Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item Spatiotemporal proteomic approaches for investigating patterning during embryonic development(2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.Item Development of single-neuron proteomics by mass spectrometry for the mammalian brain.(2021) Choi, Bok Dong; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Single-neuron proteomics holds the potential to advance our understanding of important biological processes during neuron maturation and development. However, to characterize proteins from single neurons, further technological advances are still required. This dissertation discusses the development and application of single-cell mass spectrometry (MS) technologies to investigate proteins and its role in different neurons. The work presented herein demonstrates the strategies to develop and advance single-neuron analysis using capillary electrophoresis (CE)-MS. In addition, this work features several contributions to our understanding of neuron-to-neuron heterogeneity, providing new information to advance cell biology and neuroscience.Item Towards the synthesis of PNAG crosslinkers to identify protein binding partners(2019) Mrugalski, Kevin R; Poulin, Myles; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms are an area of major concern in the medical field due to natural drug resistance. Many pathogenetic species of bacteria that infect humans including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholera form biofilms and their associated infections are becoming harder to treat. Poly β-(1→6)-N-acetyl-D-glucosamine (PNAG) is a major component of biofilms across multiple species and has been found to play a key role in the early stages of the biofilm life-cycle. However, little information is known about what proteins interact with this important polysaccharide. Our goal is to create small PNAG analogues to covalently capture and identify PNAG binding partners in E. coli, an important model organism. PNAG analogues will contain photoaffinity groups, that when activated, covalently link associated proteins to the probe. Then, using a proteomics-mass spectrometry-based approach, we will identify PNAG binding partners. Here, we describe the efforts and challenges encountered synthesizing the final PNAG probes. New synthetic routes are proposed based on literature precedent that will enable synthesis of the desired compounds.Item INTERROGATING PROTEIN CARGOES OF MDSC-DERIVED EXOSOMES ON THE BASIS OF POST-TRANSLATIONAL MODIFICATIONS(2017) Chauhan, Sitara; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells which accumulate in cancer patients and tumor-bearing mice. Their function in the tumor microenvironment is to inactivate the immune response to cancer by suppressing both the adaptive and innate immune system. Therefore, MDSC are a major obstacle in immunotherapeutic approaches designed to cure cancer. MDSC-derived from tumor bearing mice have been found to shed exosomes. Exosomes are nano-sized vesicles that carry biologically active molecules and play a role in intercellular communication. MDSC-derived exosomes have been reported to mediate the immunosuppressive functions of the parental cells by stimulating the accumulation of MDSC and also by converting macrophages to a tumor-promoting phenotype. Recent developments in government policy have launched a goal of curing cancer using immune-based therapies (Cancer MoonShot 2020). The understanding of the mechanisms and functions of MDSC immune suppression will be crucial in the success of these therapeutic endeavors. Our current study focuses on interrogating the protein cargo carried by MDSC-derived exosomes based on differential post-translational modifications (PTMs). Post-translational modifications have important roles in functions, signaling, location and interactions of proteins. Selecting proteins based on a specific post-translational modification can aid in the identification of low-abundance proteins which may not be identified in a shotgun proteomics approach. The first aim of this work was to successfully modify an existing surface chemistry method to use on exosomes. We then used a proteomic strategy to identify glycoproteins on the surface of MDSC-derived exosomes, and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. Furthermore, we also aimed at examining the ubiquitome of the MDSC-derived exosomes, using top-down and bottom-up proteomics. Since inflammation has been reported to enhance the tumor promoting activity of the MDSC, the bottom-up analysis focused on the effects of increased inflammation on the ubiquitination of the protein cargo of MDSC-derived exosomes. Spectral counting was used to estimate differences in abundance of proteins found with ubiquitinated proteoforms in high and basal levels of inflammation. The top-down analysis aimed at characterizing the length and topology of ubiquitin linkages on substrate proteins in MDSC-exosomes.Item CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY(2016) Lee, Amanda Elizabeth; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.Item PROTEOMIC CHARACTERIZATION OF EXOSOMES SHED BY MYELOID-DERIVED SUPPRESSOR CELLS(2015) Burke, Meghan Catherine; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes are a class of extracellular vesicles that have been shown to contribute to metastasis when derived from tumor cells. Myeloid-derived suppressor cells (MDSC) are an immature population of myeloid cells that accumulate in the tumor microenvironment and inhibit anti-tumor immunity. Given the role of the source cells, it is our hypothesis that MDSC-derived exosomes may contribute to or mediate the effects of MDSC in the tumor microenvironment. The goal of this work is to use mass-spectrometry based proteomics to characterize exosomes produced by MDSC that are induced by 4T1 mammary carcinoma. The protein content of the exosomes will be analyzed to determine if the exosomal proteome is representative of the parental cells or if it reflects active protein sorting. Increased inflammation in the tumor microenvironment is associated with an increased population of MDSC, which further increases the level of immune suppression. Here, the relative change in abundance of exosomal proteins under a heightened level of inflammation in the tumor microenvironment will be performed using the spectral count method. While it is known that exosomes first form through invagination at the plasma membrane, the mechanism(s) through which the protein cargo is sorted into exosomes remains poorly understood. Given the role of ubiquitination in protein localization and trafficking, immunoaffinity enrichment coupled to mass spectrometry has been employed to identify exosomal proteins that carry this modification. Identification of the substrate proteins in MDSC-derived exosomes may provide insight into exosome formation and/or function.Item Biochemical characterizations of extracellular vesicles shed by vegetative and sporulating Bacillus subtilis(2015) Kim, Yeji; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sporulation of Bacilli is a developmental process that provides long-term viability in unfavorable environments. Recently, biogenesis of extracellular vesicles (EVs) from Bacilli has also been reported to participate in various physiological and pathogenic phenomena. In this study, EVs were isolated from vegetative and sporulating Bacillus subtilis cells and characterized using mass spectrometry (MS)-based proteomics, microscopy, and fluorescence spectrophotometry. The microscopic approach demonstrated that both vegetative and sporulating cells produce EVs. In the proteomic analysis, 156 proteins were identified with statistical significance in EVs collected at the vegetative phase and 185 proteins in EVs shed during sporulation. The two EV cargos showed qualitatively and quantitatively different proteome patterns. Sporulation-associated proteins had greater abundances in EVs at the sporulation stage. Additionally, a fusion-like event of EVs with B. subtilis cells was observed by a fluorescence de-quenching assay. Based on these observations, B. subtilis EVs are proposed to support intercellular communication and sporulation.Item Alterations in the Primary Structures of Ribosomal Proteins in Acquired Drug Resistance(2012) Lohnes, Karen Lynn; Fenselau, Catherine C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Acquired drug resistance is a multifactorial process that is one of the major causes for cancer treatment failure. The anticancer drug, mitoxantrone, was recently determined to inhibit ribosome biogenesis. Changes in ribosomal protein composition and efficiency with which the ribosomes incorporate 35S-methionine has been noted in a mitoxantrone resistant MCF7 cell line when compared with a drug-susceptible parental cell line. This dissertation evaluated three proteomic workflows in order to successfully characterize the changes in the primary structures of cytoplasmic ribosomal proteins isolated from a mitoxantrone resistant breast cancer cell line that could serve some functional significance to the resistance when compared with a parental drug-susceptible cell line. A combination of the data from the three workflows allowed for the identification of 76 of the 79 human ribosomal proteins with an average sequence coverage of 76%. The N-terminal ends of 52 of the ribosomal proteins were identified using bottom-up and middle-down mass spectrometric approaches. An additional 7 N-terminal fragments were identified by top-down high resolution mass spectrometric analysis. Forty of the 52 N-terminal peptides were observed to have lost their N-terminal methionine and 19 were acetylated. Identification of the N-terminal peptides was most successful using the middle-down approach. Internal acetylations (on lysine) and phosphorylations were only noted with trypsin in-gel digestion and HPLC fraction analysis. Gel arrays of the two ribosomal populations illustrated differences in the protein compositions. Comparative densitometry imaging software indicated the presence of two novel protein spots in the drug resistant cell line as well six additional spots with increased and decreased abundances. High coverage bottom-up mass spectrometric analysis allowed for these protein spots to be assigned as isoform pairs of RPS3, RPS10, RPL11 and RPL23A. Molecular masses and top-down analyses were used to define the alterations in the ribosomal proteins in conjunction with high coverage bottom up and middle-down analyses. The change in the primary structures of these four ribosomal proteins is believed to alter access to the mRNA tunnel in the ribosome. This suggests that these ribosomes may participate in differential selective translation to allow for the cell to produce the necessary proteins during cellular stress.Item Mass Spectrometric Analysis of Cytoplasmic Ribosomal Proteins in Drug Resistant and Drug Susceptible Human Cell Lines(2006-05-30) Hays, Faith A; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This study examines changes in cytoplasmic ribosomes that accompany drug resistance in MCF-7 breast cancer cells. Differences in ribosomal protein composition between drug susceptible and drug resistant cell lines were examined. Ribosomes were isolated from mitoxantrone susceptible and mitoxantrone resistant MCF-7 cells. The acid extracted ribosomal proteins were subjected to optimized 2DGE using a "zoom" strip (pI 7-11) for the first dimension separation. Further optimization of 2DGE included the use of a 15mM DTT wick at the cathode end of the focusing tray, decreasing the protein loading amount and using large format gels for the second dimension. Forty-nine ribosomal proteins were identified in the drug susceptible cell line. Two novel protein isoforms of the proteins RPS3 and one novel isoform of RPS10 were identified in the drug resistant cell line. Methods for the extraction and detection of ribosomal proteins from the 2D gel were developed. The method of Mirza et.al. was modified and used to extract ribosomal proteins from the gel. The detection of these proteins was optimized by the use of 50% ACN/1.0% TFA to solubilize the MALDI matrix. In addition, the extracted protein solution was mixed 1:1 with 5% Triton X-100. Intact molecular weights were determined for 41 ribosomal proteins using high performance MALDI-TOF mass spectrometry. The average number of ribosomes per cell was determined for the drug susceptible, as well as the drug resistant cell line, and found to be unchanged.