Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS(2016) Adams, Katherine R.; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.Item QUANTIFICATION OF IONOPHORE ANTIMICROBIALS ASSOCIATED WITH POULTRY LITTER AND THEIR DYNAMICS IN THE SOILS OF THE MID-ATLANTIC USA(2014) Biswas, Saptashati; McGrath, Joshua M; Sapkota, Amir; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Anticoccidants, biochemically known as ionophores are added to poultry feed for growth promotion, prophylactic and therapeutic purposes to better sorb nutrients and against coccidiosis caused by parasite Eimeria sp. Ionophores belong to the class of emerging contaminants, as they are not regularly monitored in the environment and not specifically treated in the effluents. Potentially, this can cause ionophores to enter into the environment freely. There is little information regarding the dynamics of ionophores in the environment. This has been related to the lack of reliable, sensitive and robust methods that can measure their trace levels from complex environmental matrices like soil, natural water and animal manure. Studies show ionophore toxicity exhibited in flora and fauna, even reported in humans above the dose of 1 mg kg-1. Hence accumulation of ionophores in the environmental can be detrimental. Our multi-scale investigation of ionophores involved, a) method development for trace analysis of ionophores in poultry manure using liquid chromatography triple quadrupole mass spectrometry (HPLC-–MS/MS), b) batch equilibrium studies of ionophores using soils from mid-Atlantic region of the USA and c) influence of soil physico-chemical parameters on dynamics of ionophores in soil-water systems. Our HPLC-–MS/MS method was successful in quantifying ionophores ranging from (19.19 ±± 6.6) µg kg-1 to (97.86 ±± 19.19) µg kg-1 with concentrations of monensin being the highest. This method was further used to investigate partitioning of monensin in soil-ndash;water systems relevant to the occurrence of ionophores in the natural environment. Sorption and desorption isotherms were developed and influence of soil physico-chemical parameters on the sorption-desorption processes were analyzed. C-–type linear isotherms were generated with partition coefficients ranging from (6.41±± 1.34) to (343.83 ±± 5.68) LKg-1. Soil parameters such as cation exchange capacities, pH, organic matter, sand and silt content were found to correlate with sorption in different conditions. A major focus of this dissertation was to develop novel methodologies and design experiments to execute our research objectives.Item Analysis of Intact Proteins in Complex Mixtures(2013) Dhabaria, Avantika; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our goal is to develop an effective work flow for analysis of intact proteins in a complex mixture using the LC-LTQ-Orbitrap XL. Intact protein analysis makes the entire sequence available for characterization, which allows for the identification of isoforms and post translational modifications. We focus on developing a method for top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools. The complex mixtures are fractionated using 1-dimensional reversed-phase chromatography and basic reversed- phase, and open tubular electrophoresis. The analysis of intact proteins requires various fragmentation methods such as collisional induced dissociation, high energy collisional dissociation, and electron transfer dissociation. This overall method enables us to analyze intact proteins, providing a better understanding of protein expression levels and post transitional modification information. We have used standard proteins to optimize HPLC conditions and to compare three methods for ion activation and dissociation. Furthermore, we have extended the method to analyze low mass proteins in MCF7 cytosol and in E. coli lysate as a model complex mixture. We have applied this strategy to identify and characterize proteins from extracellular vesicles (EVs) shed by murine myeloid-derived suppressor cells (MDSC). MDSCs suppress both innate and adaptive immune responses to tumor growth and prevent effective immunotherapy. Recently some of the intercellular immunomodulatory effects of MDSC have been shown to be propagated by EVs. Top-down analysis of intact proteins from these EVs was undertaken to identify low mass protein cargo, and to characterize post-translational modifications.Item Identifying Molecular Functions of Heliotropic Motor Tissue Through Proteomic Analysis of Soybean Pulvini(2013) Lee, Hakme; Sullivan, Joseph; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heliotropic and nyctinastic leaf movement are mediated in soybean through turgor changes in the motor cells of the pulvinus, located at the base of the leaves. While some elements of the signaling pathways have been studied, a broad-scale protein identification has not yet been reported. In my research pulvini proteins were extracted in light- and dark-harvested soybean using the TCA/acetone method and identified by LC-MS/MS. Gene ontology analysis revealed proteins involved in proton transport were enriched in the soybean pulvinus proteome compared to a background soybean proteome. Proteins more highly expressed in the light were mostly stress response proteins, while under-expressed proteins were categorized as energy proteins. Further investigations using more sensitive extraction protocols and a multitude of sample times will build on these initial results to provide a thorough examination of heliotropic mechanisms at the molecular level.Item Microwave-Supported Acid Hydrolysis for Proteomics(2012) Cannon, Joe; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our goal is to develop, optimize and demonstrate workflows that incorporate rapid Asp-selective chemical proteolysis into proteomic studies of complex mixtures. This can be further divided into several specific aims. The first aim is to develop and optimize the sample preparation, mass spectrometric, and bioinformatic methods required for complex mixture analysis of peptides resulting from acid digestion both in solution and in polyacrylamide gels. Second, the optimized methods will be applied to three model systems. In the first application, the large peptides derived from microwave-supported acid hydrolysis of human ribosomes isolated from MCF-7 breast cancer cells are analyzed. Secondly, acid hydrolysis will be applied to characterize Lys63 linkages in polyubiquitins. Finally, all the above methods will be combined for the analysis of extracellular vesicles shed by myeloid derived suppressor cells from a murine mammary carcinoma model. After optimizing the mass spectrometric and bioinformatic methods required for analysis of peptides resulting from acid hydrolysis, the most comprehensive analysis using this digestion technique to date was achieved both for in gel and in solution analysis. In gel digestion resulted in identification of over twelve hundred peptides representing 642 proteins, and in solution digestion via mass biased partitioning allowed identification of over 300 proteins. Mass biased partitioning also resulted in two distinct peptide populations from the high and low mass analyses implemented. Nearly 90% of the predicted human ribosomal proteins were identified after acid hydrolysis. High resolution analysis of both precursor and product ions resulted in an average sequence coverage of 46% among identified proteins. It was also demonstrated that microwave-supported acid hydrolysis facilitates a more informative method for analysis of Lys63 linked polyubiquitin. After acid hydrolysis, ~629 Da mass shifts were found to be indicative of isopeptides. These isopeptides were easily identified from complex mixtures using tandem mass spectrometry and diagnostic b ions. Extracellular vesicles from a murine carcinoma model were then analyzed using in gel microwave-supported acid hydrolysis, mass biased partitioning after in solution digestion, and the sample was interrogated for the presence of ubiquitinated peptides.Item STRUCTURAL STUDIES OF THE PEANUT ALLERGEN PROTEIN ARA H 2(2010) Li, Jinxi; FENSELAU, CATHERINE; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Presented in this dissertation are comprehensive studies of the structures of the peanut allergen protein Ara h 2 and the effect of food processing (roasting) on it. A detailed elucidation of the primary structure and PTM of Ara h 2 from the raw peanuts has been described. Ara h 2 isoforms were purified and cleaved via microwave accelerated trypsin digestion. The peptide mixtures were analyzed by LC-MS/MS and targeted CID. De novo sequencing of the MS/MS spectra revealed the protein sequence of each Ara h 2 isoform. Several hydroxyproline sites have been discovered while disulfide bond structures have been partially determined. Using anti-Ara h 2 antibodies, Western blotting of 1-D gels of the raw and dark roasted peanuts was carried out in order to characterize the changes of Ara h 2 between these two samples. The result indicates that Ara h 2 may present in a much heavier form in the roasted peanuts, possibly due to crosslinking and aggregation with other proteins. Subsequent LC-MS/MS studies of trypsin digestion of five gel pieces (>100, 100-50, 50-25, 25-16 kDa) from 1-D gels of the raw and dark roasted peanuts suggests that roasting process causes the crosslinking of Ara h 2 with other proteins. This supports our results from the immunological studies.Item Proteomic Profiling and Label-Free Quantification of Bovine Milk Proteins during Experimentally Induced Escherichia coli Mastitis(2009) Boehmer, Jamie Layne; Peters, Robert R; Bannerman, Douglas D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Coliform mastitis has been a primary focus of dairy cattle disease research due to staggering affiliated losses, severe systemic complications arising from host inflammatory response to lipopolysaccharide, and the poor response of coliform pathogens to antimicrobials. Reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis, and to aid in the approval of new veterinary drugs. The aims of the current analyses were to utilize proteomic methodologies to evaluate protein expression in whey from cows with experimentally induced coliform mastitis, and to employ label-free quantification strategies to estimate changes in relative abundance of proteins identified in milk over the course of clinical infection. Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry (MS) resulted in the identification of complement factors, antimicrobial proteins, and acute phase proteins in mastitic milk. Analysis using liquid chromatography (LC) inline with electrospray ionization - quadrupole TOF tandem mass spectrometry (MS/MS) resulted primarily in the identification of abundant whey and casein proteins, and the transient detection of proteins related to host response. Nano-LC- nanospray-MS/MS using a linear ion trap, however, led to the robust discovery of over fifty inflammatory proteins in whey from mastitic milk, including the novel markers kininogen-2 and inter-alpha trypsin inhibitor heavy chain-4. Normalized spectral counts were compared to enzyme-linked immunosorbant assay (ELISA) for select proteins to assess the accuracy of the spectral count data. Similar expression patterns were detected using spectral counts and ELISA. Results indicate that proteomic methodologies can detect biomarkers of coliform mastitis in bovine milk during clinical infections, and that spectral counts are a viable means of evaluating relative changes in protein biomarkers of mastitis, including those for which no antibody currently exists.