Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item Epigenetics of Neurodegeneration: Quantification of Histone Deacetylase Isoforms and Post-translational Modifications of Histones in Alzheimer’s Disease(2015) Anderson, Kyle; Fenselau, Catherine; Turko, Illarion V; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Histone post-translational modifications have been implicated in many biological functions and diseases and serve an important role in epigenetic regulation of gene expression. Aberrant modulations in histone post-translational have been suggested to occur in the brain as part of Alzheimer’s disease (AD) pathology, consistent with the epigenetic blockade of neurodegeneration. This dissertation details the development and optimization of unique protein standards for quantification, called quantification concatamers, for the absolute quantification of histone deacetylase isoforms in human frontal cortex with AD, human neural retina with AD and age-related macular degeneration, and whole brain hemisphere of a 5XFAD mouse model of AD. Histone deacetylases are enzymes responsible for the deacetylation of histones, which can directly regulate transcription, and have been implicated in AD pathology. In addition to measuring isoforms of histone-modifying enzymes, measurements of post-translational modifications on histones were also obtained for whole hemispheres of brain from 5XFAD mice and frontal cortex from human donors affected with AD. For the changes in post-translational modifications observed, structural mechanisms were proposed to explain alterations in the DNA-histone affinity in the nucleosome, which can modulate gene expression. Measurements and structural mechanisms were consistent with the global decrease in gene expression observed in AD, which supports the data. This body of work aims to better elucidate the epigenetic pathology of AD and to aid in identification of histone-modifying enzymes involved in AD pathology for drug targets and treatment options. Currently, there are no treatments that prevent, delay, or ameliorate AD, stressing the crucial importance of AD pathology research and the promise of epigenetics as the solution.Item A Deuterium Labeling Method for the Characterization of (Chromophoric) Dissolved Organic Matter Using Ultrahigh Resolution Electrospray Ionization Mass Spectrometry(2015) Baluha, Daniel Robert; Blough, Neil; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dissolved organic matter (DOM) is a complex ensemble of naturally occurring organic compounds found in virtually all aquatic environments. The overwhelming diversity of DOM makes it extremely difficult to understand the relationship between its bulk physicochemical properties and its molecular structure and composition. This dissertation describes the development of a novel method to identify ketone/aldehyde-containing species within DOM, which are known to contribute substantially to the ultraviolet/visible (UV-vis) absorption and emission of chromophoric DOM. In this method, an aqueous sample is treated with sodium borodeuteride (NaBD4) and is analyzed via ultrahigh resolution electrospray ionization (ESI) mass spectrometry. Ketone/aldehyde-containing species (at mass m) in the untreated sample are identified by searching the mass spectrum of the reduced sample for peaks corresponding to deuterated derivatives (at mass m+3.021927n). Initial experiments demonstrated that this method reliably discriminates among mass spectral peaks in an untreated DOM sample that comprise species with zero, one, and/or two reducible moieties. The reactivity and optical properties of reducible species within Suwannee River fulvic acid (SRFA) were studied by treating an aqueous sample with several amounts of NaBD4. This study demonstrated that most species with at least one ketone/aldehyde moiety were reduced a single time under low [NaBD4], while higher [NaBD4] resulted primarily in additional reductions on multi-ketone/aldehyde species. Furthermore, the changes in UV-vis absorption and emission of the reduced aliquots relative to that of the untreated were correlated with the number of ketone/aldehyde-containing species reduced and identified by this method. The fully developed protocol was used to compare DOM extracted from several aquatic environments. Two pools of ketone/aldehyde-containing species were tentatively identified: A terrestrially-produced group of lignin/tannin-derivatives and a microbially-produced group of carboxyl-rich alicyclic molecules. While the first pool has previously been shown to contribute substantially to the absorption/emission of chromophoric DOM, the second pool most likely would not. The mass labeling method developed here revealed compositional features that are not observable by common ESI mass spectrometric analyses and may serve as a useful way to link the physicochemical properties of DOM to its structure and composition.Item Ultrasensitive CITP-MS based targeted proteomics technologies for protein identification and quantification(2014) Wang, Chenchen; Lee, Cheng S; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mass Spectrometry (MS) based technologies have enabled efficient and comprehensive proteomic profiling for biomarker discovery. However, due to sample complexity and large concentration variation, the obtained data is usually biased to endogeneous high abundance proteins while the important disease-related information went missing. Targeted proteomics enables the delivery of precise and sensitive qualitative/quantitative data of interest to researchers by focusing analysis on a preselected population of cells or proteins. This project aims to develop targeted proteomic technologies through capillary isotachophoresis (CITP)-based technique which is capable of selectively enriching trace compounds for a further improved sensitivity in both discovery and validation studies. By employing tissue microdissection and a CITP-based multidimensional separation platform, homogeneous glioma cells were isolated from unwanted cells and analyzed in search of glioblastoma biomarker. Comparative proteomic profiling of pure tumor cells from different grades of infiltrative astrocytomas revealed disease specific protein expression variation among grades. Further validation using immunohistochemistry demonstrated consistent results. This targeted tissue analyzing platform provided a sensitive and confident methodology for biomarker discovery within minute amount of samples. With the demonstrated outstanding analyzing capacity on targeted biomarker discovery, we moved on to developing ultrasensitive targeted quantitation techniques. We demonstrated online coupling of transient-CITP/CZE (capillary zone electrophoresis) with selective reaction monitoring (SRM) MS for the first time via a sheathliquid interface for improved sensitivity and selectivity. Ultrasensitive targeted quantitation was achieved through the incorporation of the selective enrichment capability of CITP/CZE with SRM MS, giving a limit of quantitation (LOQ) of 50 pM with a total sample loading of 50 attomoles. In order to further improve the sensitivity, we developed a novel sheathless interface which enables increased loading capacity and nanoflow operation by assembling a large size separation capillary and a small size porous emitter. LOQ was improved 5 times comparing to using the first sheathliquid interface, giving a LOQ of 10 pM with a total sample loading of 25 attomoles. This novel interface optimally preserved the high resolution and efficiency of CITP/CZE while improving the limited sample loading capacity, demonstrating a powerful analytic platform for targeted proteomic quantitation and validation.Item Top-Down Analysis of Bacterial Proteins by High-Resolution Mass Spectrometry(2010) Wynne, Colin Michael; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the biodefense and medical diagnostic fields, MALDI mass spectrometry-based systems are used for rapid characterization of microorganisms generally by detecting and discriminating the highly abundant protein mass-to-charge peaks. It is important that these peaks eventually are identified, but few bacteria have publicly available, annotated genome or proteome from which this identification can be made. This dissertation proposes a method of top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools to identify proteins from bacteria with unavailable genome sequences by comparison to protein sequences from closely-related microorganisms. Once these proteins are identified and a link between the unknown target bacteria and the annotated related bacteria is established, phylogenetic trees can be constructed to characterize where the target bacteria relates to other members of the same phylogenetic family. First, the top-down proteomic approach using an Orbitrap mass analyzer is tested using a well known, well studied single protein. After this is demonstrated to be successful, the approach is demonstrated on a bacterium without a sequenced genome, only matching proteins from other organisms which are thought to have 100% homology with the proteins studied by the top-down approach. Finally, the proposed method is changed slightly to be more inclusive and the proteins from two other bacteria without publicly available genomes or proteomes are matched to known proteins that differ in mass and may not be 100% homologous to the proteins of the studied bacteria. This more inclusive method is shown to also be successful in phylogenetically characterizing the bacteria lacking sequence information. Furthermore, some of the mass differences are localized to a small window of amino acids and proposed changes are made that increase confidence in identification while lowering the mass difference between the studied protein and the matched, homologous, known protein.Item SOLUTION ISOELECTRIC FOCUSING AND ITS APPLICATION IN COMPARATIVE PROTEOMIC STUDIES OF NUCLEAR PROTEINS(2005-05-27) An, Yanming; Fenselau, Catherine C; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In proteomic research, experimental and computational approaches are combined to provide global analysis of the entire proteomes of cells and tissues. The identification and quantification of multiple proteins, which constitute a specific biological system, are important for understanding complex problems in biology. The coupling of highly efficient separations and mass spectrometry instrumentation is evolving rapidly and is being widely applied to problems ranging from biological function to drug development. Development of rapid and high-resolution separation technology is an important field in proteomics. In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, integrated with mass spectrometry. An insoluble nuclear protein fraction was used for optimization and evaluation of this method. The insoluble nuclear proteins were recovered from the nuclei of human MCF-7 human cancer cells and cleaved enzymatically. The resulting peptides were analyzed by the two-dimensional separation method, which coupled solution isoelectric focusing with reversed-phase liquid chromatography interfaced with mass spectrometry. A total of 281 peptides corresponding to 167 proteins were identified by this experiment. The high sample capacity and concentration effect of isoelectric focusing make it possible to detect relatively low abundance proteins in a complex mixture. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. This method has been demonstrated to provide efficient and reproducible separation of both protein and peptides. The two-dimensional separation method was combined with proteolytic isotopic labeling for comparative analysis of protein expression in different cells. Abundances of nuclear proteins from three different drug resistant MCF-7 cancer cell lines were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of 19 proteins were found to be significantly changed. Their functions are considered in relation to potential mechanisms of in drug resistance.Item Proteomic Analysis of Plasma Membrane Proteins from Drug Susceptible and Drug Resistant Breast Cancer Cell Lines(2004-11-08) Rahbar, Amir Mikel; Fenselau, Catherine C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Drug discovery is an important field of research in the biotechnology and pharmaceutical industries. Plasma membranes are rich in drug targets and other proteins responsible for cell signaling, transport, signal transduction, and other cellular functions. Information obtained about these proteins, and the pathways they participate in, helps to facilitate the drug discovery process. Although these plasma membrane proteins play important roles in cellular function, they are usually expressed in very low abundance and are therefore hard to identify and analyze. Comparative proteomic analysis of plasma membranes in different types of cells or different disease states of the same cell or tissue type can help to design targeted therapies specific to particular cell or tissue types and can be used in the identification of biomarkers for early disease detection. In order to be able to identify proteins in the plasma membrane it is important to start out with a plasma membrane fraction that is free of contamination from other more abundant proteins from other portions of the cell. 2D gel electrophoresis is the primary protein separation tool for use with proteomics and drug discovery, however, the inability of membrane proteins to be separated using isoelectric focusing, which is the first step in the 2D gel protocol, excludes 2D gel electrophoresis as a viable technique for the separation of membrane proteins. This thesis develops and evaluates a method to identify proteins found in the plasma membranes of mammalian cells using a modified form of the cationic colloidal silica technique for plasma membrane isolation combined with analysis of these proteins using mass spectrometry. This method is then used in combination with metabolic stable isotope labeling to identify protein expression changes between the mitoxantrone drug susceptible and drug resistant MCF-7 breast cancer cell lines.