The initial steps involved in the assembly of normally soluble proteins into amyloid fibrils remain unclear, yet over 20 human diseases are associated with proteins that aggregate in this manner. Protein surface modification is a potential means of mapping the interaction sites in early oligomers that precede amyloid formation. This dissertation focuses on the use of covalent labeling combined with mass spectrometry to elucidate the structural features of Cu(II)-induced β-2-microglobulin (β2m) amyloid formation. An improved covalent modification and MS-based approach for protein surface mapping has been developed to address the need for a reliable approach that ensures protein structural integrity during labeling experiments and provides readily detectable modifications. This approach involves measuring the kinetics of the modification reactions and allows any local perturbations caused by the covalent label to be readily identified and avoided. This MS-based method has been used to study human β2m, a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. Under conditions that lead to β2m amyloid formation, reactions of β2m with three complementary covalent labels have been used to identify the Cu(II) binding site, metal-induced conformational changes, and the oligomeric interfaces. Results confirm that Cu(II) binds to His31 and the N-terminal amine. Binding to these residues causes several structural changes in the N-terminal region and ABED β-sheet which likely enables formation of oligomeric intermediates. The covalent labeling data indicate that the pre-amyloid β2m dimer has an interface that involves the antiparallel arrangement of ABED sheets from two monomers. Moreover, our covalent labeling data allowed us to develop a model for the tetramer in which the interface is mediated by interactions between D strands of one dimer unit and the G strands of another dimer unit. Lastly, the selective covalent modification approach has been used to delineate the structural changes in β2m after interaction with Cu(II), Ni(II), and Zn(II) and their effect on its aggregation. Our covalent labeling data indicates that the unique effect of Cu(II) appears to be caused by the site at which the metal binds the protein and the conformational changes it induces.

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.

A procedure based on the selective retention of either Cr(III) or Cr(VI) on alumina is evaluated and modified, and some mechanistic investigations made. Problems were encountered in selectively and quantitatively retaining either Cr(VI) or Cr(III) in the presence of the other, or in the presence of modest concentrations of various interferents. Evidence is presented that the poor recovery and selectivity were a function of chromatographic adsorption on alumina, a secondary retention mechanism which limits the practicality of the method. A modified and improved version of a plasma source atomic fluorescence spectrometer is described. Control of the spectrometer has been completely transferred to a PC-AT, which uses a control software package (LabVIEW, version 3.1) and two programmable circuit boards (AT-MIO-16DL and PC-TIO-10, both from National Instruments) to control lamp pulsing and data processing circuitry. A series of printed circuit boards have been constructed to process the photomultiplier tube current signal using a modified gated integration (boxcar integration) technique. Greater reproducibility and a two order of magnitude improvement in sensitivity were observed. Microwave assisted extraction, and sonication are compared to methanolic-HCl modified supercritical fluid extraction both quantitatively and qualitatively for the removal of organoarsenic species from National Institute of Standards (NIST) SRM1944 New York Harbor Sediment), SRM2704 (Buffalo River Sediment), and SRM2710 (Montana Soil). The extracted arsenic species were separated by ion exchange chromatography and introduced on-line into a hydride generation manifold prior to determination by plasma source mass spectrometry. Six different arsenic species were observed, and monomethyl arsonic acid (MMA) was quantified in each reference material. The determination of arsenic in soil and lake sediment samples taken in close proximity to CCA-treated wood structures revealed a highly elevated level of arsenic in soils and non-elevated arsenic level in the sediment. Though the level of arsenic in the exposed lake sediment was not elevated, the exposed sediments contained proportionally more monomethyl arsonic acid, (about 1.2% arsenic as MMA) than did the soil samples taken near pressure treated decks (from 0.02-0.30%, depending on sampling depth). The proportion of arsenic as MMA in the CCA exposed soils increased with the sampling depth.

Solid-state, wide-line, $\sp2$H Nuclear Magnetic Resonance spectroscopy was used to study the structure and dynamics of exchange of deuterated hydroxyl groups on the surface of silica gel. Spectra were obtained from three different kinds of silica gel, and detailed studies were carried out using Aldrich 70-230 mesh, high-purity silica. In ambient temperature relaxation experiments, the longitudinal relaxation time, T$\sb1$ was determined to be 70 $\pm$ 30 msec. The transverse relaxation time, T$\sb2$ was found to be inhomogeneous, with minimum and maximum values of 50 $\mu$sec and 325 $\mu$sec, respectively. By means of a quadrupolar echo modification of a Carr-Purcell-Meibloom-Gill (CPMG) spin echo experiment, it was determined that deuterium nuclei migrate slowly across the silica surface and that the inherent spectral line width of the spin packets was less than 1 KHz. At 72 K, T$\sb1$ was estimated to be 0.1-0.5 seconds, and CPMG experiments indicated that some motion still existed on the surface. In experiments at 72 K and 92 K, NMR spectra were collected from samples of silica degassed under vacuum at various temperatures. Using the technique of spectral subtraction to isolate individual spin species, we compared the experimental data with simulated spectra. We found that at least four deuterium spin species exist on the silica surface; one of these is almost certainly bound D$\sb2$O, and the others are deuterated hydroxyl groups, which were discriminated by their quadrupolar coupling constants, asymmetry parameters, and exchange energy.

Analysis of nitrogen containing compounds by gas chromatography with atomic emission detection has always been problematic. Nitrogen detection has been customarily done using the 174.53 and 174.27 nm atomic lines, with very limited selectivity and sensitivity. Nitrogen can also be detected using the cyanogen (CN) molecular emission bands at 388 nm. It has been reported that a 100-fold improvement in the selectivity using this line can be reached. Therefore, in the present work, the instrument available in the laboratory was modified to allow the use of the 388 nm line for the nitrogen detection. The modification of the instrument involved the use of a mixture of reagent gases that were optimized to produce the best nitrogen emission using the 388 nm line. Figures of merit of the detection using this line, were evaluated and compared with the commonly used nitrogen line (174 nm) and the carbon detection (193 nm). The dependence of the GC-AED response with the molecular structure of the analyte, and its application in the determination of empirical formula was also evaluated, and its application for analysis of petroleum products. The second part of the present work studied the biomarkers in deep subsurface samples and their relationship with the microbial activity. Little is known of microbial community characteristics at depths larger than 50 m below surface, information that is crucial for the clean up of contaminated underground environments. The use of classical approaches to identify microbiota, often results in the characterization of less than 10% of the biota present when analyzing deep subsurface samples, therefore, the analysis of lipid content in the samples have been proposed. Microorganisms use lipids as constituent of their cell walls or as nutritional sources. Monitoring lipids can be used to develop a picture of how, when, where and under which conditions microbes are active in the subsurface. This research was focussed in the biomarker study of a shale and sandstone from Cerro Negro, New Mexico. Biomarkers were determined to study distribution, concentration and availability of resources that support life and microbial activity and their relationship with geochemical parameters.

The Sealed Inductively Coupled Plasma Atomic Emission Spectrometer (SICP-AES) was developed for toxic and reactive gas analysis. The most important challenge in using this technique routinely to determine the metallic impurities in toxic and reactive gases is calibrating the system for metals such as Fe, Ni, Cr, and Mo. The unavailability of appropriate gaseous standards that contain these metals prevents a straightforward approach toward calibration. Two classes of organometallic Fe and Ni compounds were investigated for calibration. The first consists ferrocene, Fe(C5H5) 2, and nickelocene, Ni(C5H5)2. Corresponding diffusion tubes of these compounds were employed to calibrate for Fe and Ni in chlorine. The detection limits of Fe and Ni were 20 and 9 pg mL −1, respectively. The second class of compounds were carbonyls of Fe and Ni. Fe(CO) 5 and Ni(CO)4 are in gas phase at room temperature. This property allowed one to use them as calibration materials without significant modification to the gas handling system. However, their high toxicity required extreme caution. The detection limit of Fe was 290 pg mL−1. Iron pentacarbonyl proved to be extremely useful in calibrating the system for Fe in presence of Cl2. Unfortunately a Ni carbonyl standard mixture was not commercially available owing to its high toxicity. As a result, the production of these carbonyls as a calibration source was investigated. These carbonyls were synthesized by the reaction between Fe and Ni powders in the presence of carbon monoxide at high pressures and temperatures. The quantification was accomplished by using a number of different chemicals such as nitric acid and dilute iodine solutions. Additionally, the system was calibrated for sulfur in Cl2. Sulfur hexafluoride, SF6, was employed as the calibration material . Since 100% SF6 was used, there was a need to dilute it five orders of magnitude to obtain low ppm concentrations of S. Therefore, a gas handling system was designed that allowed the accurate dilution of SF6. Sulfur detection limit was 195 ppb (v/v). Finally, anhydrous hydrogen bromide was analyzed qualitatively by SICP-AES. All the emission lines of bromine between 200–900 nm were tabulated, the molecular features, and the impurities were identified.

Hyaluronic acid, HA, is a naturally occurring linear polysaccharide comprised of N-acetyl glucosamine and glucuronic acid. It is ubiquitous in tissues where it serves numerous biological functions. HA is polydisperse having average molecular weights that range on the order of 105 to 10 7 daltons depending upon the tissue from which it is derived and its method of purification. As with any polymer preparation, the molecular weight distribution, MWD, is important to a complete understanding of the characteristics and performance of the preparation. Size exclusion chromatography, SEC, is the most commonly employed means to determine the MWD of a polymer preparation. It is shown that SEC cannot be relied upon for the accurate and reproducible analysis of the MWD of large MW HA. Commercially available polymer standards used to calibrate SEC systems have hydrodynamic volumes that are considerably smaller than HA. HA analyzed under extreme conditions of NaCl concentration and pH that were shown to cause contraction of the molecule still had a hydrodynamic volume much greater than polymer standards. Conventional calibration with standards can be subject to error from extrapolation and unable to detect elution in the totally excluded volume. Viscosity measurements in a dual capillary viscometer demonstrated shear thinning of large MW HA at shear rates that are common inside the plumbing of SEC systems and on line viscometers. A model of shear inside an SEC column is derived in terms of the flowrate, column diameter, and particle diameter. The shear inside the column is shown to be sufficient to reduce the viscosity of large MW HA complicating the MWD analysis of HA by SEC. Chromatographic analysis based on separation in pressure driven flowing streams have fundamental limitations with shear sensitive polymers, such as HA, due to shear, which arises from parabolic flow profiles. Capillary electrophoretic methods have a flow profile that is flat and theoretically should have no shear. It is proposed that capillary electrochromatography utilizing a size exclusion stationary phase may provide the optimum tool for the analysis of large MW HA and other shear sensitive polymers.

Concerns over the production of disinfection by-products formed when drinking water is chlorinated that may be toxic to humans, has increased the interest in alternative disinfection procedures such as ozonation and chloramination. The research in this dissertation concerns the development of analytical methodologies for organic chloramines, that are formed during chloramination and chlorination, and dicarboxylic acids that are formed during oxidation processes especially ozonation. Organic chloramines may have detrimental human health effects and interfere with the measurement of inorganic chloramine. Chromatographic methodologies for their determination are limited by the difficulty in extracting them from water, lack of standards and the lack of a chromophore for HPLC analysis. The research focused on three main areas: investigation of the feasibility of gas chromatographing the organic chloramines, investigating the compound independence of the newly developed nitrogen 388 line for the atomic emission detector, and the development of an extraction procedure for removing the organic chloramines from water. The final method employed solid phase microextraction (SPME). The SPME technique was optimized for both aliphatic amines and organic N-chloramines. Measurement of dicarboxylic acids, particularly oxalic acid, in drinking water was also investigated. They contribute to microbial growth in water and may react further to form halogenated disinfection by-products. Like the organic chloramines, their hydrophilicity and lack of a suitable chromophore hinder their analysis. Furthermore, direct gas chromatographic analysis of the dicarboxylic acids is not possible as they are not volatile. A gas chromatographic procedure for the analysis of short chain dicarboxylic acids was developed. An extraction procedure for oxalic acid using tributyl phosphate, an organic base is presented. The use of chloroformate reagents, particularly trichloroethylchloroformate, for the subsequent derivatization of mono and dicarboxylic acids is investigated. Although, the derivatization clearly formed the appropriate reaction product, cleanup of reaction by-products and reagents is problematic. Several strategies for removing these matrix affects are presented. A study for the characterization of human and veterinary sodium hyaluronate products was conducted. A procedure for the supercritical fluid extraction (SFE) and determination by HPLC for miconazole nitrate is presented.