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Functional Characterization Of Microbial Symbiotic Associations By Metaproteomics, Jacque Caprio Young

Doctoral Dissertations

Rarely are microbes found in isolation in the environment, but rather form symbiotic associations with other microbes or eukaryotic hosts. The advent of the systems biology era has allowed global characterization of these symbiotic associations at levels not previously possible. However, while metagenomic studies have revealed microbial membership and potential genomic information among members taking part in the symbiosis, there is still a significant lag in the functional characterization within these symbiotic associations. Thus, in this dissertation, we utilized a metaproteomic approach to study microbial symbiotic associations. We have developed and applied this robust platform to investigate various symbiotic associations …

Direct Analysis Of Hops By Leaf Spray And Paper Spray Mass Spectrometry, Kari Blain

Masters Theses

The objective of this research is to develop a new and innovative method of hops analysis, which is much faster than standard testing methods, as well as reduce the amount of consumables and solvent used. A detailed discussion on the development of an ambient ionization mass spectrometry method called paper spray (PS-MS) and leaf spray (LS-MS) mass spectrometry will be presented. This research investigates the use of PS-MS and LS-MS techniques to determine the α- and β- acids present in hops. PS-MS and LS-MS provide a fast way to analyze hops samples by delivering data as rapidly as a UV-Vis …

New Applications Of Mass Spectrometry For Drug And Lipid Analysis, Elizabeth Lauren Emerson

Graduate Theses and Dissertations

Mass spectrometry is an important tool used in many different disciplines and settings that include forensics, drug discovery, environmental analysis, and proteomics. Gas chromatography - mass spectrometry (GC-MS) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI - TOF MS) are two of the most important instruments used for analysis of compounds. Chapters 1 and 2 of this discussion use GC-MS for the investigation of synthetic cannabinoids in `K2' incense products and the detection of metabolites in urine samples from individuals suspected of consuming these mixtures. Analytical standards were synthesized and used for identification and confirmation of structures. Detection of …

Analysis Of Adp-Ribosylation And Protein Sample Preparation By Capillary Electrophoresis, Ming Huang

Electronic Thesis and Dissertation Repository

ADP-ribosylation is one of many biologically important protein post-translational modifications (PTMs) that received considerable research interest in the past decade. The first part of this work presents the analysis of ADP-ribosylation, with special focus on the catalytic domain of an enzyme ARTD-10. The application of capillary electrophoresis (CE) is proposed in this work to separate the in vitro enzymatic mono-ADP-ribosylated ARTD-10 catalytic domain and its unmodified form. Another exciting aspect of CE application with growing interest is the sample preparation. The effectiveness and efficiency for in-vial reaction, in-capillary mixed reaction and on-capillary-tip reaction by performing methyl esterification on phophopeptides were …

Migration Behaviour Of Discontinuous Buffers In Capillary Electrophoresis During Protein Enrichment, Ting Li

Electronic Thesis and Dissertation Repository

To enrich protein at sub-microliter sample volumes, our group has developed a discontinuous buffer system which consists of an acid and a base inside a capillary. Upon voltage application, the protein moves towards and stacks at the pH junction. Prior to this work, we only focused on the protein enrichment and have not investigated the migration behaviour of various ions in this system. However, it is important to understand the mechanism of the migration of these ions, because the pH junction formation and further the protein enrichment are based on it. In this work, the ion migration patterns at the …

Biophysical And Bioanalytical Studies Of Hyaluronan And Lipids., Kristen Ann Magness 1981-

Electronic Theses and Dissertations

Hyaluronan (HA) is a major structural component of the ocular vitreous humor (VH) and lipids are essential building blocks of cell membranes. However, little is known about their possible interactions. The VH is known to undergo liquefaction throughout the lifetime that may result in complications such as posterior vitreous detachment. The possible roles of lipids in the age-dependent liquefaction of the VH are unknown. As a first step in this new area of research in our group, model studies of the repeating unit of polymeric HA were performed. This unit contains glucuronate (GlcU) linked to N-acetylglucosamine (G1cNAc) via a 1-3 …

Resolving Disulfide Bond Patterns In Snap25b Cysteine-Rich Region Using Lc Mass Spectrometry, Nozomi Ogawa

Theses and Dissertations

A global analysis of the human proteome demonstrates that there are ~5500 tryptic fragments that contain four cysteines in close proximity. Elucidating whether they form disulfide bonds in vivo under different conditions is particularly important because cysteines are known to be a vital cellular redox sensor as well as a catalytic site for important biochemical reactions. However, currently there are no methods that can resolve disulfide patterns in closely-packed cysteine residues from a complex sample. In order to address this problem, we have developed a novel mass-spectrometry-based method to identify the different disulfide bonding patterns possible, using SNAP25B cysteine-rich region …

Mass Spectrometry-Based Strategies For Protein Biophysics: Effects Of Modification, Structure, And Interactions, Richard Yu-Cheng Huang

All Theses and Dissertations (ETDs)

Two important biophysical characteristics of proteins, their interaction with ligands and their post-translational modifications, modulate various biological processes including signal transduction, chemical synthesis, and cell function. Protein-ligand interactions include the interactions with protein, peptide, DNA, and metal. Characterization of the physical properties of these interactions: binding interfaces, binding affinities, and the protein conformational changes due to the binding) is essential in understanding the mechanism of related diseases and, more importantly, in future drug design.

Mass spectrometry, with its own revolution and improvement, becomes a powerful tool in protein and peptide analysis. In this thesis, we applied two mass spectrometry-based strategies, …

Dissecting Key Determinants For Calcium And Calmodulin Regulation Of Gap Junction And Viral Protein, Yanyi Chen

Chemistry Dissertations

Calcium and calmodulin are implicated in mediating the Ca²⁺-dependent regulation of gap junctions that are essential for the intercellular transmission of molecules such as nutrients, metabolites, metal ions and signal messengers (< 1000 Da) through its specialized cell membrane channels and communication to extracellular environment. To understand the key determinants for calcium and calmodulin regulation of gap junction, in this study, we identified a calmodulin binding domain in the second half of the intracellular loop of Cxonnexin50 (the major gap junction protein found in an eye lens) using peptide fragments that encompass predicted CaM binding sites and various biophysical methods. Our study provides the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx50 in a Ca²⁺-dependent manner. Furthermore, two novel CaM binding regions in cytosolic loop and C-termini of Connexin43 (the most ubiquitous connexin) have been shown to interact with CaM with different binding modes in the presence of Ca²⁺ using high resolution NMR. Our results also elucidate the molecular determinants of regulation of gap junction by multiple CaM targeting regions and provide insight into the molecular basis of …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …

Structural Characterization Of Protein Folding Intermediates By Oxidative Labeling And Mass Spectrometry, Bradley B. Stocks

Electronic Thesis and Dissertation Repository

A key challenge associated with protein folding studies is the characterization of short-lived intermediates that become populated en route to the native state. In this work, a covalent labeling method was developed that provides insights into the structures of these transient species. Hydroxyl radical (·OH) reacts with solvent-exposed side chains, whereas buried residues are protected. Mass spectrometry is used for monitoring the locations and the extent of labeling. Pulsed ·OH labeling of proteins at selected time points during folding results in high temporal and spatial resolution when compared to existing other labeling methods.

This novel technique was validated by studying …

Identification And Quantification Of Protein Carbonylation By Mass Spectrometry, Qingyuan Liu

Theses and Dissertations

Accumulated evidence indicates oxidative stress plays important roles in disease and aging. Under oxidative stress, lipid peroxidation (LPO) leads to reactive carbonyl species (RCS) that can modify a wide range of biomolecules including protein, DNA and carbohydrate. In this dissertation, we investigate the modification of two model proteins, human serum albumin (HSA) and aconitase (ACO), by the LPO-relevant a, b-unsaturated aldehydes, acrolein (ACR) and 4-hydroxy-2-nonenal (HNE). The investigation is focused on the characterization and quantification ACR and HNE addition to the model proteins. A correlation between HNE modification and ACO activity is also determined. These results provide insights into the …

Chronic Kidney Disease: Vitamin D Treatment Regimens And Novel Assay Development For Kidney And Cardiovascular Function Biomarkers, Joe M. El-Khoury

ETD Archive

Chronic kidney disease (CKD) is highly prevalent in the US population and has high incidence of cardiovascular and all-cause mortality. A known complication of CKD is secondary hyperparathyroidism that is caused by bone and mineral imbalances, including vitamin D deficiency. Supplementation of CKD patients with vitamin D is based on guidelines issued by the Kidney Disease Quality Outcomes Initiative (K/DOQI), which recommend administration of vitamin D2 in variable doses depending on the severity of vitamin D deficiency. Retrospective and pilot studies have shown that vitamin D2 was not as effective as vitamin D3 in treating vitamin D deficiency. In Chapter …

Mass Spectrometry Applied To Problems In Lipid Biochemistry: Microchip Based Approach For Lipidomics Profiling And Analysis Of Lipid Metabolites By Lc-Ms/Ms, Tao Sun

Electronic Theses and Dissertations

Lipidomics and metabolomics are powerful tools for the examination of cellular metabolism and physiology. Methods for lipid analysis need to be developed that begin with small samples and do not overly dilute or disperse the sample in the separation process. Microchips provide a platform for interfacing lysis of small cell populations with on-chip solid phase extraction for isolating lipid samples to generate high quality mass spectra from very small samples. Chapter 1 of this dissertation presents a novel method for small scale lipidomics of bacterial cells by microchip based extraction coupled with untargeted profiling of glycerophospholipids using nanoelectrospray ionization mass …

Tandem Mass Spectrometric Analysis Of Ammonium And Sodium Oligoperoxide Adducts With The Application Of Two-Dimensional Correlation Spectroscopy And Computational Chemistry, Jessica Lynne Frisch

Electronic Theses and Dissertations

Oligoperoxides, H[OO(CH3)2C]nOOH, are formed as side products in the synthesis of the primary high explosive triacetone triperoxide (TATP). Previous tandem mass spectrometry (MSn ) experiments using a quadrupole ion trap reported that the openchained oligoperoxide adducts of ammonium or sodium resulted in different product ions in the mass spectra. Dissociation mechanisms were previously proposed based on MSn experiments, where n > 2; however, a dissociation pathway achieved by an MSn experiment, where n > 2, may not necessarily be the same pathway achieved in an MS2 experiment. For this dissertation research, the collision induced dissociation pathways were investigated for the open-chained oligoperoxides …

Noncovalent Interactions In The E. Coli Replisome: Novel Aspects Of Single-Stranded Dna-Binding Protein (Ssb) And The Β2 Sliding Clamp, Claire E. Mason

University of Wollongong Thesis Collection 1954-2016

This thesis presents analyses of key protein-protein and protein-nucleic acid interactions in the E. coli replication machinery. The E. coli single-stranded DNA-binding protein (SSB) is a central mediator of DNA metabolism, binding to regions of exposed single-stranded DNA (ssDNA) and playing crucial roles in DNA replication, recombination and repair. SSB is a stable homotetramer, held together by interactions between the N-terminal domains of its subunits. Structurally, the N-terminal domains form classic oligonucleotide-binding folds (OB-folds), and are also responsible for ssDNA binding. The SSB-ssDNA complexes may adopt a number of configurations, differing in the extent of SSB-ssDNA contact, and are heavily …

Mass Spectrometric Approaches For The Structural Characterisation Of Lipids By Ozonolysis, Michael Christopher Thomas

University of Wollongong Thesis Collection 1954-2016

Lipids are the main structural component of biological membranes and are important in complex biochemical processes including cellular signalling. Using electrospray ionisation-tandem mass spectrometry (ESI-MS/MS), information regarding fatty acid chain length and degree of unsaturation can be gained for complex lipids. Determining double bond position, however, remains challenging.

To address the limitations of collision-induced dissociation (CID) for structural characterisation, the use of ozonolysis chemistry in conjunction with mass spectrometry was explored. Initial experiments concentrated on performing ozonolysis within the electrospray source of conventional mass spectrometers; referred to here as ozone electrospray ionisation-mass spectrometry (OzESI-MS). In OzESI-MS experiments, two ozonolysis product …

New Capabilities And Applications Of Ambient Ionization Mass Spectrometry For The Surface Analysis And Imaging Of Lipids, Shane R. Ellis

University of Wollongong Thesis Collection 1954-2016

Mass spectrometry is arguably the most important technique used in lipid analysis. It provides unparalleled sensitivity and molecular specificity in the detection, quantification and structure elucidation of the diverse range of lipids present in complex biological matrices. The recent and rapid emergence of ambient ionization mass spectrometry presents new and exciting opportunities for the direct detection of lipids from surfaces, including those commonly encountered in lipid research such as tissue sections, cells and thin-layer chromatography plates. This thesis describes the application, optimization and analytical capabilities for two contemporary ambient ionization technologies - namely desorption electrospray ionization (DESI) and liquid extraction …

Infrared Laser Ablation For Biological Mass Spectrometry, Fan Huang

LSU Doctoral Dissertations

Instrument and technique development and modeling of ionization methods using infrared (IR) laser desorption and ablation for mass spectrometry (MS) analysis of chemical and biochemical samples are described. Infrared lasers are highly efficient at sample removal and post-ablation ionization of materials removed by IR laser can be used to improve ionization efficiency. Fundamental studies of the physical processes in laser ablation mass spectrometry can help elucidate the mechanism. In this research, an infrared/ultraviolet two-laser matrix-assisted laser desorption ionization (MALDI) MS was developed for analysis of biomolecules. An infrared laser was used to ablate a mixture of analyte and matrix, and …

Computational Methods For The Differential Profiling Of Triacylglycerols Using Rp-Hplc/Apci-Ms, Margaret Holbrook Broadwater

MUSC Theses and Dissertations

Reversed phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) was employed for the analysis of natural mixtures of triacylglycerols. An integrated framework for data analysis, including preprocessing, statistical analysis and automated structure identification, was implemented in the R statistical program. Raw data stored as mzXML, mzData, or mzXML files are preprocessed using a series of steps for peak detection, chromatographic alignment, and normalization. Targeted and non-targeted feature selection steps are employed to filter the data for features that are relevant and informative for a particular biological question. Triacylglycerol structures are identified by evaluating relationships between the diacylglycerol …

Ambient Ionization Mass Spectrometry Analysis Pertaining To Biofuel Process Development, Sushma Dendukuri

Online Theses and Dissertations

Mass spectrometry (MS) has undergone a revolution with the introduction of a new group of desorption/ionization (DI) techniques known collectively as Ambient Ionization mass spectrometry. Ambient ionization mass spectrometry allows for direct analysis of analytes and typically requires little or no sample preparation. Specifically, Direct Analysis in Real Time (DART) has developed rapidly and allowed investigations to occur with the analysis of biomass after various pretreatments to determine the pretreatment biomass degradation efficiency. The degradation of the initial biomass structure is a very important step before other chemical treatments can occur to generate needed sugars. Since generated sugars aid in …