Browsing by Subject "Analytical chemistry"

Currently there is a dearth of analytical techniques for studying protein-ligand interactions on the proteomic scale. Existing techniques, which rely on various calorimetry or spectroscopy methods, are limited in their application to the proteomic scale due to their need for large amounts of pure protein. Recently, several mass spectrometry-based methods have been developed to study protein-ligand interactions. These mass spectrometry-based methods overcome some of the limitations of existing techniques by enabling the analysis of unpurified protein samples. However, the existing mass spectrometry-based methodologies for the analysis of protein-ligand binding interactions are not directly compatible with current mass spectrometry-based proteomics platforms.

Described here is the development and application of a new technique designed to detect and quantify protein-ligand binding interactions with mass spectrometry-based proteomic platforms. This technique, termed SPROX (Stability of Proteins from Rates of Oxidation), uses an irreversible covalent oxidation labeling reaction to monitor the global unfolding reactions of proteins to measure protein thermodynamic stability. Two variations of the SPROX technique are established here, including one variation that utilizes chemical denaturant to induce protein unfolding and a second variation that utilizes temperature to denature proteins. The SPROX methodology is tested on five proteins including ubiquitin, ribonuclease A, bovine carbonic anhydrase II, cyclophilin A, and calmodulin. Results obtained on these model systems are used to determine the method's ability to measure the thermodynamic parameters associated with each protein's folding/unfolding reaction. Results obtained on calmodulin and cyclophilin A are used to determine the method's ability to quantify the dissociation constants of protein-ligand complexes.

The primary motivation for the development of the SPROX protocols in this work was to create a protein-ligand binding assay that could be interfaced with conventional mass spectrometry-based platforms. Two specific SPROX protocols, including a label-free approach and an oxygen-16/18 labeling approach, are developed and demonstrated using the thermal SPROX technique to analyze ligand binding in a model four-protein component mixture consisting of ubiquitin, ribonuclease A, bovine carbonic anhydrase, and cyclophilin A. The thermal SPROX technique's ability to detect cyclosporin A binding to cyclophilin A in the context of the model mixture is shown using both labeling approaches.

An application using the SPROX technique combined with a multi-dimensional protein identification technology (MudPIT)-based proteomics platform is also described. In this application, which utilized an isobaric mass tagging strategy, 325 proteins in a yeast cell lysate are simultaneously assayed for CsA-binding. This study was also used to investigate the protein targets of an already well-studied immunosuppressive drug, cyclosporin A. Two of the ten protein targets identified in this work are known to interact with CsA, one through a direct binding event and one through an indirect binding event. The eight newly discovered protein targets of CsA suggest a molecular basis for post-transplant diabetes mellitus, which is a side effect of CsA in humans.

Azobenzene disperse dyes are the fastest-growing category of commercial dyestuffs, accounting for 70% of the 9.9 million tons of industrial dye colorants used annually. Azobenzene disperse dyes are intended to be applied to synthetic fabrics such as polyester, nylon, and acrylic; however, azo dyes may also be used in cosmetic products such as hair dyes, and in fashion accessories such as leather goods. Recently, our group and others have detected azobenzene disperse dyes in dust particles collected from the indoor environment, and raising concerns about the release of these chemicals from products and human exposure. Although extensive literature characterizes these chemicals as toxic contaminants in aquatic environments, to date there exists little data on levels, exposures, and hazards associated wit exposures to azobenzene disperse dyes in the indoor environment. The presence of these dyes in the indoor environment is concerning. House dust is a sink for many contaminants that leach out or off-gas from products in the home. Due to children’s unique behaviors (e.g. crawling and hand to mouth activity) they have higher exposure to chemicals associated with dust. Azobenzene disperse dyes are implicated in literature as potentially allergenic: they are known to be present in clothing that elicits allergic reactions such as skin sensitization. Therefore, it is of crucial importance to support research that seeks to characterize children’s exposure in the home environment, and evaluate the in vitro effects of azobenzene disperse dyes. The hypothesis of this research dissertation is that azobenzene disperse dyes are prevalent in dust collected from the indoor environment at concentrations of concern for human health. In the first aim of this thesis research, azobenzene disperse dyes were characterized in commercial mixtures and in children’s polyester clothing. Azo dyes were first purified from dyestuffs by Soxhlet extraction and flash chromatography and then analyzed using ultra-high-performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS), as well as by 1H and 13C NMR for structural elucidations. Nineteen total azobenzene dyes were detected in dyestuffs via a non-targeted analysis approach, including Disperse Blue 79:1, Disperse Blue 183:1, Disperse Orange 44, Disperse Orange 73, Disperse Red 50, Disperse Red 73, and Disperse Red 354. Samples of children's polyester clothing (n=X) were then analyzed via UHPLC-HRMS. In clothing, 21 azobenzene disperse dyes were detected, 12 of which were confirmed and quantified via reference standards. Individual dyes in apparel were quantified at concentrations up to 9230 μg dye/g shirt, with geometric means ranging 7.91–300 μg dye/g shirt. Total dye load in apparel was quantified at up to 11,430 μg dye/g shirt. This research supported the development of reference standards and library mass spectra for azobenzene disperse dyes previously absent from standard and spectral libraries. This study was the first to confirm and quantify these azo compounds in children’s products, facilitating a more robust understanding of sources of azobenzene disperse dyes in the indoor environment. The second aim of this thesis research investigated the presences and quantities of azobenzene disperse dyes and related compounds in indoor house dust (n=188) collected from homes in Durham, NC. Using a targeted approach, we quantified 12 azo disperse dyes and quantified at least one dye in every house dust sample. Detection frequencies ranged from 11% to 89%; of the dyes that were detected in at least 50% of the samples, geometric mean levels ranged from 32.4 to 360 ng/g. HRMS suspect screening analysis identified an additional eight azobenzene compounds in dust that are present at high relative abundances. This study indicates that azo disperse dyes and related compounds are ubiquitous in the indoor environment. To support quality assurance and control during the analysis, a house dust Standard Reference Material (NIST SRM 2585) was extracted and analyzed with the samples. Based on the detection and abundance of azo dyes in SRM 2585, which was prepared from hundreds of dust samples collected in the mid 1990s, azo dye levels in the indoor environment may be increasing over time. To our knowledge, this is the most comprehensive quantitative study of azo disperse dyes in house dust to date. Future studies are needed to quantify additional dyes in dust, particularly those identified here via suspect screening, and to examine exposure pathways of dyes in the indoor environment where children are concerned. The third aim of this thesis research examined the binding reactivity of azobenzene disperse dyes to nucleophilic peptide residues in order to understand their potential reactivity as electrophilic allergenic sensitizers. The Direct Peptide Reactivity Assay (DPRA) was utilized via both a spectrophotometric method and a high-performance liquid chromatography (HPLC) method. Dyes isolated from the commercial dyestuffs, and several potential transformation products, were tested. All dyes were found to react with nucleophilic peptides in a dose-dependent manner with pseudo-first order (kobserved) activity, but overall to react more potently with cysteine than with lysine: EC10 values for cysteine binding were determined as low as 0.005mM and pseudo-first order rate constants as high as 0.04 hr-1 (as observed for Disperse Blue 79:1). Observed rate constants were correlated to metrics of structural features such as Hammett constants and electrophilicity indices, indicating that binding reactivity may be related to structural properties of azobenzene disperse dyes. In addition to examining dyes, the reactivity of extracts of polyester shirts were also examined; shirt extracts with high relative abundances of azobenzene disperse dyes were observed to induce greater peptide reactivity. Results suggest that azobenzene disperse dyes may function as immune sensitizers, and that clothing containing azobenzene disperse dyes may pose risks for skin sensitization. Collectively, this thesis research suggests that azobenzene disperse dyes are common in clothing, and appear to be near ubiquitous in house dust. Given their reactivity in vitro, this may present health consequences, particularly for young children.

Mass spectrometry is widely considered to be the gold standard of elemental analysis techniques due to its ability to resolve atomic and molecular and biological species. Expanding the application space of mass spectrometry often requires the need for portable or hand-held systems for use in field work or harsh environments. While only requiring “sufficient” mass resolution to meet the needs of their application space, these miniaturized systems suffer from poor signal to background ratio which limits their sensitivity as well as their usefulness in field applications.

Spatial aperture coding techniques have been used in optical spectroscopy to achieve large increases in signal intensity without compromising system resolution. In this work similar computational methods are used in the application of these techniques to the field of magnetic sector mass spectrometry. Gains in signal intensity of 10x and 4x were achieved for 1D and 2D coding techniques (respectively) using a simple 90 degree magnetic sector test setup. Initial compatibility with a higher mass resolution double focusing Mattauch-Herzog mass spectrograph is demonstrated experimentally and with high fidelity particle tracing simulations. A novel electric sector lens system was designed to stigmate high order coded aperture patterned beam which shows simulated gains in signal intensity of 50x are achievable using these techniques.

The advancement of mass spectrometry-based protein stability profiling measurements within the past twenty years has led to the development of a suite of approaches that enables the evaluation of protein folding stability on a broad range of biological mixtures with varying complexity. These approaches include chemical and thermal denaturation techniques (SPROX and TPP, respectively) as well as proteolysis strategies such as limited proteolysis (LiP) and pulse proteolysis (PP) which have all been extensively used and evaluated for small molecule protein target discovery applications. However, the capabilities of these methods have yet to be fully evaluated in the characterization of disease phenotypes and other biological events such as post-translational modifications and RNA-protein interactions. A major focus of the work included in this dissertation has been the comparative analysis of the above techniques for the characterization of biological phenotypes. The application and comparative analysis of the above techniques to the characterization of RNA-protein interactions is also described.

In the past decade, several mass spectrometry-based proteomic techniques have been developed for the large-scale analysis of protein folding stabilities. The main focus of this dissertation is to develop and apply these large-scale protein folding stability approaches in drug target identification and disease biomarker discovery. One goal of this work is to develop a novel chemo-selection strategy to improve the bottom-up proteomics readout in proteome-wide limited proteolysis experiments. Another goal of this work is to apply these methods to the target identification of two drugs with known mode of action, and to the biomarker discovery of Parkinson’s disease.

Described in the first part of the dissertation is the development of a chemo-selective enrichment strategy to isolate the semi-tryptic peptides generated in mass spectrometry-based applications of limited proteolysis methods. The method is termed Semi-Tryptic Peptide Enrichment Strategy for Proteolysis Procedures (STEPP). The STEPP-PP workflow was evaluated in two proof-of-principle drug target identification experiments involving two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow was evaluated in one proof-of-principle experiment on identification of protein conformational changes between a breast cancer cell line, MCF-7, and a normal cell line, MCF-10A. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities. When coupled to a one-pot data acquisition strategy, the one-pot STEPP-PP technique was found to have a very low false positive rate (i.e., 0.09%) in a proof-of-principle drug target identification experiments involving cyclosporin A and a yeast lysate.

The second part of this dissertation describes the application of protein folding stability approaches to the identification of protein targets of subglutinol A (a natural immunosuppressant) and manassantin A (a natural product with anti-cancer activity).

In the suglutinol A study, a combination of SPROX, TPP, CPP and STEPP-PP strategies was used to identified two consistent protein hits, deoxycytidine kinase (DCK) and exportin-2 (XPO2), from more than 2000 assayed proteins in a 2B4T cell lysate. The binding of DCK with subglutinol A was validated using a targeted gel-based pulse proteolysis experiment. A set of chemical biology experiments were performed to uncover the relation of this interaction with subglutinol A’s mode of action. It was shown that neither of the kinase activity, expression level or phosphorylation modification level of DCK was alternated by subglutinol A. However, the nuclear transportation of DCK was blocked by subgltutinol A. This reduction of DCK level in the cell nucleus possibly leads to the observed reduction of nuclear dCMP pool and the halted proliferation of sublgutinol A treated T cells.

In the manassantin A study, a combination of STEPP-LiP, STEPP-PP, one-pot STEPP-PP, one-pot SPROX and one-pot TPP strategies were performed to identify the protein target of the drug in a hypoxia-treated HEK293T cell lysate. These experiments assayed over 4000 proteins and found 4 protein hits for further validation of their interaction with manassantin A.

The third part of describes the utilization of the SPROX method to characterize the progression of PD in a mouse model of the disease in which the human α-synuclein protein with an A53T mutation was overexpressed. The thermodynamic stabilities of proteins in brain tissue cell lysates from Huα-Syn(A53T) transgenic mice were profiled at three time points including at 1 Month (n=9), at 6 Months (n=7), and at the time (between 9 and 16 Months) a mouse became Symptomatic (n=8). The thermodynamic stability profiles generated here on over 300 proteins were compared to the thermodynamic stability profiles generated on the same proteins from similarly aged wild-type mice using a two-way ANOVA analysis. A group of 22 proteins were identified with age-related protein stability changes, and a group of 11 proteins were found to be differential stabilized in the Huα-Syn(A53T) transgenic mouse model. The proteins differentially stabilized in the disease mouse model could potentially be used as Parkinson’s disease biomarkers upon further validation.

Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.

This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.

The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.

To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.

The direct link between a protein’s thermodynamic stability and function influenced the development of mass spectrometry-based methods to characterize the energetics associated with protein folding that enabled the large-scale elucidation of drug protein targets and disease state protein biomarkers. This area of structural biology is undergoing constant development and new applications are emerging. Consequently, the original contributions of this dissertation include (1) the continuation or extension of mass spectrometry and energetic-based strategies for proteome-wide characterization of protein folding stabilities in allergen-containing proteomes to discriminate allergenicity, (2) the hybridization of novel strategies with existing energetic-based approaches utilizing mass spectrometry readout for simpler and efficient characterization of protein folding stabilities and ligand binding, and (3) the development of novel mass spectrometry-based strategies for comprehensive evaluations of the thermodynamics and kinetics of protein folding.First, this dissertation describes comprehensive protein profiling methods to discriminate allergens from non-allergens. As continuation, RNA sequencing (RNA-seq) analysis served as a proxy for protein abundance, and the Stability of Proteins from Rates of Oxidation (SPROX) reported on thermodynamic stability. These techniques characterized the protein expression levels and stability of proteins in the European white birch pollen, Betula pendula (Bp), and German cockroach, Blattella germanica (Bg). The simultaneous comparison of stability and abundance confirmed that Bp and Bg allergens had significantly higher expression levels and higher stabilities compared to non-allergens from the same source. Combining the Bp and Bg results with previous studies for a robust statistical comparison of the abundance and stability of allergens and non-allergens from indoor and outdoor sources confirmed that allergens were significantly more abundant and more stable. The thermodynamic stability of the proteins in Bp was further investigated utilizing a denaturant-dependent Pulse Proteolysis (PP) strategy with thermolysin. Additionally, proteolytic susceptibility was assessed by employing a time-dependent cathepsin S digestion under native conditions. The results confirmed that allergens were significantly less susceptible to thermolysin (more thermodynamically stable) or cathepsin S digestion than the non-allergens in Bp. Additionally, no correlation resulted between the SPROX- and PP-derived thermodynamic stabilities and between the thermodynamic stabilities and proteolytic susceptibilities of selected proteins from Bp. The absence of correlation is attributed to the fundamental differences between techniques—each technique utilizes distinct probes to report on a protein’s thermodynamic stability and/or proteolytic susceptibility. Finally, the PP-derived stability for the major Bp allergen, Bet v 1, correlated with the LiP-derived proteolytic susceptibility and the generation of known T-cell epitopes connecting stability with endosomal processing having allergenic or immunogenic implications. Next, this dissertation reports the first application of the novel one-pot analysis in conjunction with the SPROX methodology for a simplified and efficient evaluation of protein folding and ligand-binding. A hybrid of the one-pot analysis with SPROX utilizing a MALDI readout enabled efficient evaluations of protein stability and ligand binding. The approach generated protein folding stabilities with similar precision to the standard curve-fitting SPROX technique. Furthermore, the one-pot analysis was coupled with the SPROX strategy for a comprehensive deconvolution of Cyclosporine A (CsA) protein targets in yeast. This novel approach identified 3 known CsA protein hits with a 0.04% false positive rate. A cross-validation between techniques (i.e., TPP, CPP, or PP, performed under similar conditions) resulted in false positive rates approaching 0 %. Finally, this dissertation showcases the development of a novel approach utilizing a native or low denaturant-based Reagent-dependent Thiolate-based Reactivity (RTR) assay utilizing mass spectrometry for the evaluation of the thermodynamics and kinetics of protein folding. An RTR strategy titled MTR utilizing a MALDI readout was performed under native conditions to report on the thermodynamics of protein folding. The MTR strategy measured the thermodynamic stability of mutants of the C domain of protein A from Staphylococcus aureus. Additionally, a low denaturant MTR approach reported the thermodynamics and kinetics of protein folding for bovine β-lactoglobulin B (LG-B). A comprehensive application of the native RTR approach was performed on yeast providing thermodynamic stability information for a subset of the proteins.

Proteins fold into well-defined three-dimensional structures to carry out their biological functions which involve non-covalent interactions with other cellular molecules. Knowledge about the thermodynamic properties of proteins and protein-ligand complexes is essential for answering fundamental biological questions and drug or biomarker discovery. Recently, chemical labeling strategies have been combined with mass spectrometry methods to generate thermodynamic information about protein folding and ligand binding interactions. The work in this thesis is focused on the development and application of two such chemical labeling protocols coupled with mass spectrometry including one termed, SUPREX (stability of unpurified proteins from rates of H/D exchange), and one termed SPROX (stability of proteins from rates of oxidation). The work described in this thesis is divided into two parts. The first part involves the application of SUPREX to the thermodynamic analysis of a protein folding chaperone, Hsp33, and its interaction with unfolded protein substrates. The second part involves the development of a new chemical labeling protocol that can be used to make protein folding and ligand binding measurements on the proteomic scale.

In the first part of this work, the SUPREX technique was used to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of the studies, which were performed at the intact protein level, suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived Kd-values for Hsp33 complexes with four different substrates were also found to be all within a range of 3-300 nM. The interaction between Hsp33 and one of its substrates, citrate synthase (CS), was characterized at a higher structural resolution by using the SUPREX technique in combination with a protease digestion protocol. Using this protocol, the thermodynamic properties for both Hsp33 and CS were evaluated at different stages of binding, including reduced Hsp33 (inactive form), oxidized Hsp33 (active form), followed by native CS and finally of Hsp33ox -CS complexes before and after reduction with DTT. The results suggest that Hsp33 binds unfolded proteins that still have a significant amount of residual higher- order structure. Structural rearrangements of the substrate protein appear to occur upon reduction of the Hsp33-substrate complexes, which may facilitate the transfer of the substrate protein to other protein folding chaperone systems.

In the second part of this dissertation, a mass spectrometry-based covalent labeling protocol, which relies on the amidination rate of globally protected protein amine groups, was designed and applied to the thermodynamic analysis of several eight protein samples including: six purified proteins (ubiquitin, BCAII, RNaseA, 4OT, and lysozyme with, and without GlcNAc), a five-protein mixture comprised of ubiquitin, BCAII, RNaseA, Cytochome C, and lysozyme, and a yeast cell lysate. The results demonstrate that in ideal cases the folding free energies of proteins and the dissociation constants of protein-ligand complexes can be accurately evaluated using the protocol. Also demonstrated is the new method's compatibility with three different mass spectrometry-based readouts including an intact protein readout using MALDI, a gel-based proteomics readout using MALDI, and an LC-MS-based proteomics readout using isobaric mass tags. The results of the cell lysate sample analysis highlight the complementarity of the labeling protocol to other chemical modification strategies that have been recently developed to make thermodynamic measurements of protein folding and stability on the proteomic scale.

Over 7 million liters of Corexit® series dispersants were applied in the Gulf of Mexico during the 2010 Deepwater Horizon oil spill to facilitate the dispersion of crude oil into the water column. At the time of application, the composition, fate, and transformation kinetics of the surfactants in Corexit®9500 were relatively unknown. Recent advances in high-resolution mass spectrometry, such as resolving power, mass accuracy, and acquisition rates, have allowed for comprehensive characterization of complex surfactant mixtures in environmental matrices. The objective of this dissertation was to develop a method for comprehensive characterization of Corexit in the marine environment using high-resolution mass spectrometry. Specifically, I assessed the kinetic and biodegradation rates and transformation products of Corexit®9500 in under environmentally relevant conditions.

In Chapter 2, I identified individual nonionic polysorbate surfactants in Corexit using ultra high-resolution mass spectrometry. The method allowed for greater confident in structural assignment and systematic differentiation of isobaric and isomeric compounds. I examined the heterogeneity of Corexit based on differences in the hydrophilic core and hydrophobic tail groups. Specifically, the nonionic surfactants exhibited variability in the degree of esterification, fatty acid chain length and saturation as well as different core groups and ethoxymer distribution. The composition of Corexit has implications on the utility, fate, and persistence during an oil spill emergency response.

In Chapter 3, I investigated Corexit degradation under biotic and abiotic conditions using temperate seawater collected from the Pivers Island Coastal Observatory in Beaufort, NC. Corexit degraded under both biotic and abiotic conditions, although biodegradation rates were two to four times faster. Kinetic degradation rates were highly dependent on the degree of esterification and fatty acid chain length. However, they were not dependent on the core group, fatty acid saturation, or degree of ethoxylation. The only observed transformation products were the nonesterified ethoxylates generated by ester hydrolysis. Abiotic hydrolysis rates were also dependent on temperature. Increased temperature had a greater influence on the monoester surfactants degradation rates than surfactants that contained two or more ester moieties. I determined the kinetic degradation rates for individual surfactants, ethoxymer series, and ester components and compared degradation rates to polysorbate mixtures.

In Chapter 4, I examined the impact of crude oil on Corexit partitioning and degradation in Arctic seawater collected from the Chuckchi Sea. Components of Corexit did not partition into the crude oil layer based on surfactant hydrophobicity. Instead, the total abundance of Corexit increased in the water column in the presence of dispersed oil. The transformation products detected were the nonesterified ethoxylates, which suggested ester hydrolysis was the main pathways of degradation. The biodegradation rates of the ester components were slightly faster with dispersed oil due to the greater concentrations in the water column. In addition, observed abiotic hydrolysis rates at cold temperatures agreed with estimated rates calculated from the kinetic variable determined in Chapter 3. Overall, Corexit followed the same degradation pathway with slower transformation kinetics in Artic seawater in the presence and absence of crude oil.

Finally, Chapter 5 presented results from the microbial community analysis in the Corexit only and Corexit dispersed crude oil treatments. In the presence of Corexit, Bacteroidetes dominated the microbial community within 24-hours. Bacteroidetes were also present of dispersed oil and untreated water, however, in lower abundance. We did not observe a dramatic change in the microbial structure after the monoester component of Corexit was removed after 24-hours. The communities maintained a large degree of diversity in the presence of Corexit and dispersed crude oil, however after 5 days Proteobacteria (specifically Oceanospirillales and Altermonadales) and Flavobacterilia accounted for 75% of the community in the Corexit treatment. The inclusion of both dispersant and oil had significant effects on microbial community structure, which can be correlated to the degradation of dispersants.

High resolution mass spectrometry analysis allowed me to perform a complete characterization of Corexit and its related polysorbates, as well as determine the degradation rates and identify transformation products under various environmental conditions. I detected individual ethoxymers and evaluated the heterogeneity of the mixtures as well as the kinetic rates of degradation. I showed that the components of Corexit degrade on varying time scales based on structural similarities. All nonionic components of Corexit were removed within 10 days in temperate seawater and after 20 days in Arctic seawater. These studies are among the most comprehensive analysis of nonionic polysorbate surfactants under environmental conditions and will help to profile other dispersants and inform the application of Corexit series dispersants in future oil spill disasters.

Semi-volatile organic compounds (SVOCs) are used in consumer products in a wide variety of applications such as flame retardants, plasticizers, pesticides, preservatives, and fragrances. Due to their extensive use in everyday products, SVOCs are widely detected in indoor environments, and human exposure is common and often chronic. As the wealth of toxicological data examining the negative health impacts of these compounds grows, the need for reliable tools to accurately measure human exposure becomes increasingly more crucial. In the past few decades, external exposure to these compounds have been evaluated through measurements in indoor air, house dust, and hand wipes, all of which have been shown to be associated with internal dose (e.g., concentrations in urine or blood). However, there are significant limitations to using each of these approaches to characterizing exposure. In recent years, silicone wristbands have been used as personal passive samplers for evaluating ambient exposure to a wide array of consumer product and industrial compounds. While over a thousand chemicals have been reported to be detected on the wristbands, very few studies have measured the concentrations on wristbands and determined how well they correlate to established biomarkers of exposure. This dissertation research sought to evaluate the use of silicone wristbands for measuring personal exposure to three classes of SVOCs- organophosphate esters (OPEs), brominated flame retardants (BFRs), and phthalates. The central hypothesis is that wristbands are an effective tool for evaluating personal exposure to SVOCs and provide more accurate measures of exposure compared to tools currently in use.

Within the first aim of this dissertation research, paired samples of polyurethane foam (collected from sofas), house dust, and serum were analyzed for flame retardants (FRs) chemicals and associations were evaluated. The detection of two FR mixtures, PentaBDE and FM 550, in foam was significantly associated with 4 to 6.5 times as high concentrations of their primary components in house dust (p<0.01). These relationships were modified by the size of the sofa footprint within the room and dust-loading rates. PentaBDE in foam was also associated with higher levels of individual PBDE congeners in serum, particularly two of the primary congeners BDE-47 and -153. Participants who lived in a home with a sofa containing PentaBDE had serum BDE-47 levels that were 2.5 times as high as participants whose sofa did not contain PentaBDE (p<0.01). This study was the first to relate a specific FR application in a consumer product with house dust and a known biomarker of exposure.

For the second aim of this research, adult exposure to OPEs and BFRs were evaluated using silicone wristbands. OPEs quantified on the wristbands were significantly associated with metabolites from pooled urine samples, and polybrominated diphenyl ethers (PBDEs) on the wristbands were similarly correlated to PBDE levels in serum (rs=0.4-0.6, p<0.05). Several novel BFR compounds which lack verified biomarkers of exposure were also measured on the wristbands and reported for the first time. These two studies were the first to evaluate FR concentrations on wristbands with known biomarkers and represent two of now four published manuscripts providing evidence that measurements on wristbands are predictive of internal dose.

In the third aim of this research, children’s exposure to OPEs, phthalates, and BFRs were examined using silicone wristbands. The ability of the wristband measurements to predict urinary metabolite levels of OPEs and phthalates was compared to that of hand wipes and house dust. Across the three classes, the children’s wristband concentrations were positively and significantly associated with a number of their corresponding biomarkers in both urine and serum, similar to observations in our adult cohort. For OPEs, phthalates, and PBDEs, the wristbands were found to have similar or an improved utility, compared to hand wipes and dust, for evaluating children’s exposures to these compounds. For instance, one of the OPEs, 4-tertbutylphenyl diphenyl phosphate (4tBPDPP), on wristbands was more strongly correlated to its urinary metabolite, tert-butyl phenyl phenyl phosphate (tb-PPP), compared to that on hand wipes and dust (rs=0.35, p<0.01, compared to rs=0.16 and 0.05 for hand wipes and dust, respectively). For the phthalate benzyl butyl phthalate (BBP), wristbands and hand wipes were similar associated with the urinary metabolite, mono benzyl phthalate (MBzP), but both were stronger than the dust correlation (rs=0.56 for wristbands and hand wipes, p<0.001; rs=0.23 for dust, p<0.05). Similar results were observed among the PBDEs on the three exposure mediums and their serum biomarkers, although the magnitudes of correlation with serum were more similar for wristbands and dust.

Taken together this dissertation research provides some of the first insights on the evaluation of personal exposures to SVOCs using silicone wristbands. It includes six distinct studies evaluating human exposure to sixty-five chemicals from three classes of compounds. Further, this research offers novel contributions to the field of exposure science, evaluating the relationship between wristbands and established biomarkers of exposure and comparing them to the existing tools used in standard exposure assessments. Wristbands have the potential to serve as an inexpensive and non-invasive medium for evaluating human exposure to chemical mixtures, and this work provides support for their use in large-scale research efforts to characterize SVOC exposures. Additional research should continue to assess wristbands for their ability to measure meaningful exposures for additional classes of chemicals, and importantly, identify the pathways of exposure (e.g., dermal absorption, inhalation, etc.) that are captured by the wristbands.

Detection and quantification of protein-ligand binding interactions is extremely important for understanding interactions that occur in biological systems. Since traditional techniques for characterizing these types of interactions cannot be performed in complex systems such as cell lysates, a series of energetics-based techniques that are capable of assessing protein stability and measuring ligand binding affinities have been developed to overcome some of the limitations of previous techniques. Now that the capabilities of the energetics-based techniques have been exhibited in model systems, the false-positive rates of the techniques, the range of biological questions to which the techniques can be addressed, and the use of the techniques to discover novel interactions in unknown systems remained to be shown. The Stability of Proteins from Rates of Oxidation (SPROX) technique and the Pulse Proteolysis (PP) technique were applied to a wide range of biological questions in both yeast and human cell lysates to evaluate the scope of these experimental workflows. The false-positive rate of iTRAQ-SPROX protein target discovery on orbitrap mass spectrometer systems was determined to be < 0.8 %. The iTRAQ-SPROX technique was successfully applied to the discovery of both known and novel protein-protein, protein-ATP, and protein-drug interactions, leading to the quantification of protein-ligand binding affinities in each of these studies. In the pursuit of discovering geldanamycin protein interactors, the use of iTRAQ-SPROX and SILAC-PP in combination was determined to be advantageous for confirming protein-ligand interactions since the techniques utilize different quantitation strategies that are subject to separate technical errors in quantitation. Finally, the iTRAQ-SPROX and SILAC-PP techniques were used to evaluate the interactions of manassantin A in a human cell lysate. In this work, a previously unknown protein target of manassantin A, Filamin A, was detected as a hit protein using both the iTRAQ-SPROX and SILAC-PP protocols. The work completed in this dissertation has expanded the understanding of the limitations of energetics-based techniques and shown that biological replicate analyses are essential to confirm ligand interactions with novel protein targets.

Following the phase-out of polybrominated diphenyl ethers (PBDEs) in the early 2000s, organophosphate esters (OPEs) emerged as PBDE substitutes and have been applied to furniture foam, electronics, building materials, and some plastics to reduce their flammability. Although they have been used for quite some time in hydraulic fluids, isopropylated and tert-butylated triaryl phosphate esters (ITPs & TBPPs) have been more recently introduced as flame retardant (FR) replacements for the pentaBDE mixture in polyurethane foam (PUF). In addition to their use as FRs, ITPs and TBPPs are also used as plasticizers.

ITPs and TBPPs comprise a family of aryl organophosphate esters with varying degrees of isopropylation and tert-butylation. Individual ITP and TBPP isomers have been widely detected in indoor house dust, and recent biomonitoring studies demonstrate that human exposure to these compounds is widespread. Due to concerns about their persistence, bioaccumulation, and potential toxicity (P, B, & T), the U.S. Environmental Protection Agency (EPA) listed ITPs as one of five high priority chemicals fast-tracked for expedited risk assessment under the 2016 Toxic Substances Control Act (TSCA) reform.

As such, studying the exposure, metabolism, and in vitro effects of these compounds is especially timely. The hypothesis of this research dissertation is that ITP and TBPP isomers may exhibit some of the same P, B, & T properties that motivated the phase out of PBDEs. The main objectives of this research project were to generate meaningful data to fill gaps in our knowledge of ITP and TBPP isomers, and to contribute to the ongoing risk assessment of these compounds.

In the first aim of this thesis research, the maternal transfer of Firemaster® 550 (FM 550), a commercial mixture containing ITP isomers and brominated FRs, was investigated in dosed Wistar rats. Gestational and lactational transfer were examined separately, with dams orally exposed to 300 or 1000 µg of FM 550 for 10 consecutive days during gestation (gestational day [GD] 9-18) or lactation (postnatal day [PND] 3-12). Levels of parent compounds were measured in dam and pup urine. The two brominated components of FM 500, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis (2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP), underwent both gestational and lactational transfer. Triphenyl phosphate (TPHP) and ITPs were rapidly metabolized by the dams and were not detected in whole tissue homogenates. However, diphenyl phosphate (DPHP) and mono-isopropylphenyl phenyl phosphate (ip-PPP) were detected in urine from the dosed animals. This study was the first to confirm ip-PPP as a urinary metabolite of ITPs and establish a pharmacokinetic profile of FM 550 in a mammalian model.

In the second aim of this thesis research, the contribution of individual ITP and TBPP isomers was quantified in four commercial flame retardant mixtures: FM 550, Firemaster® 600 (FM 600), an ITP mixture, and a TBPP mixture. Findings suggested similarities between FM 550 and the ITP mixture, with 2-isopropylphenyl diphenyl phosphate (2IPPDPP), 2,4-diisopropylphenyl diphenyl phosphate (24DIPPDPP), and bis(2-isopropylphenyl) phenyl phosphate (B2IPPPP) being the most prevalent ITP isomer in both mixtures. FM 600 differed from FM 550 in that it contained TBPP isomers rather than ITP isomers. ITP and TBPP isomers were also detected and quantified in house dust standard reference material, SRM 2585, demonstrating their environmental relevance.

The third aim of this thesis research investigated phase I and II in vitro metabolism of TPHP, 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) at 1 and 10 µM doses using human liver subcellular fractions. Parent depletion and the formation of known metabolites, including DPHP, hydroxyl-triphenyl phosphate (OH-TPHP), ip-PPP, and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored via gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS). Tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP, accounting for up to 33% of the initial parent dose. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry (HRMS) and screened for in recently collected human urine. This study provided insight into recent human biomonitoring and epidemiological studies and contributed to our understanding of the biological fate of ITP and TBPP isomers.

Finally, the fourth aim of this thesis research evaluated ITPs, TBPPs, and related commercial mixtures for their effect on the activity of purified human liver carboxylesterase (hCE1). Four of the 15 OPEs tested had IC50 values lower than 100 nM, including TPHP, 2-ethylhexyl diphenyl phosphate (EHDPP), 4-isopropylphenyl diphenyl phosphate (4IPPDPP), and 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), as did four commercial flame retardant mixtures tested. Because hCE1 is critical for the activation of imidapril, an ACE-inhibitor prodrug prescribed to treat hypertension, the most potent inhibitors, TPHP and 4tBPDPP, and an environmentally relevant mixture (house dust) were further evaluated for their effect on imidapril bioactivation in vitro. TPHP and 4tBPDPP were potent inhibitors of hCE1-mediated imidapril activation (Ki = 49.0 and 17.9 nM, respectively), as were extracts of house dust (100 µg/ml), which caused significant reductions in imidapril activation. Combined, these data suggest that exposure to OPEs can affect pharmacotherapy.

Collectively and in context of other recently published findings, this thesis research suggests that ITPs and TBPPs may be regrettable substitutes for PBDEs.

Human activities represent a significant burden on the global water cycle, with large and increasing demands placed on limited water resources by manufacturing, energy production and domestic water use. In addition to changing the quantity of available water resources, human activities lead to changes in water quality by introducing a large and often poorly-characterized array of chemical pollutants, which may negatively impact biodiversity in aquatic ecosystems, leading to impairment of valuable ecosystem functions and services. Domestic and industrial wastewaters represent a significant source of pollution to the aquatic environment due to inadequate or incomplete removal of chemicals introduced into waters by human activities. Currently, incomplete chemical characterization of treated wastewaters limits comprehensive risk assessment of this ubiquitous impact to water. In particular, a significant fraction of the organic chemical composition of treated industrial and domestic wastewaters remains uncharacterized at the molecular level. Efforts aimed at reducing the impacts of water pollution on aquatic ecosystems critically require knowledge of the composition of wastewaters to develop interventions capable of protecting our precious natural water resources.

The goal of this dissertation was to develop a robust, extensible and high-throughput framework for the comprehensive characterization of organic micropollutants in wastewaters by high-resolution accurate-mass mass spectrometry. High-resolution mass spectrometry provides the most powerful analytical technique available for assessing the occurrence and fate of organic pollutants in the water cycle. However, significant limitations in data processing, analysis and interpretation have limited this technique in achieving comprehensive characterization of organic pollutants occurring in natural and built environments. My work aimed to address these challenges by development of automated workflows for the structural characterization of organic pollutants in wastewater and wastewater impacted environments by high-resolution mass spectrometry, and to apply these methods in combination with novel data handling routines to conduct detailed fate studies of wastewater-derived organic micropollutants in the aquatic environment.

In Chapter 2, chemoinformatic tools were implemented along with novel non-targeted mass spectrometric analytical methods to characterize, map, and explore an environmentally-relevant “chemical space” in municipal wastewater. This was accomplished by characterizing the molecular composition of known wastewater-derived organic pollutants and substances that are prioritized as potential wastewater contaminants, using these databases to evaluate the pollutant-likeness of structures postulated for unknown organic compounds that I detected in wastewater extracts using high-resolution mass spectrometry approaches. Results showed that application of multiple computational mass spectrometric tools to structural elucidation of unknown organic pollutants arising in wastewaters improved the efficiency and veracity of screening approaches based on high-resolution mass spectrometry. Furthermore, structural similarity searching was essential for prioritizing substances sharing structural features with known organic pollutants or industrial and consumer chemicals that could enter the environment through use or disposal.

I then applied this comprehensive methodological and computational non-targeted analysis workflow to micropollutant fate analysis in domestic wastewaters (Chapter 3), surface waters impacted by water reuse activities (Chapter 4) and effluents of wastewater treatment facilities receiving wastewater from oil and gas extraction activities (Chapter 5). In Chapter 3, I showed that application of chemometric tools aided in the prioritization of non-targeted compounds arising at various stages of conventional wastewater treatment by partitioning high dimensional data into rational chemical categories based on knowledge of organic chemical fate processes, resulting in the classification of organic micropollutants based on their occurrence and/or removal during treatment. Similarly, in Chapter 4, high-resolution sampling and broad-spectrum targeted and non-targeted chemical analysis were applied to assess the occurrence and fate of organic micropollutants in a water reuse application, wherein reclaimed wastewater was applied for irrigation of turf grass. Results showed that organic micropollutant composition of surface waters receiving runoff from wastewater irrigated areas appeared to be minimally impacted by wastewater-derived organic micropollutants. Finally, Chapter 5 presents results of the comprehensive organic chemical composition of oil and gas wastewaters treated for surface water discharge. Concurrent analysis of effluent samples by complementary, broad-spectrum analytical techniques, revealed that low-levels of hydrophobic organic contaminants, but elevated concentrations of polymeric surfactants, which may effect the fate and analysis of contaminants of concern in oil and gas wastewaters.

Taken together, my work represents significant progress in the characterization of polar organic chemical pollutants associated with wastewater-impacted environments by high-resolution mass spectrometry. Application of these comprehensive methods to examine micropollutant fate processes in wastewater treatment systems, water reuse environments, and water applications in oil/gas exploration yielded new insights into the factors that influence transport, transformation, and persistence of organic micropollutants in these systems across an unprecedented breadth of chemical space.

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Unfortunately, these studies have not been as useful as expected at identifying disease-related proteins that can be exploited for diagnostic and therapeutic purposes, presumably due to the indirect link between a protein’s expression level and its function. Investigated here is the use of thermodynamic stability measurements to probe a more biologically relevant dimension of the proteome. It has the potential to become a new strategy for disease state characterization and to help elucidate the molecular basis of the disease. This thesis outlines the use of two discovery based techniques and one validation based technique to study protein folding and stability changes associated with breast cancer.

The first part of this dissertation describes the application of a mass spectrometry-based technique, stable isotope labeling with amino acids in cell culture and stability of proteins from rates of oxidation (SILAC-SPROX), in a comparison of the MCF-7 versus BT-474 breast cancer cell lines and a comparison of the MCF-7 versus MDA-MB-468 breast cancer cell lines. This work enabled ~1000 proteins to be assayed for breast cancer-related thermodynamic stability differences. The 242 and 445 protein hits identified with altered stabilities in these comparative analyses created distinct molecular markers to differentiate the three cell lines.

The second part of this dissertation describes the development of a SILAC-based limited proteolysis (SILAC-LiP) strategy. The applicability of the protocol was demonstrated in a proof-of-principle study using proteins from a yeast cell lysate and a ubiquitous ligand. The SILAC-LiP protocol was further applied in a comparison of the MCF-7 versus MCF-10A cell lines. This work identified ∼200 proteins with cell line dependent conformational changes, as determined by their differential susceptibility to proteolytic digestion using the nonspecific protease, proteinase K. The overlap between the SILAC-LiP hits reported here and the SILAC-SPROX hits previously identified in these same cell lines was relatively small (~20%). Thus, this work indicates that the SILAC-SPROX and SILAC-LiP techniques can be used together to provide complementary information on the disease states.

Furthermore, the protein hits identified in both the SILAC-SPROX and SILAC- LiP experiments included a large fraction (∼70%) with no significant expression level changes. This suggests protein folding and stability measurements can provide information about disease states that is orthogonal to that obtained in protein expression level analyses.

The last part of this dissertation focuses on the establishment of targeted mass spectrometry-based validation assays for the protein biomarker candidates with altered thermodynamic stabilities identified in the SILAC-SPROX experiments. Application of the PAB-SPROX protocol on the MCF-7 cell lysate enabled reproducible identification and quantitation of a subset of prioritized target peptides.

Over the past two decades, a toolbox of mass spectrometry-based proteomic methods has been developed that enables the conformational properties of proteins and protein-ligand complexes to be probed in complex biological mixtures, from cell lysates to whole cells. The focus of this dissertation is the extension of these methodologies to the study of protein-gas and protein-metal interactions, an area of limited application. The goals of this work are two-fold. The first is to improve current mass spectrometry-based proteomic methods that measure protein folding stability, which is accomplished by the development of a chemo-selection strategy for proteolysis procedures and a “one-pot” approach that increases statistical significance while decreasing experiment costs. The second goal of this work is the application of these methodologies and others to the study of protein-gas and protein-metal interactions in complex biological mixtures (i.e., cell lysates), in which insights could be gained about gas and metal biological activities by surveying their interactions within a proteome. The first part of this dissertation describes in more detail the development and application of a semitryptic peptide enrichment strategy for proteolysis procedures (STEPP) that enables the isolation of information-rich semitryptic peptides. With the STEPP protocol, the number of semitryptic peptides increased by 5- to 10-fold and the amount of structural information was maximized in limited proteolysis experiments. The combination of the pulse proteolysis technique with a novel “one-pot” approach for data acquisition and analysis (one-pot STEPP-PP), resulted in false positive rates reaching close to zero (i.e., 0.09%) for a proof-of-principle drug target identification experiment for cyclosporine A and a yeast lysate. Described in the second part of this dissertation is the application of the improved proteolysis methodologies and others to multiple studies of protein-gas and protein-metal interactions on the proteomic scale. First, the development and application of protein stability measurements to the study of protein-gas interactions, specifically protein-xenon interactions, is described. A sample preparation protocol that was conducive to protein-gas binding studies is developed and validated against a known xenon-binding protein, metmyoglobin. Ultimately, this sample preparation protocol was employed in large-scale, proteome-wide SPROX and limited proteolysis experiments to identify xenon-interacting proteins in a yeast lysate. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known. Our survey of the proteome revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes and revealed correlations between the mechanisms by which ATP and Xe target proteins. Next, the application of one-pot STEPP-PP is described in the context of two research areas, both related to identifying the protein targets of metal-associated cell death processes. First described is the utilization of this technique in combination with protein expression level analysis to identify bacterial protein targets of copper delivered by small molecule ionophores. The protein folding stability and expression level profiles generated in this work enabled the effects of ionophore vs. copper to be distinguished and revealed copper-driven stability changes in proteins from processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (by 3-fold) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies. The second application of the one-pot STEPP-PP methodology is to the study of Fe- and Zn-mediated sensitization to erastin-induced ferroptotic cell death. Our approach enabled differential protein expression and protein folding stability measurements to be made on RCC4 cells exposed to excess iron and zinc along with the ferroptosis-inducing molecule erastin. Of the protein targets identified, a few have known ties to pathways involved in ferroptotic cell death, while others have not been previously linked with ferroptosis. Future work aims at assaying the potential metal binding properties of these proteins, to connect them to their metal-enhancing ferroptosis effects. The final research area described in this dissertation is the development and application of a novel metal-induced protein precipitation (MiPP) approach which exploits the protein precipitation properties of metals to study proteins that are susceptible to metal overload. Total protein precipitation as a function of metal concentration was assayed across various proteomes (bacterial, fungal, and mammalian) and metals (copper, zinc, iron, etc). Copper-induced protein precipitation was measured within E. coli and C. albicans proteomes by coupling precipitation curves with a bottom-up proteomics readout. Proteome-wide precipitation studies revealed a wide distribution of copper precipitation midpoints for the identified proteins within these species. A fundamental understanding of the biophysical basis of susceptibility or tolerance to metal precipitation can potentially be garnered through more in-depth analysis of the proteins that fall significantly outside the average precipitation midpoint of each proteome.

The detection and quantitation of protein-ligand binding interactions is important not only for understanding biological functions but also for the characterization of novel protein ligands. Because protein ligands can range from small molecules to other proteins, general techniques that can detect and quantitate the many classes of protein-ligand interactions are especially attractive. Additionally, the ability to detect and quantify protein-ligand interactions in complex biological mixtures would more accurately represent the protein-ligand interactions that occur in vivo, where differential protein expression and protein complexes can significantly affect a protein's ability to bind to a ligand of interest.

The work in this dissertation is focused on the development of new methodologies for the detection and measurement of protein-ligand interactions in complex mixtures using multiplex analyses. Methodologies for two types of multiplexed analyses of protein-ligand binding interactions are investigated here. The first type of multiplex analysis involves characterizing the binding of one protein target to many potential ligands, and the second type involves characterizing the binding of one ligand to many proteins. The described methodologies are derived from the SUPREX (stability of unpurified proteins from rates of H/D exchange) and SPROX (stability of proteins from rates of oxidation) techniques, which are chemical modification strategies that measure thermodynamic stabilities of proteins using a relationship between a protein's folding equilibrium and the extent of chemical modification. These two techniques were utilized in the development and application of several different experimental strategies designed to multiplex the analysis of protein-ligand interactions.

The first strategy that was developed involved a pooled compound approach for making SUPREX-based measurements of multiple ligands binding to a target protein. Screening rates of 6 s/ligand were demonstrated in a high-throughput screening project that involved the screening of two chemical libraries against human cyclophilin A (CypA), a protein commonly overexpressed in types of cancer. This study identified eight novel ligands to CypA with micromolar dissociation constants. Second, an affinity-based protein purification strategy was developed for the detection and quantitation of specific protein-ligand binding interactions in the context of complex protein mixtures. It involved performing SPROX in cell lysates and selecting the protein of interest using immunoprecipitation or affinity tag purification. A third strategy developed here involved a SPROX-based stable isotope labeling method for measuring protein-ligand interactions in multi-protein mixtures. This strategy was used in a proof-of-principle experiment designed to detect and quantify the indirect binding between yeast cyclophilin and calcineurin in a multi-component protein mixture. Finally, a quantitative proteomics platform was developed for the detection and quantitation of protein-ligand binding interactions on the proteomic scale. The platform was used to profile interactions of the proteins in a yeast cell lysate to several ligands, including the bioactive small molecules resveratrol and manassantin A, the cofactor nicotinamide adenine dinucleotide (NAD+), and two proteins, phosphoglycerate kinase (Pgk1) and pyruvate kinase (Pyk1). The above approaches should have broad application for use as discovery tools in the development of new therapeutic agents.

This work examines the characterization of supramolecular assemblies and, more specifically, host-guest complexes involved in molecular recognition events. The supramolecular assemblies studied take root from metal ion delivery in biological uptake pathways, specifically the delivery of iron to microbial cells. These assemblies are studied in an effort to further understand the nature of molecular recognition events, specifically the nature and strength of interactions between a host and a guest, and possible applications of these systems.

The development of a mass spectral method by which to characterize supramolecular assemblies involving the cation binding hosts 18-crown-6, benzo-18-crown-6, dicyclohexano-18-crown-6, and dibenzo-18-crown-6 macrocycles, and the linear ionophore lasalocid with cationic guests, including substituted protonated amines and the iron siderophore ferrioxamine B is presented. Methodology was developed using ESI-MS to successfully quantitate host-guest interactions in binary and complex mixtures. Binding constants were obtained in the range of log Ka = 3 - 5 and correspond to similar systems previously studied in the literature. The studies presented here further our understanding of the molecular recognition events that must occur between a siderophore and a receptor and provide an improved method by which to measure the strength of their interaction.

The effects of redox hosts on host-guest complex formation with ferrioxamine B and the characterization of the host-guest complexes formed and the strength of the interactions between them were studied using cyclic voltammetry, ESI-MS, FAB-MS and ITC. A shift in redox potential towards more positive values is observed upon addition of a cationic siderophore guest to a solution of a redox-active para-Wurster's aza crown or mono-substituted Wurster's aza crown macrocycle. Mass spectral evidence indicates the formation of a host-guest complex between the cationic siderophore and the redox host. A redox switch mechanism is proposed, whereby the redox state of the host influences the binding affinity between the host and guest and, consequently, host-guest complex formation. These systems offer a unique means by which to modulate the uptake or release of ionic guests from a cavity by using externally controlled methods and can be applied to selective metal ion compartmentalization.

Finally, the application of supramolecular assemblies as a tool in the field of drug delivery is presented. The covalent attachment of an antimalarial drug, artemisinin, by our collaborators to a siderophore produced by M. Tuberculosis, mycobactin, facilitates the subsequent delivery of the drug into the microbial cell by taking advantage of the natural biological iron uptake pathway. Here, the molecular recognition event and supramolecular assembly of interest is that occurring between the siderophore-drug assembly and the microbial receptor. Characterization of the siderophore-drug assembly using cyclic voltammetry shows that there is an interaction between the Fe-mycobactin and artemisinin when these are covalently attached in the form of a conjugate. Increased current output is observed due to an intramolecular electron transfer between the two components. Based on these in vitro data, we propose a redox mechanism by which the drug-siderophore conjugate exhibits a therapeutic effect in vivo.

In the last 10 years, several mass spectrometry-based proteomic techniques have been developed for the large-scale characterization of protein conformations, thermodynamic stabilities, and protein−ligand interactions. The main focus of this dissertation involves the development and application of several mass spectrometry-based-methods in this current suite of proteomics techniques for the large-scale analysis of protein folding and stability measurement. One goal of this work is to investigate the use of protein folding and stability measurements to better detect and understand the biophysical properties of post-translational modifications. Another goal of this work is to develop a novel protein stability measurement technique for making thermodynamic measurements of protein folding and ligand binding interactions. This technique, which involves a combination chemical denaturant and protein precipitation yields significantly better proteomic coverage and a largely reduced false discovery rate compared to its sister technique, Stability of Proteins from Rates of Oxidation (SPROX).

The first part of this dissertation describes the application of the stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) on comparing the conformational properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. A total of 168 and 251 protein hits were identified with dephosphorylation-induced stability changes using the SPROX and LiP techniques, respectively. The SPROX results revealed that the magnitudes of the destabilizing effects of dephosphorylation on the different aaRSs were directly correlated with their previously reported aminoacylation activity change upon dephosphorylation. The example of these aaRSs substantiates the close link between protein folding thermodynamic and function and helps establish the utility of thermodynamic stability measurements for understanding protein function.

The second part of this dissertation describes the development of a new protein-stability based proteomic method for identification and quantification of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔGf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein−drug binding affinities. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast lysate. The technique was also used to identify protein targets of sinefungin in a human MCF-7 cell lysate. The CPP technique yielded dissociation constant (Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values.

The third part of this dissertation describes two protein target discovery applications of the CPP approach including one involving subglutinol A (a natural product with immunosuppressive activity) and one involving clemastine fumarate (an existing anti-histamine drug with recently discovered anti-malarial activity). As part of this work, about 800 proteins in a mouse 2B4 T cell lysate were assayed for subglutinol A-induced stability changes, and deoxycytidine kinase was identified as the protein hit. The magnitude of the ligand induced stability change was used to calculate a Kd value of 250 M, which is close to the reported cell based IC50. In the protein target discovery study on clemastine fumarate, a total of 800 yeast proteins were assayed for drug-induced stability changes and 8 protein with clemastine-induced stability changes were identified, including SEC14 cytosolic factor, glycerol kinase, asparagine--tRNA ligase, and inosine triphosphate pyrophosphatase. The latter two applications demonstrate that CPP can reliably identify and quantify protein-drug interactions in a complex biological mixture, making it a valuable addition to the current suite of proteomic methods for the large-scale detection and quantitation of protein-ligand binding interactions.

Polycyclic aromatic hydrocarbons (PAHs) are compounds ubiquitous in the environment and are harmful to human health. PAHs can easily enter the human body through the consumption of edible oils due to their high lipophilicity. Most of the published papers investigating PAH concentrations in edible oils focused on virgin (i.e., unheated) samples instead of those being used in cooking at temperatures higher than room temperature. This study aimed to provide a comprehensive landscape of PAH concentration in soybean oil by simulating a more realistic scenario of oil consumption—eating oil being cooked at different temperatures. I quantified the concentration of PAHs in soybean oil after being heated to four temperatures (100°C, 150°C, 190°C, and 222°C) with three durations (5 minutes, 30 minutes, and 60 minutes). Liquid liquid extraction (LLE), tandemly followed by solid phase extraction (SPE), were used to extract PAHs from the matrix and remove interferences from the extract. PAH concentrations were determined by a gas chromatography mass spectrometer (GC-MS). With extended heating time, concentrations of ACE and FLU showed a rising trend in 100°C and 150°C data groups but a falling trend at higher temperature data groups. Concentrations of PHEN and ANTH had a sharp increase when the oil was heated to 222°C and held one hour. Concentrations of B[a]A, CHRY, and B[a]P rose slightly with extended heating time at almost all temperatures. Concentrations of ACY, FLTH, B[b]F, B[k]F, IND, D[ah]A, and B[ghi]P were low and nearly undetectable. Only NAP concentrations in 100°C-oil samples were over the maximum residue level (MRL) set by China’s national standard. Diverse concentration changes of different PAHs after the oil was heated suggested that B[a]P alone or a combination of several PAHs are not sufficiently representative to be the marker or surrogate for PAH exposure. This diversity also resulted in an undetermined correlation between the PAH remaining in the oil and heating temperatures and hold time. The information of PAHs increment caused by heating soybean oil was incomplete because of data unavailability for the vaporized part.

Nanoceria, or cerium (IV) oxide, is used widely in industry for its catalytic and physical properties, thus its release into the environment is eminent. The environmental risk of nanoceria is still unclear, but understanding its environmental fate could help to inform future studies. To understand its fate, large scale simulated wetlands were constructed and dosed weekly. Nanoceria of primary particle sizes ~4nm (sm-CeO2) and ~140nm (lg-CeO2) were dosed into these mesocosms, 750 mg total over nine months. Single particle ICP-MS (spICP-MS) was employed with microsecond dwell times (0.1ms) to understand particulate cerium in surface water. spICP-MS proved ineffective as a comparison tool between treatments, since sm-CeO2 was below instrument detection, and further data processing could not amend the issue. However, comparisons between nanoceria stocks and mesocosm samples could be conducted. Mesocosm water dosed with lg-CeO2 exhibited some aggregation of its smaller fraction.

At the end of 9-months, elemental analyses showed that nanoceria size did not affect the mass of cerium in surface water, as mesocosms treated with lg-CeO2 contained 4.0 ± 1.4 mg of Ce and sm-CeO2 treated waters contained 5.6± 6.7mg Ce. Greater biological uptake did occur when treated with smaller particles, as shown in elevated root concentration (sm-CeO2: 67 ± 14 ng g-1 ; lg-CeO2: 21 ± 10 ng g-1) and total Ce in Egeria densa biomass (sm-CeO2: 22 ± 1.4 ; lg-CeO2: 2.9 ± 0.5 mg). The environmental compartments in this study accounted for a small fraction of dosed nanoceria (< 5%), so assumed nanoceria fate is the sediment.