Biochemistry Commons^™

High Resolution Mass Spectrometry As A Platform For The Analysis Of Polyoxometalates, Their Solution Phase Dynamics, And Their Biological Interactions., Daniel T. Favre

Doctoral Dissertations

Polyoxometalates (POMs) are a class of inorganic molecule of increasing interest to the inorganic, bioinorganic and catalytic communities among many others. While their prevalence in research has increased, tools and methodologies for the analysis of their fundamental characteristics still need further development. Decavanadate (V10) specifically has been postulated to have several unique properties that have not been confirmed independently. Mass spectrometry (MS) and its ability to determine the composition of solution phase species by both mass and charge is uniquely well suited to the analysis of POMs. In this work we utilized high-resolution mass spectrometry to characterize V10 in aqueous …

Protein Stability In Solution And In The Gas Phase., Yousef Haidar

Electronic Thesis and Dissertation Repository

Electrospray Ionization mass spectrometry (ESI-MS) is widely used for probing proteins, yet many aspects of this technique remain elusive. Using MS, ion mobility spectrometry (IMS), and circular dichroism (CD) spectroscopy, this thesis sheds light on the stability differences of proteins in the gas phase and solution. After a general introduction (Chapter 1), Chapter 2 scrutinizes some aspects of native ESI. Our data highlight the significance of cone voltage in maintaining a native-like fold and show the advantage of using NH₄Ac in protein experiments. Chapter 3 focuses on hydrogen/deuterium exchange (HDX)-MS. Several studies have reported that D₂O …

Eluication Of Lipid Metabolic Pathways In Differentiating Giardia Lamblia Using High Resolution Mass Spectrometry, Cameron Ellis

Open Access Theses & Dissertations

Giardia lamblia is an intestinal protozoan found worldwide, including the U.S. This parasite exists in two morphologic stages - a replicative trophozoite and a relatively dormant yet viable cyst. While exposures of cysts to gastric acid during passage through the human stomach induces excystation, factors in the small intestine, where trophozoites colonize trigger encystation or cyst formation. Transformation into cyst stage is essential for Giardia to survive in the environment for months before infecting new hosts. Because of its small genome size (11.7 Mb), metabolic pathways in Giardia are highly reduced. As far as lipid metabolism is concerned, only limited …

Deciphering Phosphoprotein Phosphatase Signaling Networks Using Proteomics Approaches, Brooke Brauer

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a highly regulated mechanism of cell signaling control and its deregulation is implicated in disease. The kinases that catalyze the addition of phosphate groups onto their substrate proteins have been well studied, their signaling pathways mapped, and their effects on cell and organismal health observed. Knowledge of the phosphatases that reverse the reaction only recently began to come into focus. Phosphoprotein phosphatases (PPPs), long thought to be housekeeping enzymes, are now known to be exquisitely specific towards their substrates, but the exact nature of phosphatase regulation—both upstream and downstream of the phosphatase—is unclear.

PPPs recognize substrates through …

Deciphering Protein Higher-Order Structure And Interactions Via Diethylpyrocarbonate Labeling-Mass Spectrometry, Xiao Pan

Doctoral Dissertations

The study of protein higher-order structures is vital because it is closely related to the investigation of protein folding, aggregation, interaction and protein therapeutics. Consequently, numerous biochemical and biophysical tools have been developed to study protein higher-order structures in many different situations. The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a powerful tool for studying protein structures and offers many advantages over other traditional techniques, such as better structural coverage, high throughput, high sensitivity, and the ability to study proteins in mixtures. This dissertation focuses on diethylpyrocarbonate (DEPC) as an effective CL reagent that can …

Amyloidogenesis Of Β-2-Microglobulin Studied By Mass Spectrometry And Covalent Labeling, Blaise G. Arden

Doctoral Dissertations

Amyloid-forming proteins are implicated in a number of debilitating diseases. While many amyloid-forming proteins are well studied, the early stages of amyloidosis are still not well understood on a molecular level. Covalent labeling, combined with mass spectrometry (CL-MS), is uniquely well suited to provide molecular-level insight into the factors governing the early stages of amyloidosis. This dissertation leverages CL-MS techniques to examine the early stages of β-2-microglobulin (β2m) amyloidosis. β2m is the protein that forms amyloids in the condition known as dialysis-related amyloidosis. An automated CL-MS technique that uses dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide as a labeling reagent was developed and used …

Lcms-Based Analysis Explains The Basis Of Oxidative Resistance In Selenium-Containing Thioredoxin Reductase, Daniel Haupt

Graduate College Dissertations and Theses

Selenocysteine (Sec) is referred to as the 21st proteogenic amino acid and is found in place of the redox-sensitive amino acid cysteine (Cys) in a small number of proteins. Sec and Cys carry out similar chemistry and are structural isomers save for a single atom difference; the former contains selenium (Se), while the latter contains sulfur (S) in the identical position. Sec poses a high bioenergetic cost for its synthesis and subsequent incorporation into protein not shared by Cys. Since Sec’s discovery in 1976, scientists have debated why certain proteins express Sec while others express Cys. In recent years, it …

Development And Application Of Mass Spectrometry-Based Approaches For Protein Higher Order Structure Analysis And Protein-Protein Interaction Characterization, Mengru Zhang

Arts & Sciences Electronic Theses and Dissertations

Proteins, one of the most fundamental biomolecules, adopt unique higher order structures (HOS) to enable diverse biological functions. Deciphering protein HOS is crucial to gain deeper insights of their working mechanisms and to develop biotherapeutics. Mass spectrometry (MS)-based approaches evolved rapidly in the past 30 years and are now playing critical roles in protein HOS characterization. One of those approaches is MS-based footprinting whose principle is to map the solvent accessible surface area (SASA) to deliver structural information. Protein footprinting can be achieved by reversible labeling, e.g., hydrogen-deuterium exchange (HDX), and by irreversible labeling using radical-based reagents or other targeted …

Intrinsic Buffer Hydroxyl Radical Dosimetry For Hydroxyl Radical Protein Footprinting, Addison Roush

Honors Theses

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry is a powerful technique for the analysis of protein topography as it generates covalent mass labels that can survive downstream sample handling, and it is sensitive to the solvent accessibility of amino acid sidechains. Of the multiple platforms for HRPF, fast photochemical oxidation of proteins (FPOP) utilizes a pulsed 248 nm KrF excimer laser to label proteins by photolyzing hydrogen peroxide. FPOP is the most widely used HRPF platform because it labels proteins faster than unfolding can occur. Variations in FPOP sample conditions make it difficult to compare results between experiments …

Covalent Labeling-Mass Spectrometry For Characterizing Protein-Ligand Complexes, Tianying Liu

Doctoral Dissertations

This dissertation focuses on applying covalent labeling (CL) and mass spectrometry (MS) for characterizing protein-ligand complexes. Understanding protein-ligand interactions has both fundamental and applied significance. Covalent labeling is a protein surface modification technique that selectively modifies solvent-exposed amino acid side chains of proteins. A covalent bond is formed between the functional groups of labeling reagent and protein’s side chain. One of the key factors that affects CL reactivity is a side chain’s solvent accessibility. Ligand binding protects residues on the protein surface from being labeled, and residues involved in ligand binding can be indicated via decreases in labeling extents. The …

Investigating The Rotary Mechanism Of Atp Synthase Using Molecular Dynamics Simulations, Angela Marcela Murcia Rios

Electronic Thesis and Dissertation Repository

F₁-ATPase is a motor protein that can use ATP hydrolysis to drive rotation of the central subunit. The γ C-terminal helix constitutes of the rotor tip that is seated in an apical bearing formed by the α₃β₃ head. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we develop a molecular dynamics (MD) strategy to model the off-axis forces acting on γ in F₁-ATPase. MD runs showed stalling of the …

Nbpmf: Novel Network-Based Inference Methods For Peptide Mass Fingerprinting, Zhewei Liang

Electronic Thesis and Dissertation Repository

Proteins are large, complex molecules that perform a vast array of functions in every living cell. A proteome is a set of proteins produced in an organism, and proteomics is the large-scale study of proteomes. Several high-throughput technologies have been developed in proteomics, where the most commonly applied are mass spectrometry (MS) based approaches. MS is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification, quantification, and post translational modification (PTM) characterization in proteomics research. There are usually two different ways to identify proteins: top-down and bottom-up. Top-down approaches …

An Investigation Of The Ck2-Dependent Phosphoproteome Using Inhibitor Refractory Ck2-Alpha, Edward Cruise

Electronic Thesis and Dissertation Repository

Protein kinase CK2 is a constitutively active serine/threonine kinase that is overexpressed in several human cancers, and by virtue of the vast number of putative substrates in the phosphoproteome, is implicated in the regulation of numerous cellular processes. Consequently, CK2 is an emerging therapeutic target with many CK2 inhibitors having been developed. An example of one such inhibitor is the clinical stage compound CX-4945. Although highly selective for CK2, the ATP competitive CX-4945 has demonstrated affinity for other kinases. Unique features of the catalytic pocket of CK2 have allowed for the development of inhibitor refractory mutants, which have since been …

Mass Spectrometric Analysis And Machine Learning Enable Microorganism Classification Based On Rna Posttranscriptional Modifications, Colin Christopher Aldrich

Legacy Theses & Dissertations (2009 - 2024)

RNA post-transcriptional modifications (PTMs) are dynamic features that can be up- or down-regulated by the health and metabolic state of a cell. These covalent modifications are installed and removed on RNA nucleosides by enzymes controlled by the activation and deactivation of specific genes. The goal of this research was to demonstrate that RNA PTMs can serve as a unique feature for the classification/identification of microorganisms. We utilized a scheme based on electrospray ionization mass spectrometry (ESI-MS) to obtain global PTM profiles from total RNA extracted from various microorganisms in optimal growth conditions as well as Salmonella typhimurium (S. typhimurium) spiked …

The Application Of Hydrogen/Deuterium Exchange And Covalent Labeling Coupled With Mass Spectrometry To Examine Protein Structure, Nicholas B. Borotto

Doctoral Dissertations

Thorough insight into a protein’s structure is necessary to understand how it functions and what goes wrong when it malfunctions. The structure of proteins, however, is not easily analyzed. The analysis must take place under a narrow range of conditions or risk perturbing the very structure being probed. Furthermore, the wide diversity in size and chemistry possible in proteins significantly complicates this analysis. Despite this numerous methods have been developed in order to analyze protein structure. In this work, we demonstrate that mass spectrometry (MS)-based techniques are capable of characterizing the structure of particularly challenging proteins. This is done through …

Towards Structural Determination Of Human Α1-Glycine Receptor Allostery, Rathna Jyothi Veeramachaneni

Electronic Theses and Dissertations

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in …

Characterization Of Protein-Protein Interactions For Therapeutic Drug Design Utilizing Mass Spectrometry, Alex J. Johnson

Masters Theses

The number of transferrin based therapeutics progressing to clinical trials remains disappointingly small despite promising capabilities of transporting therapeutic payloads to cancer cells and across the blood brain barrier. This meager success record is largely due to the complexity and heterogeneity of all protein conjugation products that generates difficulties for their analytical characterization. Discussed in this work, transferrin is conjugated to lysozyme as a model therapeutic to deliver this bacteriostatic protein to target central nervous system infections. In this work ESI- and MALDI-MS were used to characterize the modification sites at lysine residues in hopes of characterizing heterogeneity within the …

Characterization Of The Desorption Electrospray Ionization Mechanism Using Microscopic Imaging Of The Sample Surface, Michael Craig Wood

Theses and Dissertations

Desorption electrospray ionization (DESI) is an ambient ionization technique for mass spectrometry. This solvent based desorption ion source has wide applicability in surface analysis with minimal sample preparation. Interest in improving detection limits, broadening applications, and increasing the spatial resolution for chemical imaging has led to studies of the DESI mechanism. An inverted microscope has been used to image interactions between the DESI spray and test analytes on a glass surface. Microscopic images recorded with millisecond time resolution have provided important insights into the processes governing analyte transport and desorption. These insights are the basis of a rivulet-based model for …

Explorations In Homeoviscous Adaptation And Mass Spectral Analysis Of Membrane Lipids, Michael Douglas Timmons

University of Kentucky Doctoral Dissertations

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment.

The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass …

Counter-Flow Ion Mobility Analysis: Design, Instrumentation, And Characterization, Nosa Agbonkonkon

Theses and Dissertations

The quest to achieve high resolution in ion mobility spectrometry (IMS) has continued to challenge scientist and engineers in the field of separation science. The low resolution presently attainable in IMS has continued to negatively impact its utility and acceptance. Until now, efforts to improve the resolution have mainly focused on better instrumentation and detection methods. However, since the resolution of IMS is diffusion limited, it makes sense to address this limitation in order to attain high resolution. This dissertation presents a new IMS technique, which utilizes a high electric field and opposing high gas flow velocity with the aim …