Orientador: Maria Aparecida Carvalho de Medeiros / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-27T04:43:14Z (GMT). No. of bitstreams: 1 Vilella_KellyAdrianaRibeiroTagliaferro_M.pdf: 2403765 bytes, checksum: 91f29c165c5f2b7217d7fc43cb9b5daf (MD5) Previous issue date: 2015 / Resumo: Os resíduos de herbicidas triazínicos são compostos com moderada toxicidade, altamente persistentes no ambiente, contaminando os mananciais e águas subterrâneas e são muito utilizados em várias culturas, inclusive da cana-de-açúcar. Os herbicidas atrazina e simazina foram oxidados com 'H IND.2O IND.2' na presença de catalisadores biomiméticos (metaloporfirinas de ferro e rutênio) e os produtos gerados na reação foram analisados via cromatografia gasosa (GC, do inglês gás chromatography) associada à espectrometria de massas (MS, do inglês mass spectrometry), buscando elucidar os subprodutos. As reações de oxidação dos herbicidas triazínicos e os subprodutos gerados foram monitoradas por espectrofotometria na região do ultra violeta e visível (UV-Vis) e cromatografia gasosa (GC), utilizando-se o detector de captura de elétrons (ECD, do inglês elétron capture detector). Os rendimentos das reações de oxidação das triazinas com 'H IND.2O IND.2' e catalisadas pelas metaloporfinas de ferro (Fe(FTTPCl)) e rutênio (Ru(OCTTPP)), variaram de acordo com as condições de reações catalíticas. Foi observado que houve degradação significativa dos analitos (94,70% para a atrazina e 92,60% para a simazina utilizando a (Fe(FTTPCl)) e; 94,38% para a atrazina e 67,19% para a simazina utilizando a (Ru(OCTTPP))) e também foi observado a transformação dos herbicidas nos subprodutos desetilatrazina (DEA) e o deisopropilatrazina (DIA). Os dados de monitoramento das reações catalíticas por UV-Vis revelaram as estabilidades dos catalisadores (Fe(FTTPCl)) e (Ru(OCTTPP)), nas condições oxidantes das reações. Os resultados obtidos com a cromatografia gasosa acoplada com a espectrometria de massas (GC-MS), utilizando a técnica de ionização por impacto de elétrons ¿ (EI, do inglês electron ionization), full scan, com o modo positivo (EI+), associado a este pico revelaram o pico do íon molecular (m/z= 215, associado ao herbicida atrazina [M]+ e os principais fragmentos (m/z): 200(associado ao íon [M ¿ CH3]+), 173 e 138; sendo que o espectro de massa obtido após a reação de oxidação revelou o desaparecimento do pico associado aos herbicidas e formação de novos picos, associados a fragmentos de subprodutos. Similarmente, a identificação da simazina foi obtida com o modo positivo (EI+), tendo sido revelado o pico do íon molecular (m/z= 201) e os principais fragmentos (m/z): 186, 173 e 138 / Abstract: Waste triazine herbicides are compounds with moderate toxicity, highly persistent in the environment, contaminating water sources and groundwater are widely used in various cultures, including the cane sugar. The atrazine and simazine herbicides were oxidized with 'H IND.2O IND.2' in the presence of biomimetic catalysts (iron and ruthenium metalloporphyrins) and the products generated in the reaction were analyzed by gas chromatography (GC) associated with mass spectrometry (MS), to elucidate the by-products. The oxidation reaction of the triazine herbicide and by-products generated were monitored by spectrophotometry in the ultraviolet region and visible (UV-Vis) and gas chromatography (GC), using electron capture detector (ECD). Proceeds from the triazines oxidation reactions with 'H IND.2O IND.2' catalyzed by iron and metalloporphyrins (Fe (FTTPCl)) and ruthenium (Ru (OCTTPP)), varied according to the conditions of catalytic reactions. It was observed that there was significant degradation of the analytes (94.70% to 92.60% for atrazine and simazine using the (Fe(FTTPCl)) and; 94.38% to 67.19% for atrazine and simazine using the (Ru(OCTTPP))) and was also observed the transformation of herbicides in desethyl atrazine products (DEA) and the deisopropil atrazine (DIA). The monitoring data of catalytic reactions by UV-Vis revealed the stability of the catalysts (Fe(FTTPCl)) and (Ru(OCTTPP)) in oxidizing conditions of the reactions. The results obtained with gas chromatography coupled with mass spectrometry (GC-MS) using the electron impact ionization technique (EI), full scan, positive mode (EI+), associated this peak revealed molecular ion peak (m/z = 215, associated with atrazine [M]+ and major fragments (m/z): 200 (associated with the ion [M - CH3]+), 173 and 138, and the mass spectrum obtained after the oxidation reaction revealed the disappearance of the peak associated with the herbicides and formation of new peaks associated with byproducts fragments. Similarly, the identification of simazine was obtained in the positive mode (EI+) and been revealed molecular ion peak (m / z = 201) and the principal fragments (m / z): 186, 173 and 138 / Mestrado / Tecnologia e Inovação / Mestra em Tecnologia

Orientador: Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T06:50:00Z (GMT). No. of bitstreams: 1 Pudenzi_MarcosAlbieri_M.pdf: 6732140 bytes, checksum: 0b8176e8c477d9dca874d72376dfcae5 (MD5) Previous issue date: 2015 / Resumo: Esse trabalho tem como objetivo conhecer e avaliar a ionização dos compostos polares no petróleo for Espectrometria de Massas por Ressonância Ciclotrônica de Íons com Transformada de Fourier (FTICR MS do inglês Fourier Transform Ion Cyclotron Resonance Mass Spectrometry), relacionando essa ionização com parâmetros experimentais e características intrínsecas das moléculas. No Capítulo 1 o tipo de amostra de interesse (petróleo) é abordado, tanto em suas características quanto em sua análise, definindo petroleômica e sua função. A espectrometria de massas é apresentada, então, como ferramenta de grande utilidade nos estudos petroleômicos, em especial aquela de ultra alta resolução como FTICR MS (Fourier Transform Ion Cyclotron Resonance Mass Spectrometry) acoplada a fonte de ionização eletrospray (ESI). No Capítulo 2 a ferramenta estatística do planejamento de experimentos é apresentada e, então, utilizada em uma amostra real de petróleo de forma a relacionar os parâmetros operacionais da fonte de ESI com respostas avaliadas comumente em petroleômica. Aspectos interessantes das relações parâmetro-resposta surgem durante o estudo, mostrando que a análise de petróleo por ESI-FTICR MS, apesar de largamente utilizada na literatura, não é trivial e precauções devem ser tomadas durante a otimização dos parâmetros experimentais. No Capítulo 3 as técnicas de APCI (Atmospheric Pressure Chemical Ionization) e APPI (Atmospheric Pressure PhotoIonization) são abordadas e utilizadas, juntamente com ESI, para avaliar a ionização de padrões de compostos similares àqueles encontrados nas principais classes de compostos polares do petróleo. A análise pelas técnicas que ionizam de forma mais abrangente os compostos apresentaram comportamentos que tornam tais análises inviáveis. Uma reação de metilação que melhora a ionização de compostos sulfurados também é realizada em duas condições, comparando-se tais condições a partir dos espectros obtidos após suas análises. Conhecendo a ionização desses compostos padrões, é possível entender a petroleômica de forma mais ampla dando maior confiabilidade para estabelecer as relações composição-comportamento do petróleo / Abstract: The aim of this work is to know and evaluate the crude oil polar compounds ionization through Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, relating this ionization with experimental parameters and intrinsic characteristics of molecules. On Chapter 1 the sample of interest (crude oil) is addressed, both in its characteristics and its analysis, defining petroleomics and its function. Mass spectrometry is then presented as a tool of great utility in petroleomic studies, in special the ultra high resolution one as FTICR MS coupled to eletrospray Ionization (ESI). On Chapter 2 the design of experiment statistic tool is presented and used in a real crude oil sample in order to relate operational parameters of ESI source to commonly evaluated responses in petroleomics. Interesting aspects regarding parameter-response relationships arises during the study, showing that the crude oil analysis using ESI-FTICR MS, while it is largely used in literature, it is not trivial and precautions have to be taken during the optimization of experimental parameters. On Chapter 3 APCI (Atmospheric Pressure Chemical Ionization) and APPI (Atmospheric Pressure PhotoIonization) technics are addressed and used, along with ESI, to evaluate the ionization of standard compounds similar to those polar compounds found in crude oil. Analysis using sources that ionizes in the most comprehensive way has shown such behavior that makes the analysis unviable. A methylation reaction that improves the ionization of sulfur compounds is also made on two conditions, comparing these conditions through the obtained spectra after their analysis. By knowing the ionization of these standard compounds it is possible to understand the petroleomics in a much comprehensive way, providing reliability to stablish composition-behavior relations of crude oil / Mestrado / Quimica Organica / Mestre em Química

Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene, chlorinated solvents often used in plastics and dry cleaning solvent manufacturing. The treatment of VC-contaminated sites by bacteria that can biodegrade VC has been demonstrated to be a practical and potentially cost-effective alternative to traditional "pump and treat" site cleanup options. However, little is known about the biochemical pathways involved in VC-assimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. The results of these studies demonstrated the successful extraction and identification of proteins involved in VC-assimilation from ethene-enriched groundwater samples. The presence and diversity of VC-assimilating bacteria in several ethene-enriched groundwater samples were examined using tandem mass spectrometry analysis to identify the protein biomarkers EtnC and EtnE. VC-assimilating organisms can evolve in vitro from bacteria that grow on ethene but very little is known about the molecular changes involved. Proteomic investigations comparing three strains of Mycobacterium strain JS623, a wild type and two VC-adapted strains, validated previous studies indicating that protein expression changes are involved in VC-adaptation. Tandem mass spectrometry and spectral counting were used to identify proteins and semi-quantitatively estimate protein expression levels in the three ethene-grown JS623 variants. The results of this study suggest that multiple VC-adaptation mechanisms are involved in the two VC-adapted strains

<p>This thesis covers the four topics discussed in each of the following paragraphs. It is unified by the dual ability of ambient ionization mass spectrometry as a useful analytical tool allowing for monitoring of chemical reactions, in addition to its capability to accelerate reaction rates using the same equipment under accelerating or non-accelerating conditions. The ability to manipulate reactions and monitor the subsequent effects to the rate of the reactions can provide vital information for many industrial arenas. Current process analytical technology (PAT) is extremely time-consuming, and typically costly due to dependence on analysis conducted at the end stage of production. Additionally, many chemical reactions found to be useful in pharmaceutical or manufacturing industries are labor intensive and require harsh conditions such as heat or expensive catalysts. Several methods have been developed to overcome these current limitations, while providing vital information on short-lived intermediates, degradation products, and accelerated reaction rates. A sampling device was developed and coupled with nESI allowing for monitoring of heterogeneous chemical reactions by mass spectrometry without the additional requirement of separation (filters, chromatography, etc.) In addition, this technique maintains the high sensitivity, specificity, speed and structural elucidation provided by mass spectrometry analysis. The analysis provided kinetic profiles of all reactants, intermediates, products and coproducts throughout the course of the reaction.</p><p> The ability to effectively control chemical reactions and their rates is a priority across several fields of study. Several factors affecting reaction rates, such as heat and catalysts selected, have been well studied. However, there has been recent interest in exploring the capabilities for reaction acceleration in charged microdroplets. It is known that reaction rates on the surface of a droplet greatly differ from reactions occurring in the droplet. The Katritzky transamination reaction was used as a model to identify the effects of the air-solution interface on reaction acceleration by varying the air-liquid surface to volume ratio. The significant increase in reaction rate constants was further enhanced by solid–solution interfacial effects observed after addition of glass nanoparticles.</p><p> The effective degradation of non-polar hydrocarbons is an environmental concern as they are the main composition of waste generated from petroleum processing. Saturated alkanes are relatively stable molecules which present a challenge for analysis by mass spectrometry without the use of extreme experimental conditions. A rapid analysis method by paper spray ionization was developed that allows for the oxidation products of saturated alkanes to be monitored by MS in under two minutes. This method relies on the generation of a hydroxyl radical by reacting iron (III) chloride with aqueous hydrogen peroxide on the principle of Fenton’s chemistry. The presence of this radical in direct contact with an alkane produces several oxidation products which can be easily monitored by MS. The reagents are added to a paper triangle sequentially, creating a thin film which allows reaction acceleration in relatively small volumes analyzed directly from paper at atmospheric pressure.</p><p> The dimerization of 4-ethynylaniline derivatives in acetonitrile was monitored by nano electrospray ionization mass spectrometry. Dimer products formed by electrocyclization and radical processes were observed that are not detected as a corresponding bulk reaction. This gas-phase reaction has been interrogated in a solution phase analog with radical initiators and characterized by ¹H NMR. This work demonstrates that compounds can be synthesized by the electrospray process. Future studies may reveal how this observation affects the interpretation of the MS results involving electrospray.</p>

Condensed phase membrane introduction mass spectrometry (CP-MIMS) is a direct, in situ analysis technique that is well suited to persistent organic pollutants, pesticides, and other small molecules. In CP-MIMS, neutral analytes permeate a hollow fibre membrane, typically polydimethylsiloxane (PDMS), driven by a concentration gradient. Analytes are subsequently dissolved by a liquid (condensed) solvent acceptor phase that is continuously flowed through the membrane lumen, which finally entrains the analytes to a mass spectrometer for detection. The membrane rejects charged and particulate matrix components, therefore eliminating sample cleanup that is otherwise necessary for conventional (i.e., chromatographic) techniques. However, larger analytes may suffer from relatively lengthy response times and lower sensitivity. A heptane cosolvent was therefore doped into the PDMS membrane, resulting in a polymer inclusion membrane (PIM). Through a system coupling CP-MIMS to electrospray ionization (ESI), the use of a PIM for model compounds resulted in faster response (~3×) and improved sensitivity (~3.5×, parts per trillion level detection limits). While effective for the demonstration of the PIM, pairing ESI with CP-MIMS represents an inherent incongruity: ESI is effective for polar, hydrophilic analytes, whereas CP-MIMS (i.e., PDMS membranes) is effective for hydrophobic analytes. CP-MIMS was therefore coupled with liquid electron ionization (LEI) as a more suitable ionization strategy. In LEI, the post-membrane solvent flow is entrained at nanolitre per minute flowrates to a LEI source, where the liquid is sequentially nebulized, vaporized, and ionized. The CP-MIMS-LEI coupling was optimized for the measurements of polycyclic aromatic hydrocarbon (PAH) isomer classes from aqueous samples, demonstrating low ng/L detection limits and response times (≤1.6 min). CP-MIMS-LEI was also applied to PAH isomer classes from soil samples, demonstrating rapid sample throughput (15 samples/hr) and low μg/kg detection limits, and additionally was quantitatively comparable to conventional techniques. A similar CP-MIMS-LEI system was applied to online monitoring of catalytic oxidation and alkylation reactions, demonstrating quantitative, real-time results for harsh, complex organic reaction mixtures. A significant analytical improvement was conducted by intentionally exploiting the already present liquid acceptor phase as an in situ means of providing liquid chemical ionization (CI) reagents for improved analyte sensitivity and selectivity (i.e., CP-MIMS-LEI/CI). Acetonitrile and diethyl ether were used as a combination acceptor phase/CI reagent system (i.e., proton transfer reagents) for the direct analysis of bis(2-ethylhexyl)phthalate from house dust (6 mg/kg detection limit). CP-MIMS-LEI/CI was then applied to PAHs from soils. Using methanol and dichloromethane combination acceptor phase/CI reagents, CP-MIMS-LEI/CI was shown to quantify and resolve PAH isomers from direct soil analyses via diagnostic PAH adduct ions: [M+CH2Cl+CH3OH-HCl]+ or [M+CHCl2-HCl]+. Using these selective ions, CP-MIMS-LEI/CI was again shown to be rapid (15 soils/hr), sensitive (ng/g detection limits) and quantitatively comparable to gas chromatography-MS for PAH measurements (average percent difference of -9% across 9 PAHs in 8 soil samples). The results across this thesis present a compelling argument for direct, quantitative screening from complex samples using CP-MIMS-LEI/CI, particularly given the simple workflow and short analytical duty cycle. / Graduate

Tissues are heterogeneous ecosystems comprised of various cell types. For example, in tumor tissues, malignant cancer cells are surround by various non-malignant stromal cells. Proteins, especially N-linked glycoproteins, are key players in tumor microenvironment and respond to many extracellular stimuli for involving and regulating intercellular signaling. Understanding the human proteome and glycoproteome in heterogeneous tissues with spatial resolution are meaningful for exploring intercellular signaling networks and discovering protein biomarkers for various diseases, such as cancer. In this study, we aimed to develop new liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analytical methods for spatially-resolved proteome and glycoproteome profiling in tissue samples, and apply them for profiling potential biomarkers for pancreatic cancer. We first systematically and synchronously optimized the LC-MS parameters to increase peptide sequencing efficiency in data dependent proteomics. Taking advantage of its hybrid instrument design with various mass analyzer and fragmentation strageties, the Orbitrap Fusion mass spectrometer was used for systematically comparing the popular high-high approach by using orbitrap for both MS1 and MS2 scans and high-low approach by using orbitrap for MS1 scan and ion trap for MS2 scans. High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. We then systematically optimized various MS parameters for high-high approach. We investigated the influence of isolation window and injection time on scan speed and MS2 identification rate. We then explored how to properly set dynamic exclusion time according to the chromatography peak width. Furthermore, we found that the orbitrap analyzer, rather than the analytical column, was easily saturated with higher peptide loading amount, thus limited the dynamic range of MS1-based quantification. Finally, by using the optimized LC-MS parameters, more than 9000 proteins and 110,000 unique peptides were identified by using 10 hours of effective LC gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor targeting and high-resolution fragment detection for high-efficient data dependent proteomics. Understanding the tumor heterogeneity through spatially resolved proteome profiling is meaningful for biomedical research. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was stained by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance with traditional H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT was achieved by combining with data independent proteomic analysis. This IHC-SISPROT workflow was successfully applied for identifying 6660 and 6052 protein groups from cancer cells and cancer- associated fibroblasts (CAFs) by using only 5 mm 2 and 12 μm thickness of hepatocellular carcinoma tissue section. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues. N-linked glycoproteins are promising candidates for diagnostic and prognostic biomarkers and therapeutic targets. They often locate at plasma membrane and extracellular space with distinct cell type distribution in tissue microenvironment. Due to access to only low microgram of proteins and low abundance of glycoproteins in tissue sections harvested by LCM, region- and cell type-resolved glycoproteome analysis of tissue sections remains challenging. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieved all the steps for glycoprotein enrichment, digestion, deglycosylation and desalting in a single spintip device. Sample loss is significantly reduced and the total processing time is reduced to 4 hours, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 μg of mouse brain proteins. By seamlessly combining with LCM, the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1,875, 1,794, 1,801, and 1,417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of tissue sections. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with five year survival rate of around 8%. No effective biomarkers and targeted therapy are one of the major reasons for this urgent clinical situation. To explore potential protein biomarkers and drug targets located at intercellular space of pancreatic tumor microenvironment, we established chemical proteomic approach for deep glycoproteome profiling of PDAC clinical tissue samples based on the above- mentioned new proteomic methods. Taking advantage of a long chain biotin- hydrazide probe with less space hindrance, the new method outperformed traditional hydrazide chemistry method in terms of sensitivity, time efficiency and glycoproteome coverage. The method was successfully applied to enrich and validate LIF and its receptors as potential biomarkers for PDAC. In addition, to explore the full map of pancreatic tumor microenvironment glycoproteome with diagnostic and therapeutic values, we collected 114 pancreatic tissues, including 30 PDAC tumor tissues, 30 adjacent non-tumor (NT) tissues, 32 chronic pancreatitis tissues and 22 normal pancreatic tissues, and systematically profiled their glycoprotein expression pattern by using the developed glycoproteomic strategy. The deepest glycoproteome of PDAC was achieved, which covered the majority of previously reported glycoprotein biomarkers and drug targets for PDAC. Importantly, we discovered many new glycoproteins with differential expression in PDAC and normal tissue types. Moreover, LCM-based cell-type proteome profiling was achieved for 13 PDAC tissue samples, which covered more than 8000 proteins for both pancreatic stromal cells and pancreatic cancer cells in each sample. We therefore provided a valuable resource for screening novel and cancer specific glycoprotein biomarkers for pancreatic cancer with spatial resolution

No description available.

<p>Mass spectrometry-based techniques have been widely used in reaction monitoring due to their high sensitivity and ability to offer structure information by tandem mass spectrometry. We applied nanoelectrospray mass spectrometry (nanoESI-MS) to simultaneously monitor pre-catalysts, catalytic intermediates, reagents, and products of palladium catalyzed Suzuki-Miyaura cross-coupling reactions. A set of Pd cluster ions related to the monoligated Pd (0) active catalyst is detected, and its deconvoluted isotopic distribution reveals contributions from two neutral molecules. One is assigned to the generally accepted Pd (0) active catalyst, seen in MS as the protonated molecule, while the other is suggested to correspond to a deactivated form of Pd catalyst. Oxidative stress testing of the synthetic model catalyst XPhos Pd cyclo-octadiene, performed using oxygen and Fe(III), supported this assignment. Thus, the make-up of the monoligated set of Pd (0) ions appears to indicate the oxidation state of the system. The formation and removal of the oxidative addition intermediate during the catalytic cycle was monitored to provide information on the progress of the transmetalation step. </p> <p> </p> <p><a>Recently, microdroplets created by ambient ionization source have been used as reaction vessels to accelerate organic reactions. Field desorption mass spectrometry under ambient conditions is applied to study solution-phase organic reactions in micro-volumes. Compared to nanoelectrospray, it is noteworthy that radical cations and formation of radical cation products are observed. Three reactions, the hydrazone formation by phenyl hydrazine and indoline-2,3-dione, the Katritzky reaction between a pyrylium salt and anisidine, and the Hantzsch synthesis of 1,4-dihydropyridine, were investigated by this system and reaction acceleration was observed to different extents. The increase in rate relative to that for the corresponding bulk reactions is attributed to solvent evaporation which increases concentration, and to the increase of surface-to-volume ratio with enhanced interfacial reaction rate constants. Later work in this thesis describes explicit solvent calculations to study the energies and structures of the hydrazone formation reaction from phenylhydrazine and indoline-2,3-dione in acidic methanol with density functional tight binding (DFTB) methods. Additionally, the thesis covers MS based methods for determination of isoaspartate and aspartate in peptide by gas-phase chemistry and detection of S-nitrosoglutathione in exhaled breath condensate sample.</a></p>

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which an RNA-binding protein, TDP-43, mislocalizes and pathologically accumulates from its normal nuclear locale to the cytosol. Given that the subcellular localization and expression of TDP-43 is tightly regulated, we posit that identifying novel interactors of wild-type and mutant TDP-43 could reveal insight into networks involved in regulating its localization, ultimately driving neurodegeneration in ALS. Using CRISPR/Cas9, our lab previously generated knock-in cell lines expressing GFP in the endogenous TARDBP locus (encoding for TDP-43) for both wildtype (WT) and an ALS-causing mutant (Q331K). We have shown that the Q331K mutation causes loss-of-function and mislocalization of TDP-43. I performed immunoprecipitation coupled to mass spectrometry (IP-MS) on this cell model to elucidate interactors of WT- and Q331K- TDP-43. Our data show that there is an overall loss of TDP-43 interactors in cells with the TDP-43Q331K mutation. By setting statistical cut-offs for significance, we identified 34 shared and 12 unique interactors of TDP-43WT. We used bioinformatic approaches to identify enriched pathways and literature searches to look for interactors relevant to TDP-43 and ALS pathobiology. Our shortlist of 14 candidates for validation included proteins involved in the nuclear mRNA export pathways, RNA binding proteins and proteins identified in other interactome studies and TDP-43 based screens. Using orthogonal approaches, we show evidence of robust interaction of four top hits (PABPC1, HNRNPC, DDX39b and ELAVL1) with TDP-43WT, and a significant decrease in the degree of interaction of HNRNPC, DDX39b and ELAVL1 with TDP-43Q331K. Importantly, this decrease in interaction was only observed at the endogenous level, highlighting the importance of maintaining the steady state levels of TDP-43 in the cell for these assays. We characterized the effects of knockdown and overexpression of these four hits using protein-specific overexpression constructs and shRNAs and observed a significant increase in TDP-43 nuclear localization upon knockdown of these four hits, suggesting that there is a functional effect associated with hit knockdown. Overexpression or knockdown of the top hits in a splicing assay did not identify significant changes in TDP-43’s splicing or RNA binding abilities, suggesting that these hits do not affect splicing function in our hit characterization studies. Using this novel experimental tool and unbiased screen, we identified alterations in TDP-43 protein-protein interactions in the context of ALS and have generated tools to characterize their roles in cellular functions using knockdown and overexpression approaches. Together with the knock-in cells, these tools will allow us to gain insight into pathways involved in driving neurodegeneration, in the context of ALS.

Identification of polysaccharides is not a straightforward task due to the high degree of stereochemistry present in their isobaric monomers. Their isobaric nature causes traditional mass spectrometry to fall short when trying to differentiate not only the conformation of the monomers but the position of the glycosidic bonds that bind them. This structural information is important for biochemists as they study the role of different glycans in biological processes. Tandem mass spectrometry (MS/MS) allows the study of the fragment ions formed during collision induced dissociation (CID), the fragments formed depend on the structure and stability of the precursor molecule and can be used to identify the compounds. These fragmentation pathways will be as complex as the species that form them. To date, typical saccharide fragments are separated into three groups that represent the major fragments: Cross-ring cleavages (A/X), and those resulting from cleaving different sides of the glycosidic bond (B/Y) and (C/Z). Ion mobility separation (IMS) has shown to have some success at discerning polysaccharide conformers and those of other biopolymers such as proteins and polynucleotides. Ion mobility separates gas-phase ions by colliding them with non-reactive gases and relating respective increase in flight time to their collision cross-section (CCS). In this study, the relative energetics of the first steps of the cross-ring cleavage and both glycosidic bond cleavage channels for isomaltotriose [glc(α1-6)glc(α1-6)glc] as well as a minor water loss channel were explored using density functional theory (DFT) calculations at the B3LYP/6-31+g(d) level of theory. It was demonstrated that charge-remote mechanisms are a viable alternative to charge-directed mechanisms when under the high energy short time scale conditions present during an ESI-MS/MS experiment. To verify the efficiency of ion mobility for isomeric separation, the relative experimental CCS of isomaltotriose [glc(α1-6)glc(α1-6)glc], maltotriose [glc(α1-4)glc(α1-4)glc], panose [glc(α1-6)glc(α1-4)glc] and raffinose [gal(α1-6)glc(α1-2)fru] were determined by comparison with literature CCS values for dextran, a variable-length oligomer of α1-6 linked glucose was used as an external calibrant. The experimental CCS of the precursor ions were compared to literature values when available as well as the calculated effective values of the optimized DFT geometries using the trajectory method of the MOBCAL computational suite. As phosphate is often used as an adducting agent to increase the intensity of the precursor ion when running an IMS experiment, the effect of its presence on the fragmentation of isomaltotriose and large isomaltooligosaccharides was studied. It was seen that depending on the location of the phosphate ion, it will preferentially dissociate leaving behind a neutral glycan. This explains the low abundance of fragment ions observed when selecting a phosphate-adducted precursor ion during an MS/MS experiment. IMS and MS-MS are complementary methods that can be used to identify monomers within a polysaccharide and how they are bound.