Physical Sciences and Mathematics Commons^™

Functionalized Plasmonic Nanostructures For Ultrasensitive Single Cell Analysis, Priya Rathi

Arts & Sciences Electronic Theses and Dissertations

Ultrasensitive detection and quantification of soluble, secreted and cell surface-bound proteins is critical for advancing our understanding of cellular systems, enabling effective drug development, novel therapies, and bio-diagnostics. However, exiting technologies are largely limited by their sensitivity, making the detection and quantification of low-abundant proteins extremely challenging. This forms a major barrier in various fields of biology and biomedical sciences. In this work, we introduce novel cellular analysis methodologies based on plasmon-enhanced fluorescence for analyzing cell structure and probing surface and secreted proteins from cells. In the first part, we introduce plasmon-enhanced expansion microscopy and demonstrate the effectiveness of employing …

Integrated Mass Spectrometry-Based Method In Protein Chemistry: Metal-Binding Protein And Integral Membrane Protein, Chunyang Guo

Arts & Sciences Electronic Theses and Dissertations

Most biological processes are associated with protein catalysis. Characterizing protein structure, therefore, is crucial for understanding biological function. Mass spectrometry (MS)-based methods have emerged as a pivotal biophysical tool to interrogate protein structures; they can characterize protein-ligand/protein complexes that are refractory to conventional high-resolution means such as X-ray crystallography, Cryo-EM, and nuclear magnetic resonance (NMR) spectroscopy, in part owing to high molecular weight and significant flexibility. The integrated MS-based platform will provide topology and structural information of proteins from sketch to detail; native MS identifies protein complexes’ composition and stoichiometry; hydrogen-deuterium exchange MS (HDX-MS) detects the protein complexes’ dynamic change …

Mass Spectrometry-Based Strategies In Protein Higher Order Structure Analysis: Fundamentals And Applications In Protein-Ligand Interactions, Xiaoran Liu

Arts & Sciences Electronic Theses and Dissertations

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which …

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 …

Mass Spectrometry-Based Strategies For Protein Characterization: Amyloid Formation, Protein-Ligand Interactions And Structures Of Membrane Proteins In Live Cells, Ke Li

Arts & Sciences Electronic Theses and Dissertations

Mass spectrometry (MS)-based protein footprinting characterizes protein structure and protein-ligand interactions by interrogating protein solvent-accessible surfaces by using chemical reagents as probes. The method is highly applicable to protein or protein-ligand complexes that are difficult to study by conventional means such as X-ray crystallography and nuclear magnetic resonance. In this dissertation, we describe the development and application of MS-based protein footprinting from three perspectives, including I) protein aggregation and amyloid formation (Chapter 2-3), II) protein-ligand interactions (Chapter 4-5), and III) in-cellulo structures and dynamic motion of membrane proteins (Chapter 6). Fast Photochemical Oxidation of Proteins (FPOP) is the main methodology …

Mapping Analyte-Signal Relations In Lc-Ms Based Untargeted Metabolomics, Nathaniel Guy Mahieu

Arts & Sciences Electronic Theses and Dissertations

The goal of untargeted metabolomics is to profile metabolism by measuring as many metabolites as possible. A major advantage of the untargeted approach is the detection of unexpected or unknown metabolites. These metabolites have chemical structures, metabolic pathways, or cellular functions that have not been previously described. Hence, they represent exciting opportunities to advance our understanding of biology. This beneficial approach, however, also adds considerable complexity to the analysis of metabolomics data - an individual signal cannot be readily identified as a unique metabolite. As such, a major challenge faced by the untargeted metabolomic workflow is extracting the analyte content …

Investigation Of 89zr-Siderophores As Molecular Imaging Agents For Positron Emission Tomography Imaging Of Bacterial Infections, Nora C. M. Goscinski

Arts & Sciences Electronic Theses and Dissertations

Siderophores are small molecules synthesized by bacteria to harvest Fe³⁺ from their environment. In infection scenarios, their production can increase infection virulence by increasing the ability of bacteria to obtain Fe³⁺ and therefore grow more rapidly. The selective uptake of siderophores in vivo in multi-bacteria environments indicates that this class of molecules has a potential use as selective imaging agents. In this work, DFO-NCS and a library of trihydroxamate siderophores were evaluated as vehicles to deliver ⁸⁹Zr selectively to bacteria for Positron Emission Tomography (PET) imaging of bacterial infections.

Productive work with radiometals involves thorough knowledge of …