Browsing by Subject "Mass Spectrometry"

Now showing 1 - 12 of 12
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    ADVACING ORGANELLE ANALYSIS: DEVELOPMENT OF TECHNIQUES FOR THE ENRICHMENT OF ENDOCYTIC ORGANELLES AND TO DETERMINE AUTOPHAGOSOME PROPERTIES
    (2013-07) Satori, Chad Patrick
    Lysosomes and endocytic organelles are intracellular bodies present in eukaryotic cells responsible for the degradation of endocytosed extracellular targets. Autophagosomes traffic proteins, organelles, and other intracellular components to lysosomes to facilitate degradation during the degradative process of autophagy. Multiple disorders have been connected to malfunctions in lysosomes (Nieman pick, galactosialidosis, Danon disease) and autophagosomes (Alzheimers, Parkinson's, Huntington's).Methods are needed to enrich organelles in order to study their properties without contamination from unwanted organelles. Current methods to enrich endocytic organelles do not result in highly enriched organelles (differential centrifugation), are time consuming and tedious (density gradient centrifugation), and can damage membranes. Methods are also needed to determine endocytic and autophagy organelle properties such as organelle molecular composition, organelle-specific biotransformation of anti-cancer drugs, individual organelle surface properties and marker protein levels, and pH. The work described in this thesis develops new techniques to improve our ability to enrich endocytic organelles and determine their properties. This work includes: (1) the magnetic enrichment of endocytic organelles and determination of pH by capillary cytometry, (2) the determination of the biotransformation of N-L-leucyldoxorubicin to doxorubicin, (3) the development of a workflow to determine preliminary identifications of enriched autophagosome samples and (4) determine temporal changes in individual autophagy organelle numbers, surface charge, and LC3-II levels from basal and rapamycin enhanced autophagy levels. These methods will improve our understanding of how lysosomes and autophagosomes contribute to disease, leading to better therapeutic strategies that may improve and lengthen people's lives. Endocytic organelle enrichment was done by trafficking dextran coated magnetic iron oxide nanoparticles to lysosomes and endocytic organelles prior to magnetic separation. No detectable enzymatic activity from mitochondria and peroxisomes were observed in the enriched endocytic organelle fractions suggesting that the enriched lysosomes were in high enrichment. A majority of enriched, individual endocytic organelles had an acidic pH as determined by capillary cytometry suggesting the enriched endocytic organelle fraction had intact membranes. Enriched endocytic organelle fractions were then used to determine the biotransformation of N-L-leucyldoxorubicin to doxorubicin. Previous reports had suggested endocytic organelles may be important for intracellular biotransformation. About 45% of the biotransformation from uterine sarcoma cell post nuclear fraction occurred in the enriched endocytic organelle fraction suggesting intracellular biotransformation may be more critical to prodrug activation than previously believed.Ultra high performance liquid chromatography coupled to near-simultaneous low- and high-collision energy mass spectrometry was used to determine preliminary identifications of compounds enriched or unique to enriched autophagosome fractions. A workflow was developed to detect and confirm features (unidentified compounds with a characteristic chromatographic tR and m/z value) in the enriched sample as well as making and confirming identifications from online databases. Multiple high-relevancy preliminary identifications were made that are relevant to autophagy as supported by literature searches. Following validation, these preliminary identifications could prove to be important to maintain autophagosome function and autophagy. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) was used to determine temporal changes in the detected number of individual autophagy organelle events (phagophores, autophagosomes, amphisomes, and autolysosomes), of GFP-LC3-II levels, and of surface charge by CE-LIF. Pharmacological treatment with vinblastine was used to accumulate autophagosomes and phagophores from basal and rapamycin enhanced autophagy do detect temporal changes in autophagy organelles characteristic of the autophagy level and its autophagy flux. The dramatic contrast between time dependent changes in individual organelle properties between basal and rapamycin enhanced autophagy conditions demonstrates an anticipated complexity of autophagy flux which likely plays critical role in response to drug treatments, aging, and disease.
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    Analysis of pyridyloxobutyl and pyridylhydroxybutyl DNA adducts in extra-hepatic tissues of rats treated with tobacco-specific nitrosamines.
    (2009-07) Zhang, Siyi
    The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) and N’-nitrosonornicotine (NNN) are potent carcinogens in tobacco products, and are believed to play a major role as causes of tobacco-related cancers. NNK and NNN require metabolic activation to exert their carcinogenic effects. Cytochrome P450-catalyzed α-hydroxylation of NNK and NNN generates a reactive intermediate, which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. NNK is reduced to its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reversible and stereoselective manner. NNAL similarly undergoes α- hydroxylation and produces pyridylhydroxybutyl (PHB)-DNA adducts. In this thesis, we used liquid chromatography-electrospray ionization-tandem mass spectrometry (LCESI- MS/MS) to analyze POB- and PHB-DNA adducts in extra-hepatic tissues of F344 rats treated chronically with NNK and enantiomers of NNAL and NNN. POB- and PHB-DNA adduct levels were remarkably similar in NNK- and (S)-NNAL-treated rats, while distinctively different from those in (R)-NNAL-treated rats. These data indicate extensive retention of (S)-NNAL in various tissues of NNK-treated rats, and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis. (S)-NNN treatment produced more POB-DNA adducts in the rat oral mucosa, whereas adduct formation from (R)-NNN treatment was more favored in the nasal olfactory and respiratory mucosa. These results suggest that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues, and that (S)-NNN might be an oral carcinogen in rats. In additional studies of this thesis, LC-ESI-MS/MS methods were developed for the quantitative analysis of 1, N2-propanodeoxyguanosine adducts derived from acrolein and crotonaldehyde (Acr-dGuo and Cro-dGuo). Acrolein and crotonaldehyde are widely-spread environmental pollutants, are present in cigarette smoke, and are formed endogenously through lipid peroxidation. Our methods for the analysis of Acr-dGuo and Cro-dGuo adducts are sensitive, accurate, and precise. These adducts were detected for the first time in the human lung. However, no differences were observed in adduct levels between self-reported smokers and non-smokers. The potential importance of these adducts in the human lung requires further study.
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    Development, Characterization, and Applications of a 3D Printed micro Free-Flow Electrophoresis Device
    (2017-02) Anciaux, Sarah
    Micro free-flow electrophoresis (μFFE) is a unique separation technique because of its continuous nature. Analytes are pressure driven through a planar separation channel, and an electric field applied laterally to the flow producing a spatial separation. Fabrication methods associated with μFFE devices hinder our ability to address the limitations of μFFE. This work focuses on a novel fabrication method to reduce the overall fabrication cost and time, followed by validating and characterizing the device. A novel μFFE device is fabricated in acrylonitrile butadiene styrene (ABS) by 3D printing two sides of the device and then acetone vapor bonding them while simultaneously inserting electrodes and clarifying the device. Fluorescent dyes are separated, and their limit of detection determined. After validation of the new fabrication method, a new device design is made with the sample inlet modified so that 2D nLC × μFFE separations can be performed. 2D nLC × μFFE separations of fluorescent dyes, proteins, and tryptic BSA digest are demonstrated. These samples allow comparison between the surface properties of glass and 3D printed devices. Peak asymmetries, widths, and the interface were investigated. Minimal surface adsorption is observed for fluorescent dyes, proteins, and peptides, unlike in glass devices. After investigating surface properties, an open edge device for coupling to mass spectrometry is designed and compared to its glass counterpart. A novel ionization method is demonstrated from a hydrophobic membrane and the open edge device is shown to have stable flow.
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    Discovery, Dereplication, And Functional Assignment Of Natural Products In Streptomyces Coelicolor M145
    (2021-11) Marshall, Andrew
    Natural products are structurally complex molecules produced from numerous organisms such as fungi, plants, and bacteria. Sought after primarily for their therapeutic properties, especially as antibacterial agents, screening of natural product-producing organisms extracts from 1920-1960s resulted in a “golden age” of antibiotic discovery. However, this did not last as antibacterial resistance and rediscovery of known molecules became rampant. Re-invigoration of natural product screening efforts occurred as genome sequencing became commonplace and the biosynthetic potential of natural product producers was revealed to be much greater than previously thought. This has led to new efforts to exploit their biosynthetic potential. Key improvements in analytical tools, namely mass spectrometry, has accelerated efforts to sensitively examine natural product mixtures. Additionally, computational tools have enabled the analysis of the large datasets that are a result of these sensitive analytical tools, including quantitation and identification of many molecules, providing a more comprehensive view of natural product extracts than ever before. Application of these tools towards discovery and functional annotation of natural products enables greater insight into the production of these important molecules. This thesis is focused on developing and applying modern mass spectrometry-based approaches to investigate the natural products produced from Streptomyces coelicolor M145, the most well-studied streptomycete. First, ion mobility mass spectrometry is investigated and shown to be an effective approach to separate and distinguish isobaric cyclic-prodiginines that are produced by S. coelicolor. Second, discovery and characterization of a novel actinorhodin-related molecule, θ-actinorhodin, is accomplished using a combined metabolomics and proteomics approach. This represents the first known trimeric benzoisochromanequinone natural product and illustrates the effective use of metabolomics and proteomics on the same liquid chromatography-mass spectrometer. Third, a comprehensive analysis of the time-resolved relationship between gene expression and metabolite abundance of S. coelicolor is enabled by the use of mass spectrometry-based tools and correlation network analysis. This resulted in important conclusions about the concerted expression of biosynthetic gene clusters and the resulting production of the linked metabolite. Finally, several strategies are presented for future analysis of natural product extracts including the use of proteomics to identify putative Streptomyces using a collated list of natural product synthases and an approach combining modern dereplication tools and chemoselective probes for prioritization of natural products for purification.
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    DNA Modifications As Biomarkers For Precision Medicine In Patients Treated With Alkylating Drugs
    (2023) Guidolin, Valeria
    DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and oftentimes severe side effects in patients undergoing alkylating-drug based therapies. This observation supports the need for the development of biomarkers able to identify patients who effectively respond to certain therapies and recognize those who instead will develop serious side effects. The use of DNA adducts as predictive biomarkers has been proposed by several studies and herein reviewed. This dissertation focuses on the development and application of analytical methods able to comprehensively screen DNA adducts produced by alkylating drugs. The first study of this dissertation evaluates busulfan reactivity with DNA. Busulfan can promptly react with DNA, therefore, taking advantage of our DNA adductomic approach, DNA adducts formed by reacting busulfan with calf-thymus DNA were characterized. Samples collected from 6 patients undergoing busulfan-based chemotherapy prior to allogeneic hematopoietic cell transplantation were analyzed for the presence of busulfan-derived DNA adducts. Among the 15 adducts detected in vitro, 12 were observed in the patient blood confirming the presence of a large profile of DNA adducts in vivo. Two of the detected adducts were structurally confirmed by comparison with synthetic standards and quantified in patients. Similarly, in the second study, an extensive profile of DNA adducts generated by cyclophosphamide was characterized. Cyclophosphamide is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity. Our DNA adductomic method has been optimized and tailored to maximize the detection of cyclophosphamide-derived adducts. Furthermore, the use of 15N-bacterial DNA served as further confirmation for DNA adduct identification and structural elucidation. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with cyclophosphamide. The last study focused on the synthesis of a cyclophosphamide-derived DNA adduct to be used for quantitation and as an internal standard for future studies. Departing from reported synthetic schemes, several different approaches were tested. This study is currently ongoing, but the reaction schemes tested allowed for a better understanding of dGuo-alkylation regioselectivity. Overall, the work described in this thesis set the stage for the evaluation of a relationship between busulfan and cyclophosphamide DNA adducts and therapy outcome to identify DNA adducts to be used to stratify patients and distinguish who will benefit from therapies from those who may experience severe adverse toxic outcomes.
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    DNA-protein crossing-linking by BIS-electrophiles
    (2008-10) Loeber, Rachel Lea
    A key carcinogenic metabolite of the important industrial chemical 1,3-butadiene (BD), DEB is a bifunctional alkylating agent capable of reacting with both DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)- guanine monoadducts, which can then react with a second nucleophilic site to form crosslinked adducts. A recent report revealed a strong correlation between expression of the human DNA repair protein AGT in bacteria and the cytotoxic and mutagenic activity of DEB (J. G. Valadez et al., Chem. Res. Toxicol. 17 (2004) 972-982). As AGT expression appeared to enhance the toxic effects of this bis-electrophile, the authors proposed that DEB induces AGT-DNA cross-links. The purpose of our study was to structurally characterize DEB-induced AGT-DNA conjugates and to identify amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was first detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of both wild-type AGT or a C145A AGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol- dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+- MS/MS sequencing of tryptic peptides obtained from dG monoepoxide-treated AGT indicated that the two cross linking sites were located at the alkyl acceptor site, Cys145 39 and a neighboring active site residue, Cys150. The same two active site cysteines became cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+-MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of AGT (C = Cys145 ). Replacement of the catalytic cysteine residue with alanine (C145A AGT) abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified AGT analyzed in parallel with an authentic standard. Based upon these results, the formation of AGT-DNA cross-links is a likely mechanism to explain the enhanced cytotoxicity of DEB in cells expressing this important repair protein.
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    Expanding Computational Resources for the Discovery of Molecular Biomarkers of Exposure
    (2024-02) Murray, Kevin
    Recurrent exposure to genotoxic chemicals and other harmful environmental agents is directly related to the development of adverse phenotypes, carcinogenesis, and developmental disorders through chemical-mediated toxicity. Systems toxicology aims to chemically profile and computationally model the toxicity pathways to gain mechanistic insights into the outcomes of environmental exposure and develop modern safety guidelines. Analytical methods are being developed to perform broad, unbiased characterization of molecular biomarkers of exposure to improve the understanding of the molecular consequences and physiological responses following the introduction of reactive electrophilic chemicals. There are few computational workflows available to support this rapid development of new analytical technology. This thesis introduces novel computational resources for the parameterized analysis of discovery-driven, systems toxicology results generated by liquid chromatography-coupled mass spectrometry. Chapter 1 of this thesis presents a comprehensive review of existing analytical technology for the molecular profiling of exposure biomarkers. This review focuses on discovering toxicologically relevant compounds using constant ion monitoring, fragmentation filtering, in-source collision-induced dissociation, mass defect filtering, or isotope pattern filtering. The mechanism of discovery of each analytical method is examined and the strengths and limitations of each approach are discussed. Chapter 2 of this thesis presents a novel computational workflow for discovering molecular biomarkers of exposure using fragmentation filtering. In this study, an original module named DFBuilder for the metabolomics data processing software MZmine is presented. The application of this software tool for the discovery of covalently modified DNA nucleosides is evaluated. In this work, a novel colibactin-derived, E. coli associated DNA adduct product discovery is highlighted. Chapter 3 of this thesis extends the application of the DFBuilder module to discover urinary metabolites produced from detoxifying reactive electrophilic chemicals in tobacco cigarette smoke. This work presents the first reported application of a high-resolution mass spectrometry method for profiling mercapturic acid conjugates in positive ion mode. The combination of this novel analytical method and computational workflow discovers numerous prospective mercapturic acid signatures never reported in human urine. Statistical evaluation of these results demonstrates that many of these products are associated with cigarette usage. Chapter 4 of this thesis reports a novel mass spectral library of conjugated mercapturic acids. Using multiple fragmentation strategies, this library represents thousands of mass spectra collected in positive and negative ion polarity. This work serves as a foundation of resources necessary for verifying discovery results produced from the analytical methods presented in this thesis. Metadata insights from this library that will help future efforts to characterize mercapturic acid conjugates are discussed. This thesis concludes with a summary and future perspective evaluating the remaining computational challenges in exposomics and analytical chemistry-based approaches for systems toxicology. The areas that most need support are highlighted, and the capacity of emerging computational solutions to improve experimental outcomes is discussed.
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    Functional Proteomics Analysis To Discover And Characterize Oxygen-Dependent Cellular Pathways
    (2019-02) Erber, Luke
    Oxygen and iron homeostasis are a critical components for the maintenance of cellular biology. These metabolites are essential substrates in cellular metabolism, signaling and bioenergetics, thereby inseparably linked to the normal physiology of all metazoans. To adapt to changes in the microenvironment, cells dynamically modulate hypoxia response pathways. Lack of oxygen reduces the post-translational modification proline hydroxylation and altering key transcription factors such as hypoxia-inducible factor 1alpha and prevents its hydroxyproline-dependent degradation. Stabilized HIF proteins activate the expression of hypoxiaresponse genes to sustain growth under hypoxia condition. The studies herein focus on the hypothesis that post-translational modifications of hydroxylation and phosphorylation are a mechanistic link between oxygen and iron availability and the cellular physiological response. This research has focused on the characterization of oxygen and iron-sensing pathways dependent on proline hydroxylation and phosphorylation. Through a system-wide proteomics survey, I identified Brd4 as a novel proline hydroxylation protein substrate in cancer cells. Specific prolyl hydroxylase activity significantly regulates the Brd4-mediated transcriptional function and strongly induced acute myeloid leukemia cell proliferation and apoptosis. This study integrates molecular biology and quantitative proteomics approaches to discover and characterize cellular oxygen and iron-sensing physiology and reveal novel cellular pathways that may have broad impact in cancer biology and metabolic diseases.
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    Mass Spectrometry Based Analysis Of Electrophile Induced Adducts Of Biomolecules
    (2019-08) Degner, Amanda
    Humans are exposed to a wide range of electrophilic agents, which can form covalent adducts at nucleophilic sites in biomolecules including DNA and proteins. If not repaired, DNA adducts can inhibit biological processes such as DNA replication and transcription, which can lead to mutagenesis, carcinogenesis, and toxicity. DNA repair pathways exist to remove these lesions, but the role of different pathways in the repair of different DNA adducts is not fully understood. Additionally, electrophiles can be detoxified through further metabolism such as glutathione conjugation before they are able to form adducts. Interindividual differences in the balance between detoxification, adduct formation, and repair ultimately determines the risk a person has of developing cancer after carcinogen exposure. In Chapter 2, we investigated the role of nucleotide excision repair (NER) and Fanconi Anemia (FA) repair pathways in repair of a 1,3-butadiene (BD) induced DNA-DNA interstrand crosslink (ICL), bis-N7G-BD. NanoLC-nanoESI+-MS/MS methodology was employed to quantify the amount of crosslinks present after human cell lines deficient in components of NER or FA and their isogenic controls were treated with an electrophilic metabolite of BD, 1,2,3,4-diepoxybutane (DEB), and allowed to repair for up to 72 h. Despite observing increased sensitivity to DEB treatment in repair deficient cell lines in cytotoxicity assays, no change in repair kinetics was observed between repair deficient and control cells, suggesting that in humans, there is a redundancy in DNA repair pathways. The same nanoLC-nanoESI+-MS/MS methodology was employed to measure bis-N7G-BD formation in HL60 cells, where crosslink levels correlated with cytotoxicity and micronuclei formation. In Chapter 3, we investigated the roll of a glutathione S-transferase theta 1 (GSTT1) in detoxification of another epoxide metabolite of BD, 3,4-epoxy-1-butene (EB). In a previous genome wide association study, differences in GSTT1 copy number explained a fraction of ethnic differences in BD metabolism, which could contribute to ethnic differences in smoking-induced lung cancer risk. A HPLC-ESI+-MS/MS method was developed for the quantitation of EB-GSH conjugates in cells that did or did not express GSTT1 after treatment of EB. No difference in EB-GSH conjugates or EB-GII DNA adduct levels were observed between GSTT1-/- and GSTT1+/+ cell lines. However, the expression of GSTT1 was had a protective effect against EB-induced apoptosis. In Chapter 4, an unknown electrophile-induced adduct to hemoglobin (Hb) was identified. Previous adductomic screens of human blood samples using the FIRE procedure to screen for adducts to N-terminal valine in Hb found an unknown adduct corresponding to an added mass of 106.042 Da. This adduct was identified as a 4-OHBn adduct to N-terminal valine, which was confirmed by HPLC-ESI+-HRMS analysis of authentic synthesized standard. Accurate mass, retention time, and MS/MS fragmentation patterns between the standard and unknown adduct matched. Levels of the 4OHBn-Val adduct were quantified to be 380 ± 160 pmol/g Hb in 12 human blood samples. Both 4-quinone methide and 4-hydroxybenzaldehyde were found to be capable of forming this adduct and thus were identified as possible sources.
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    Mass Spectrometry-Centered Multi-Omic Applications In The Analysis Of Inflammation And Exposure
    (2022-10) Rajczewski, Andrew
    Bottom-up proteomics represents an exciting technology which has found great utility across multiple fields of biological research. Using high-resolution mass spectrometry coupled with sophisticated bioinformatic software applications, bottom-up proteomics affords qualitative and quantitative information that reflects the actual molecular phenotype of a system in ways that next-generation sequencing technologies do not. Despite this, there are also blind spots in conventional bottom-up proteomics experiments; many of these limitations can be abrogated via the integration of bottom-up proteomics with other forms of ‘omics technologies and data. Through supplemental bioinformatic workflows, putative identifications of non-canonical peptides or non-host peptides (e.g microbial, viral) can be validated. The use of RNA-Seq data can be used to generate protein sequence databases files for proteogenomics, where non-canonical peptide sequences arising from genomic mutations, translocations, aberrant splicing events, etc. which are invisible to conventional proteomics experiments can be readily detected. In addition, by integrating and directly comparing proteomics data with transcriptomic data, levels of epigenetic and/or post-transcriptional control can be examined in a system in response to stimuli of interest that are invisible to both technologies. These supplemental approaches expand the power of bottom-up proteomics to where it becomes a highly useful tool for studying systems in which multiple levels of gene product expression response are regulated, including viral infections, tissues undergoing long-term inflammation, and exposure to endogenous and exogenous electrophiles.The first chapter of this thesis provides an overview of the current state of proteomics-centered multi-omic technologies and their potential utility in biological research. The review begins with outlining the improvements to bottom-up proteomics technologies which have enabled a greater depth of information, such as isobaric peptide tagging, data-independent acquisition, ion mobility applications, and other instrumental advances. From there, bioinformatic tools are discussed that are of use in the analysis of proteomics data, with a focus on integrating mass spectrometry-based data with other forms of ‘omics data. Specific applications of using RNA-Seq data to inform the data analysis of proteomics, proteogenomics, are also addressed. The chapter concludes with notable instances of proteomics-centered multi-omics analysis as well as potential future applications of these technologies. The second chapter of this thesis addresses the analysis of open-source proteomics datasets with customized multi-omic bioinformatic tools to determine the optimal targets for the detection of SARS-CoV-2 infections in patients. Through the use of in vitro and patient datasets, a panel of potential viral peptides were established, was used to search patient datasets. Ultimately, we found four peptides in the viral nucleocapsid which were reliably detected in patients and were unique to the SARS-CoV-2 virus. The third chapter of this thesis utilizes proteogenomics workflows to examine the consequences of long-term inflammation in the proximal colon tissue of a murine model of inflammatory bowel disease. In this model, Rag2-/-Il-10-/- mice were subjected to five months of Helicobacter hepaticus infection in their colon to trigger chronic infection. For these analyses, RNA-Seq data acquired from these test subjects in an earlier study were converted into a FASTA protein sequence database containing variant sequences stemming from this treatment. Through quantitative proteogenomic analysis we noted significant changes in abundances of proteins consistent with an inflammatory response; through bioinformatic analysis of our data, we also validated and confirmed the presence of 39 non-canonical peptides across our infected and control samples, demonstrating the importance of validation of targets of interest in proteogenomic studies. The fourth chapter of this thesis integrates multiple levels of ‘omic analysis to examine the effects of inflammation on murine Type II pneumocytes, a constituent cell within alveoli which serve as the source of lung adenocarcinomas. Mice were exposed to intranasal dosages of LPS or to whole-body cigarette smoke exposure for variable amounts of time before being sacrificed and the Type II cells isolated for analysis. Bottom-up proteomics of cells subjected to LPS for 3 weeks revealed a phenotype consistent with inflammation; this was reinforced when compared to transcriptomic data from the same cells, as these showed. Global proteomics analyses of Type II pneumocytes of mice subjected to exposure to cigarette smoke revealed significant changes in protein abundances occurring after after 10 weeks of exposure with a 4-week recovery period post exposure, encompassing biological processes such as nucleotide and amide metabolism as well as synthesis and acetyl CoA synthesis, which demonstrated a greater degree of disjuncture with the associated RNA-Seq data as compared to our LPS study. The fifth chapter of this thesis examines the utility of bottom-up proteomics in examining the formation of amino acid adducts in hemoglobin, which serves as a valuable reservoir for exposome studies due to its longevity and high concentration within the blood. We were able to validate the presence of 4-hydroxybenzyl adducts at the N-terminal valine of hemoglobin and demonstrate their formation at nucleophilic side chains within the protein. In addition, we compared bottom-up proteomics to the FIRE method, an experimental procedure which serves to isolate N-terminal adducts in hemoglobin for LC-MS detection, with a panel of electrophilic compounds incubated with blood at various concentrations and incubation times. Ultimately, we found that a proteomics-based approach to untargeted adductomics allowed for the detection of novel adducts at a number of sites within hemoglobin. In this thesis we have applied mass spectrometry-based ‘omics technologies to complicated biological systems. We have utilized publicly available proteomics datasets to determine the optimal targets for MS-based detection of SARS-CoV-2 in patient samples. Using RNA-Seq data, we performed quantitative proteogenomic analysis of a murine model of IBD and validated the presence of several non-canonical peptide sequences. We also used multi-omic analyses to compare LPS-driven and cigarette smoke-driven inflammation of murine Type II pneumocytes. Finally, we demonstrated the utility of bottom-up proteomics in detecting and characterizing adducts in human hemoglobin as a record of the exposome. Overall, this work expands the utility of proteomics-centered analyses in characterizing systems subjected to viral infection, inflammatory stimuli, and exposure to environmental contaminants.
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    New particle formation: sulfuric acid and amine Chemical nucleation photochemical reaction chamber studies and the laboratory cluster-CIMS.
    (2012-02) Titcombe, Mari E.
    The formation of new particles from gas phase condensation has been shown to significantly enhance concentrations of cloud condensation nuclei (CCN) in the Earth's atmosphere. Particles that have grown to CCN size contribute significantly to Earth's radiation balance. And particle nucleation has been observed throughout the atmosphere in varying meteorological conditions. Yet the chemical processes involved in particle nucleation are not well understood. Sulfuric acid has long been recognized as a contributor to new particle formation. However, sulfuric acid condensation alone cannot account for high particle production and growth rates observed in many regions of the atmosphere. Scientific understanding of these processes has been limited by available instrumentation and the chemical complexity of the atmosphere. A novel Chemical Ionization Mass Spectrometer (cluster-CIMS) has been developed, in collaboration with colleagues at the National Center for Atmospheric Research, to characterize homogeneously nucleated molecular clusters produced in a controlled laboratory environment. The cluster-CIMS gently ionizes neutral molecular clusters for quadrupole mass filtration with a minimum of disturbance to cluster composition. It is capable of characterizing particles from molecular sizes up to 1.5 nm in diameter with a resolution of +/- 1 amu. A climate controlled photochemical reaction chamber, designed as a 1000 L batch reactor, was built to produce nucleated molecular clusters at atmospherically relevant conditions. Laboratory experiments were conducted to elucidate potential molecular candidates for particle nucleation. The role of amines in particle formation was experimentally examined after atmospheric observations revealed enhanced sulfuric acid nucleation rates in the presence of amine compounds. Experimental results obtained with the cluster-CIMS, as well as other aerosol instrumentation, support the hypothesis that amines enhance sulfuric acid nucleation rates.
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    Plasma Extracellular Vessicles, Exercise Resistnace and Cancer
    (2021-07) Vanderboom, Patrick
    Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size exclusion chromatography (SEC)-based method to purify EVs from platelet poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identified more proteins with higher precision compared to several conventional EV isolation approaches. We applied the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Further, we applied the SEC-MS approach to evaluate the effects of endurance and resistance exercise on EV proteomic cargo in human plasma. A prerequisite to studying the role of EVs in metabolic disorders such as type 2 diabetes is an in-depth understanding of the molecular mechanisms involved in dysregulation of insulin sensitive tissues such as skeletal muscle. In line with this, we utilized a multi-omics approach to characterize mechanisms underpinning exercise resistance in obese individuals. In this study we characterized the difference in the response to acute exercise between healthy subjects and individuals with chronic inflammation using skeletal muscle biopsy samples. This study identified dysregulation of diverse signaling pathways, including glycogen synthesis, myogenesis and AMPK signaling in obese individuals. Finally, we utilized the biomarker potential of SEC-MS to identify differences in protein abundance and composition in EVs isolated from individuals with the hematological malignancy multiple myeloma (MM) and its precursor disease multiple myeloma of undetermined significance (MGUS). This analysis found that CD71 was upregulated in small and large EVs in individuals with MM relative to MGUS, suggesting that iron utilization and homeostasis in the tumor micro environment may play a role in disease progression. Additionally, CD40 was upregulated in individuals with standard cytogenetik risk MM relative to MGUS but was not identified in individuals with high risk MM suggesting that adaptive immune function is correlated to cytogenetic risk. These proteins hold the potential to be useful biomarkers to monitor disease progression in the clinic.