Thesis advisor: Jianmin . Gao / Cysteine residues perform many essential cellular functions, including nucleophilic and redox catalysis, metal coordination, structural stabilization and cellular protection. Cysteine-related mutations are oftentimes related to diseases due to the amino acid’s functional importance. This has led cysteine to become a focus of small molecule drug discovery. A comparison of the cysteine proteome of diseased cells versus healthy cells can elucidate novel cysteine residues that play an important role in progressing the disease state. Two disease models were chosen to be the focus of this proteomic study; breast cancer through the human epithelial MCF10 progression series and immunoactivation through the Raw 246.7 mouse macrophage cell line. Comparative proteomics with mass spectrometry revealed several changes within the cysteine proteome when the cells were diseased. Some cysteines had changes in reactivity, most likely indicating a loss or gain of a modification or disulfide bond. Other cysteines showed increased labeling due to an increase in the overall expression of the protein encompassing the cysteine residue. Further follow-up of an interesting hit from the Raw cell comparison, immune responsive gene 1 (IRG1), was conducted. IRG1 produces itaconate from cis-aconitate under inflammatory conditions, disrupting the citric acid cycle. IRG1 was confirmed to have increased expression following activation of the macrophage cells by lipopolysaccharides. It was also successfully recombinantly expressed in and purified from Escherichia coli for use in an activity assay to determine if the cysteine labeled in the mass spectrometry experiment is essential for the protein function. With additional knowledge of cysteines that help progress disease states, new small molecule inhibitors can be developed to target these cysteines and impede the function that is beneficial for the disease. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Much important work has been done to understand reaction pathways and identify products, yields, and reaction rates for atmospheric oxidation processes. Non-methane hydrocarbons (NMHCs) are the most significant of the organic compounds present in the atmosphere from a chemical perspective and are released into the atmosphere from both natural and anthropogenic sources. The oxidation of these hydrocarbons by hydroxyl radical HO generates products that may themselves be toxic, that play a major role in the formation of photochemical smog, and that to a lesser extent contribute to the formation of acid precipitation. NMHCs have chemical reactivities many times that of methane, the most abundant HC in the atmosphere. However, the atmospheric oxidation processes of less than 50% of atmospheric NMHCs are known. A new experimental technique is needed that can provide insight into atmospheric oxidation products, reaction intermediates, and the relative importance of secondary reaction pathways that follow the initial attack of HO upon a hydrocarbon. The technique should operate at atmospheric pressure to better represent natural reaction processes and conditions, and provide a rapid and direct measure of product identities and yields. In this study we will describe the development and application of a technique that we believe meets these requirements, a technique we call High Resolution Kinetic Atmospheric Pressure Ionization Mass Spectrometry (HRKAPIMS). We begin with the use of atmospheric pressure ionization mass spectrometry in studies of atmospheric oxidation processes. We first describe a potential pitfall in the use of APIMS for the analysis of smog chamber experiments, a common APIMS application, discussing methods to eliminate interference reactions that would otherwise make interpretation difficult. A new experimental approach to the use of APIMS for the analysis of oxidation processes is next described and its use demonstrated. The oxidation of toluene by API source-generated HO produces oxidation products that are protonated and detected by the mass spectrometer. With this approach, we observe all the products found in a variety of previous studies employing a large array of experimental setups and analytical instrumentation. This is significant because our experiments are carried out in a far simpler experimental environment. Toluene is chosen for these experiments because it is an important constituent in polluted urban atmospheres with a complex oxidation mechanism that remains poorly understood. We describe the development of HRKAPIMS, a powerful new approach that allows the simultaneous detection of stable products along with free radical intermediates. The use of nitric oxide to affect product yields is demonstrated, giving valuable insights into reaction kinetics and mechanisms. We also address the theoretical aspects of HRKAPIMS, describing semiempirical calculations to estimate gas-phase basicities for a wide variety of compounds and discuss the errors implicit in this approach. The use of gas-phase basicities is discussed in terms of mass spectrometric analysis and analyte response. Kinetic and thermodynamic modeling is used to address the issues of APIMS and HRKAPIMS sensitivity and response and gain insights into the conditions necessary for linear response and quantitative detection of analytes.

Hemoglobin and serum albumin, two prevalent proteins in human blood, contain unbound cysteine thiol moieties, creating a nucleophilic site with the potential for covalent modification by reactive chemical species. These covalent modifications, called “adducts”, are stable entities that accumulate during acute and chronic exposure and remain covalently bound for the life-span of the protein. Despite their current use as exposure markers for a variety of compounds, the use of adducts in assessing exposure to drugs of abuse has not yet been explored. The goal of this work was to examine the in vitro adduct forming capability of selected drugs of abuse with hemoglobin to provide additional proof of principle for the development of a real-world detection and monitoring analysis method. This goal was accomplished by first analyzing the binding capabilities of the drugs of interest with glutathione, a smaller tripeptide. Use of protein adducts as biomarkers of drug exposure may allow for an increased window of detection, from several days to several months, as compared to current blood analysis methods. In total, there were 16 drugs analyzed in the research, and they covered a wide range of abused drugs, including cocaine, methamphetamine, and Δ 9 -THC. Results from the glutathione trials showed that 10 of the 16 the drugs of interest were able to form covalent adducts with the free thiol moiety, with four drugs forming more than one novel adduct. The MS results for hemoglobin showed 11 adducts formed for five of the drugs under investigation. Additional MS/MS confirmatory data was obtained for two of those 11 adducts. I successfully identifyied adducts formed between drugs of abuse and glutathione and hemoglobin, which have the potential to be used as long-term biomarkers of exposure.

Indiana University-Purdue University Indianapolis (IUPUI) / Paper spray mass spectrometry is an alternative technique for toxicological screening that is able to quickly and adequately screen for compounds encountered in postmortem investigations with little sample handling and no sample preparation. For analysis of dried blood spots using a triple quadrupole mass spectrometer, detection criteria were defined to align with relevant regulatory guidelines while considering how fragment ion selection, method sensitivity, and fragment ion ratio tolerances are best utilized in paper spray mass spectrometry. For analysis, drugs and drug metabolites relevant to postmortem investigations were spiked into drug-free blood, and by monitoring two fragment ion channels in selected reaction monitoring mode, as well as the ratio between the two fragment ions, a method was developed capable of detecting over 120 drug and drug metabolites at concentrations relevant to postmortem drug screening. Total analysis time for the developed method is less than 8 minutes, and less than 50µL of sample and 5mL of solvent are consumed during analysis.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy, School of Physics. Johannesburg, 2017. / Accelerator Mass Spectrometry (AMS) is an ultra-sensitive isotopic analysis technique that allows for the determination of isotopic ratios of rare long-lived radionuclides such as radiocarbon. AMS has become an important tool in many scientific disciplines, due to its sensitivity of detecting isotopic ratios at the level of 10-15 by making use of nuclear physics techniques and methods. The objective of the present work was to design and implement a new AMS system at iThemba LABS, the first of its kind on the African continent. The system is described in detail along with the relevant ion optics simulations using TRACE-3D. Beam optics calculations were performed for carbon isotopes, using the TRACE-3D code, in order to optimize the design of the new spectrometer and assess its overall performance. The AMS technique was applied in two unique South African research projects in relation to archaeology and environmental air pollution studies. The AMS technique, combined with the Proton-Induced X-Ray Emission (PIXE) technique, was also applied in an environmental study with respect to the contribution of contemporary and fossil carbon in air pollution in the Lephalale District, close to both the newly built Medupi coal-fired power station (~5 GW, the largest ever build in South Africa), and the existing Matimba coal-fired power station. The discrimination of contemporary carbon and fossil carbon is accomplished by using the AMS technique in measurements of the 14C/C ratios of aerosol particulate matter. The absence of 14C in fossil carbon material and the known 14C/C ratio levels in contemporary carbon material allows us to distinguish between contemporary carbon and fossil carbon and decipher in this manner different anthropogenic contributions. iv The contemporary carbon throughout our sampling campaign in the Lephalale District has been measured to be approximately 53% of carbon aerosol. As many studies have been performed of contemporary carbon and fossil carbon, no other contemporary and fossil carbon source assessment method provides the definitive results that can be obtained from radiocarbon measurements. PIXE analysis for the determination of the elemental composition of particulate matter in samples near the Medupi coal-fired power station in the Lephalale District was also performed for 6 elements, namely, K, Ca, Ti, Mn, Fe, and Zn. In the samples that were analyzed the particulate matter concentrations did not exceed the air quality standards regulation at Lephalale. The recommended daily limit air quality standard by South African legislation is 75 µg/m3. Enrichment Factor (EF) analysis of soil with respect to Fe shows anomalously high values for Zn. AMS was also applied to archaeological studies of early herding camps of the khoe khoe people at Kasteelberg, situated on the southwest coast in South Africa, and are among the best preserved sites of their kind in the world. Sea-shell samples from the Kasteelberg B (KBB) site have been dated with AMS at Lawrence Livermore National Laboratory (LLNL) in an effort to elucidate the relationship between the herder-foragers of the inland and shoreline sites in terms of migration patterns. The radiocarbon dates obtained are in general agreement with the other studies that have been performed on the site, and show that the ages of artifacts are less than 2000 years. The samples for this study originate from various well defined stratigraphic-levels at square A3 at KBB. It was evident from excavation that the artefacts seem to be of the same period and there is no evidence of mixing from different stratigraphic layers. v Radiocarbon dates were calibrated using Calib 6.1 and each was corrected for marine reservoir effect. The date range between the earliest and most recent dates that were obtained span gap is approximately 400 years from AD 825 to AD 1209. The majority of the radiocarbon dates of the KBB site belong to dates of 1002-1100 AD, the other few belong to 825-958 AD, and the last single date of 1209 AD. The new AMS dates from this work suggest the high probability that indeed there was a hiatus between the two occupations designated as lower and the upper KBB. The significant changes seen in material culture styles as well as in the nature of occupation and change in accumulation rate of deposits therefore do not necessarily indicate a cultural replacement caused by the arrival of a new population. This implies that the occupants of lower KBB may also have been Khoe-speakers, and not local San. / GR2018

Globalization and the rise of fish importation has led to an increase in mislabeling. To combat this problem, analytical and molecular methods have been employed. First, nitrofuran metabolites were extracted, hydrolyzed, and derivatized in channel catfish, swai, and tilapia. Utilizing high performance liquid chromatography coupled with triple quadrupole mass spectrometry, derivatized metabolites were detected at levels of 1 ng/mL with coefficients of determination greater than 0.998. Recoveries greater than 90% and relative standard deviation less than 17% indicate that the method is successful. Secondly, chip based electrophoresis coupled with restriction fragment length polymorphism was used for the species differentiation. By analyzing restriction digestion products, fragmentation patterns from fin-clip and muscle could consistently differentiate different species requiring two or fewer endonucleases for positive identification. This method of screening reduces the expertise, time, and expense required to reduce fish mislabeling. In tandem, these methodologies could significantly reduce the dangers of fish mislabeling.

Deformed Epidermal Autoregulatory Factor-1 (DEAF-1) is a transcription factor that binds TTCG motifs and has roles in fetal development, clinical depression and cancer. In order to further our understanding of the DEAF-1 protein, this study characterizes previously unidentified DEAF-1 interacting proteins and post-translational modifications of DEAF-1. A region encompassing the DNA binding domain of DEAF-1 interacts with the C-terminal Bax interacting domain of the Ku70 subunit of the DNA-PK holoenzyme. Ku70 acts as an anti-apoptotic protein through C-terminal domain and so DEAF-1 was assessed for its ability to influence apoptosis after various stimuli. DEAF-1 acted as a pro-apoptotic protein after intrinsic stimuli. Apoptotic activities occurred through a nuclear, DNA independent mechanism and a mutation that eliminated Ku70 interactions also inhibited DEAF-1 pro-apoptotic activities. Analysis of mammalian purified DEAF-1 indicated a number of phosphorylation sites and also a methylated arginine residue. Various assays were performed on mutated forms of DEAF-1 to determine the significance of the modified sites on DEAF-1 functions and properties. Lysine mutation of the methylated arginine site appeared to augment protein-protein interactions with itself and also Ku70. Alanine mutations at three of the identified phosphorylation sites increased DEAF-1 pro-apoptotic activities. In vitro kinase assays identified CDK5 as potential kinase that can phosphorylate DEAF-1. These studies provide new insight into potential functions, properties, and regulation of DEAF-1.

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