Analytical Chemistry Commons^™

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 …

Isolation Of Proteins And Extracellular Vesicles Via Capillary-Channeled Polymer Fiber Stationary Phases, Lacey Billotto

All Theses

Capillary-channeled polymer (C-CP) fiber stationary phases have been developed by the Marcus lab as an alternative approach to traditional, commercially-available silica-based supports for biomacromolecule separations. C-CP columns are created with natural and synthetic base polymers in a variety of chemistries and shapes, allowing for customization of separations, while providing low material costs and ease of construction. The stationary phase is virtually nonporous with respect to the size of proteins and can operate under high linear velocities (~100 mm s^-1) while maintaining relatively low backpressures. As a result of these hydrodynamic benefits, low flow resistance at high volume flow …

Capillary Electrophoresis: Method Development And Application In Biomolecule Analysis, Lindsay Marie Veltri

Graduate Theses, Dissertations, and Problem Reports

Abstract

Capillary Electrophoresis: Method Development and Application in Biomolecule Analysis

Lindsay Veltri

Biomolecule analysis is essential in disease diagnostics and the development of therapeutic agents. There is a vast array of methods available for biomolecule analysis, however, capillary electrophoresis is well-suited due to minimal sample consumption (~nanoliters of solution) and fast analysis times. In this report, capillary electrophoresis was used to analyze glycosylation of therapeutic proteins. Additionally, a capillary electrophoresis-mass spectrometry method is reported for the analysis of proteins.

Post-translation protein modifications like glycosylation, play an important in protein structure and function. Modifications of protein glycosylation can lead to improved …

Investigations Of The Structure And Protein-Protein Interactions Of Chlamydia Trachomatis Scc4, Thilini Oshadhi Senarath Ukwaththage

LSU Doctoral Dissertations

Chlamydia trachomatis (CT) is the most common, sexually transmitted bacterial disease (STD) in the world. In the developmental cycle of CT, specific chlamydia chaperone 4 (Scc4) is a unique protein with essential and multiple roles. Hence, Scc4 is significant as a virulence target for therapeutic approaches to treat chlamydial infections. A novel approach was discovered to purify tag free Scc4 by utilizing a 6X-histidine-tag on Scc1 in the co-expressed Scc4:Scc1 complex by capturing the complex on nickel-charged immobilized metal affinity chromatography resin, followed by dissociation of Scc4 with sarkosyl. Using triple resonance NMR experiments, backbone and sidechain resonances …

Capillary Electrophoresis Analysis Of Proteins Using Phospholipid Based Materials, Cassandra L. Crihfield

Graduate Theses, Dissertations, and Problem Reports

Capillary electrophoresis has many advantages that make it a powerful technique for the analysis of proteins, which is challenging due to the natural heterogeneity of proteins. The small sample volume required for each analysis (nano- and pico-liter), fast separation times (<30 >min), and ability to couple the separation to mass spectrometry are all factors that make capillary electrophoresis an ideal technology for protein separations. Capillary zone electrophoresis and capillary gel electrophoresis are the two most commonly employed modes for protein analysis and allow for rapid, high resolution separations that can be automated. This makes capillary electrophoresis ideal for meeting the …

An Exploration Of Protein And Dna Components In Fingerprint Residue, Ashley Borrego

Student Theses

The main focus of this project was to investigate the protein and DNA components in both sebaceous and eccrine fingerprints. This study investigated the relative content of DNA and proteins in eccrine fingerprints to sebaceous fingerprints. All volunteers were instructed to wash and dry their hands prior to depositing parallel thumbprints. Twenty volunteers were instructed to touch their face to produce sebaceous prints, and 5 volunteers were instructed to wear gloves over a heat source to produce sweaty or eccrine prints. Microscopy was used to score the cellular debris of the right fingerprint on a scale of 1-4 based on …

Precipitation And Coacervation In Polyelectrolyte-Colloid Systems, Fatih Comert

Doctoral Dissertations

Complex coacervation between oppositely charged polyelectrolytes (PEs) and colloids (i.e. micelles and proteins) has increasingly become popular due to the use of these materials in a range of applications including but not limited to personal care, biomaterials, protein purification, and food science. In taking advantage of complex coacervation, which is a liquid-liquid phase separation, precipitation has always represented a problem by introducing inhomogeneities, irreversibility and irreproducible kinetics. Therefore, understanding the dominant factors driving the formation of precipitates is important to control outcomes. In this work, we have performed comparative studies of coacervation and precipitation in model PE-colloid systems to investigate …

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 …

Distinguishing Macrophage Activation States By Mass Spectrometry, Matthias Manfred Knust

Graduate Theses and Dissertations

Macrophages are versatile and highly adaptive cells that are involved in a wide range of physiological processes including host defense, homeostasis or regeneration, as well as pathogenesis. They react to their microenvironment, assuming various roles based on chemical and/or physical cues, and can reversibly shift between these so-called activation states. Concurrently, the technique of immunohistochemistry is used to gain spatial information on activated macrophages on tissue sections. The aim of this work was to find mass spectral biomarkers that allow the differentiation of activation states, and establish conditions that can be used in imaging mass spectrometry (IMS) experiments to investigate …

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 …

Micro Bca Analysis Of Proteins, Anusha Chennupati

All Capstone Projects

The main objective of this project was to develop and validate methods for detection and analysis of proteins using a newly introduced microdrop plate reader equipped with a 16 well microdrop reader (2 uL sample volume). We used commercially available BSA, egg albumin, trypsinogen, Pepsin, β-lactoblobulin and lysozyme as out test moieties. Protein drop size, time, BCA reagent quantity, was assessed at various levels using a microdrop micro-BCA protocol (Pierce Chemical Company) with the appropriate controls (PBS blank) the method validated using the appropriate statistical and method parameters (i.e %RSD, and regression analysis of the standard curves). The micro method …

Elucidating The Structure Of Protein Aggregates By Raman Spectroscopy, Ludmila A. Popova

Legacy Theses & Dissertations (2009 - 2024)

The structures and properties of amyloid fibrils are of considerable interest due to their associations with numerous neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and transmissible spongiform encephalopaties (prion diseases). Understanding fibrillogenesis at a molecular level requires detailed structural characterization of amyloid fibrils. However amyloid fibrils are difficult objects to study due to their non-crystalline and insoluble nature. These properties make the application of classical tools of structural biology, such as X-Ray crystallography and solution Nuclear Magnetic Resonance spectroscopy, impractical for structural characterization of protein fibrils.

Incorporation Of Capillary-Channeled Polymer (C-Cp) Fibers Into Micropipette Tips For Solid Phase Extraction With Applications In Bioanalysis, Donald Fornea

All Theses

Solid phase extraction (SPE), is a widely used sample preparation method that is currently experiencing a surge in research interest, clever architecture/format development, and stationary phase characterization.1-3 SPE devices commonly exist in syringe, cartridge, disk, and micropipette tip formats.4 Micropipette tip SPE has enhanced the determination of proteins by electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) by handling micro-liter volumes of sample and buffer removal/solvent exchange.5-7 Buffers have deleterious effects on ionization taking place in MS sources due to preferential ionization, ion suppression effects, and the formation of adduct species competing for ionization in …

Characterization Of Capillary-Channeled Polymer Fibers As Stationary Phases For High-Performance Liquid Chromatography Of Macromolecules, Dwella Nelson

All Dissertations

Capillary-channeled polymer (C-CP) fibers are being developed and characterized as high-performance liquid chromatography (HPLC) stationary phases for reversed-phase separations of proteins. Conventional porous phases are not well suited for separating large macromolecules due to the slow diffusivity of the molecules, large diffusional distances, and stagnant mobile phase zones within the column. Stationary phases are continually being developed to address the challenges associated with HPLC of proteins and other macromolecules in general. C-CP fiber stationary phases are an alternative to conventional bead technology and offer a variety of chemical and physical advantages. The fibers can be selected for their chemical functionality …