Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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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 DEVELOPMENT OF A PROTEOMIC STRATEGY FOR ANALYSIS OF PLASMA MEMBRANE PROTEINS(2013) Choksawangkarn, Waeowalee; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plasma membrane (PM) proteins play crucial roles in cell signaling and communications, and they are the targets of more than two thirds of drugs currently under development. Studies on changes in protein content, quantity and modifications of the PM proteins indicate metabolic alteration of disease related cells; therefore, mass spectrometry-based proteomic studies may lead to improved understanding of the pathology, the characterization of novel biomarkers, and discovery of future drug targets. The main objectives of my research are to develop an effective enrichment strategy and to optimize the proteomic workflow for analysis of PM proteins from cells in suspension. Strategies were optimized with human multiple myeloma cells cultured in suspension, and optimized strategies were applied to study the PM proteome of myeloid-derived suppressor cells (MDSC) collected from an animal model. We focus on optimization of the cationic nanoparticle pellicle method for enrichment of PM proteins. The principle of this method is to attach cationic nanoparticles to the cell surface by electrostatic interaction between the positively charged nanoparticles and the negatively charged cell surface. Thus, the heavier coated-plasma membrane sheets can be separated more easily from cellular organelles by centrifugation after cell lysis. The isolated PM proteins were identified by LC-MS/MS analysis. We have also optimized the workflow for proteolysis to enhance identification of hydrophobic PM proteins. Our studies reveal that higher density nanoparticle pellicles provide higher enrichment efficiency of the PM proteins and that a procedure using digestion in the gel matrix enhances the analysis of highly hydrophobic proteins. The most effective enrichment technique and optimized proteomic procedures were applied to characterize the PM proteins from MDSC obtained from BALB/c mice carrying 4T1 mammary carcinomas. These cells are known to accumulate in individuals with cancer and suppress anti-tumor immunity. Their accumulation and activity are increased with heightened-levels of inflammation. Comparative studies of the PM proteins expressed in the cells derived from basal- and heightened- levels of inflammation were performed using the spectral counting method. This work reveals a set of protein candidates that have a high potential to be involved in the inflammation-driven immunosuppressive activity of the MDSC.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.