• Login
    View Item 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Development of Fast Photochemical Oxidation of Proteins for in Vivo Modification in Caenorhabditis elegans

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Thumbnail
    Name:
    EspinoPerez_umaryland_0373D_11 ...
    Size:
    13.44Mb
    Format:
    PDF
    Download
    Author
    Espino, Jessica cc
    0000-0002-7203-9145
    Advisor
    Jones, Lisa M.
    Date
    2020
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    Mass spectrometry (MS) has become widely used for the characterization of protein structure and protein-protein interactions (PPI). Unlike many commonly used structural methods, MS is not limited by the size of molecules, thus allowing for the study of a wide range of purified protein complexes, cells, tissues, and complex organisms. Instrumentation advancements have also decreased the need for large sample concentrations and have increased mass accuracy and resolution. In the past decade, MS-based protein footprinting has become increasingly utilized for the determination of higher-order protein structure and provides residue-level analysis on PPI interaction sites, protein-ligand interactions, and regions of conformational change by covalently modifying the solvent-accessible surface area (SASA) of proteins through the use of a small chemical label. The hydroxyl radical protein footprinting (HRPF) method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals (•OH) to oxidatively modify solvent-accessible amino acid side chains. These radicals are generated via hydrogen peroxide photolysis using a KrF excimer laser at a 248 nm wavelength. To date, most applications of FPOP have been performed in vitro in relatively pure protein systems. Most notably, it has been applied for antibody epitope mapping, protein folding, and protein aggregation. This work focuses on the extension of FPOP for in vivo protein structural analysis in Caenorhabditis elegans, a method entitled in vivo FPOP (IV-FPOP). FPOP is particular suited for in vivo protein studies because of the irreversible nature of the modification, which mitigates time constraints with respect to sample preparation, proteomic digestion, and sample processing. Additionally, the •OH generated can label 19 out of 20 amino acids allowing for the study of multiple proteins regardless of protein sequence or cellular location. Given the complexity of the platform, numerous parameters required optimization for maximum labeling efficiency including the development of a microfluidic flow system for the labeling of worms by IV-FPOP, hydrogen peroxide concentration optimization, and the addition of chemical penetration enhancers to increase hydrogen peroxide uptake by the worm, and the implementation of a multiplexing proteomics platform increased throughput of IV-FPOP oxidatively modified peptides.
    Description
    Pharmaceutical Sciences
    University of Maryland, Baltimore
    Ph.D.
    Keyword
    Caenorhabditis elegans
    Mass Spectrometry
    Protein Footprinting
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/14473
    Collections
    Theses and Dissertations School of Pharmacy
    Theses and Dissertations All Schools

    entitlement

     
    DSpace software (copyright © 2002 - 2023)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.