absorbed on the test strip. To speed up the arsine generation, continuous stirring condition can be applied. It also made more arsine absorbed on the test strip. The SEM study proves the structure of the darker colored compound. For the lighter colored compounds, the information is not enough to make a conclusion.

The ability to monitor protein aggregation at the molecular level is critical for progress in many areas of life sciences ranging from exploring mechanisms of amyloidosis and etiology of conformational diseases to development of safe and efficient biopharmaceutical products. Despite the spectacular progress in understanding the mechanisms of protein aggregation in recent years, many aspects of the aggregating proteins' behavior remain unclear because of the extreme difficulty in tracking evolution of these notoriously complex and heterogeneous systems. Here, we introduce an electrospray ionization mass spectrometry (ESI MS)-based methodology that allows the early stages of heat-induced aggregation to be studied by monitoring both conformational changes and formation of oligomers as a function of temperature or stress duration. The new approach allows biopolymer behavior (both reversible and irreversible processes) to be monitored in great detail over a wide temperature range. Validation of the methodology is carried out by comparing temperature profiles of model proteins and nucleic acids deduced from MS measurements and differential scanning calorimetry. In order to evaluate the suitability of ESI MS for direct profiling of soluble glycoprotein aggregates, we used heat-stressed human antithrombin, to compare size-exclusion chromatography (SEC) and ESI MS as a means to probe composition of the complex mixture of soluble oligomeric species generated by heat-induced aggregation. Once the appropriate corrections are made, the abundance of the small aggregates derived from ESI MS becomes remarkably close to that calculated based on SEC data, suggesting that ESI MS may be directly applied for at least semi-quantitative characterization of soluble protein aggregates. Application of the methodology to study heat-induced aggregation of human glucocerebrosidase and antithrombin unequivocally links loss of conformational fidelity to formation of soluble oligomers, which serve as precursors to aggregation. Sequential conformational transition of a monoclonal antibody can also be sensitively probed with this method. The ability to make a distinction between various biopolymeric species (based on the differences in their masses) and their conformers (based on the differences in their charge state distributions) allows the temperature-controlled ESI-MS measurements to be carried out in complex systems with very high degree of specificity. This unique feature of the new experimental technique makes it very appealing to the biotech and biopharmaceutical sectors, where the need to engineer/formulate stable biopolymer-based products (e.g., protein drugs) places a premium on the ability to characterize their behavior as a function of temperature with a high degree of precision and accuracy. One of the unique advantages of hydrogen/deuterium exchange mass spectrometry (HDX MS) as a tool to probe protein higher order structure and dynamics is its ability to detect distinct conformational states under certain conditions. When the exchange follows the so-called EX1 or EXX regime, a distinction among various conformers can be made based on the different levels of deuterium incorporation, which manifest themselves in the form of bi- or multi-modal isotopic distributions of protein ions. In this work we exploit this unique advantage of HDX MS and the ability of mass spectrometers to select narrow populations of protein ions to develop a method which allows structure of distinct conformers to be probed at high spatial resolution. Ubiquitin is selected as the model. Validation of the method is carried out by comparing relative magnitude of protection of individual backbone amides deduced from MS and NMR measurements. The two conformers coexisting in the model system exhibit remarkable difference in deuterium incorporation at expected portions of protein sequence. The comparison to reference conformational states of ubiquitin reveals the structural nature of these conformers. These results demonstrate the capability of the top-down HDX MS/MS to specifically capture the conformational features of individual intermediates co-existing at equilibrium.

This dissertation focuses on the mass spectrometric based methods for studying protein-metal binding. Identifying metal-protein interaction is a key step in understanding metal-binding protein structure and function. A phenomenon associated with gas phase dissociation behavior of metal-peptide complexes has been investigated. A positive correlation was found between the number of strong coordination groups in the peptide sequence and the degree of c and z ion formation after electron transfer dissociation of the peptide-metal complexes. Establishing thermochemical cycle enables a theoretical understanding of the process. A new mass spectrometric method has been developed to identify Zn-bound His residues in Zn-metalloproteins relies on variations in the hydrogen deuterium exchange of the C2 hydrogen of His side chains. We show that this approach can be used to study the Zn-bound His residue in human &beta-2-microglobulin; (&beta2m;), a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. The different effect of three divalent transition metals including Cu(II), Ni(II) and Zn(II) on &beta2m; oligomerization and fibril formation under physiological conditions is described. We found that Cu(II) can induced &beta2m; oligomerization and amyloidosis. In contrast, no oligomeric species can be formed with Ni(II), and only oligomers can be formed with Zn(II). A combination of metal catalyzed oxidation (MCO)-MS, hydrogen deuterium exchange (HDX)-MS and other spectroscopic techniques is utilized to obtain insights into the mechanism of Cu(II)-induced &beta2m; amyloidosis. We elucidate the different ways that these metals bind &beta2m;, thereby identifying key features of the &beta2m-Cu;(II) interaction that are essential for enabling this protein to form amyloid fibrils.

Transition metals play an important role in marine environments. They can be both essential and toxic for marine organisms. These metals are complexed by organic ligands, and their complexes are present at trace levels and in complicated mixtures making their analysis difficult. For this reason, sources and chemical compositions of these species remain unknown. This dissertation is centered on studying the dissociation dynamics of metal complexes in order to use mass spectrometry to get structural information for metal complexes at low concentration levels. This work describes the study of the collision-induced dissociation (CID) patterns and energetics exhibited by different metal complexes in a quadrupole ion trap mass spectrometer. A variety of model ligands containing different donor atoms were synthesized and complexed with first-row transition metals. It was found that differences in the electronic structure of the metal ion, the coordination number of the complex, and the nature of the donor groups bound to the metal are reflected in the types of product ions observed in CID spectra. Upon dissociation, pentacoordinate complexes of Cu and Zn exhibited differences in the preference to remain coordinated to different donor group. For Cu, this preference reflected a balance between the inherent binding strength of the donor group and its flexibility. For Zn complexes, the inherent donor group binding strength and stability of the product ions were more important. The metal electronic structure also impacted the types of dissociation products observed in penta-, and hexacoordinate complexes. Upon CID Cu tends to be reduced. For Zn H2 is the dominant dissociation pathway. Mn, Fe, Co, and Ni Complexes predominantly lose a single binding upon dissociation and sometimes Ni is reduced. Relative dissociation energies were determined by a variable energy CID approach. Metal complexes having metal centers with smaller ionic radii had the lower dissociation energies, except for Cu(II) complexes because its easy reduction. In summary, CID seems to have some promise for helping to distinguish among complexes with different coordinating functional groups, coordination numbers and/or metal centers and thus may some potential for providing coordination structure information for complexes present at trace concentration levels.

A thorough understanding of the chemical and physical properties of small molecules involves spectroscopy, thermodynamics, kinetics and dynamics of the system. This thesis highlights the use of photochemistry to measure these properties for Au+(C2H4), Pt+ (C2H4) and ethylbenzene cation as well as for deposition of zeolite films. Chapter 1 highlights the principles behind photodissociation and the information that can be obtained from spectroscopy. Also discussed is what the appearance of the photodissociation spectrum and time-of-flight profile implies about the molecular dynamics and kinetics. Chapter 2 examines gas-phase photodissociation of the classic π complexes Au+(C2H4) and Pt+(C 2H4). Spectroscopic onsets provide upper limits to the metal-ligand bond strengths of 344 kJmol−1 and 230 kJmol−1 for Au+(C2H4) and Pt +(C2H4) respectively. The spectrum of Au +(C2H4) features an extended progression in the metal-ligand stretch with a frequency of 176 cm−1 that drops to 160 cm−1 in Au+(C 2D4). Hybrid density functional theory (DFT) calculations and TD-DFT calculations are used to explore the structures, bonding and electronic spectroscopy of the two complexes. The photodissociation pathways and kinetics of the ethylbenzene radical cation are investigated in Chapter 3. The energy dependence of the various pathways are investigated. The most abundant dissociation channel is C 7H7+ + CH3 at all wavelengths, but C6H4+ + C2H6 and C6H6+ + C2H4 are also important in the near-UV. The C6H4+ + C2H6 pathway is especially interesting as it exhibits a significant peak broadening with ∼0.6 eV kinetic energy release. Later studies using vibrationally cold ions examined the dissociation rate, k(E), at various photon energies. Simulations of the time of flight profile show that the k(E) increases from 0.97 × 106 s−1 at 2.38 eV to 2.6 × 106 s −1 at 2.67 eV internal energy. An alternate application of molecule-light interaction to surface science is explored in Chapter 4. Here, the technique of pulsed laser deposition has been employed in efforts to improve thin film growth for ETS-4 molecular sieves. Characterization using X-Ray Diffraction and Transmission Electron Microscopy confirm that the zeolite structure is unaltered during the deposition process. Recommendations for further studies are discussed in Chapter 5.

Sulfur and selenium participate in a variety of essential biological functions. The investigation of two non-heme iron dioxygenases with sulfur-containing substrates is reported along with studies of seleno-compounds by gas chromatography with atomic emission detection (GC-AED). Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in E. coli as a 26.8 kDa fusion protein bearing a poly-histidine tag. Kinetics studies revealed a Km value of 2.5 ± 0.4 mM at pH 7.5 and 37 °C, with no requirement for secondary proteins or cofactors. Fe was demonstrated to be the only metal that is essential for activity. Inhibition studies with cysteine analogs along with the use of x-ray absorption spectroscopy (XAS) allowed for the characterization of the active site. Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway. Klebsiella produces two ARD enzymes that share a common polypeptide sequence and differ only in the metal ion present. In the presence of Fe-containing ARD (ARD'), reaction of acireductone with dioxygen produces formate and the ketoacid precursor of methionine. In the presence of the Ni-bound form (ARD) the same substrate produces formate, methylthiopropionate and CO, an off-pathway shunt. XAS study of the structure of the catalytic Fe center in resting state enzyme shows a six coordinate Fe site composed of N/O-donor ligands including 3-4 histidine residues. The substrate binds to the Fe center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand. Cancer prevention attributed to the properties of selenium is widely recognized, but the mechanism of tumor inhibition by this element is still not known. The determination of Se-metabolites and the understanding of their fate are indispensable. Liver extracts from rats administered with selenized yeast were examined by GC-AED. Organoseleno compounds have been observed, including selenomethionine, and possibly the newly-discovered S-(methylseleno)-cysteine molecule. Aromatic Se-compounds have been recognized as antitumorigenic alternatives with lower toxicity. Gas chromatographic behavior of several aryl diselenide derivatives were analyzed.