• 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

    Method Optimization of a New Automated Platform for Proteome-Wide Structural Biology

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Thumbnail
    Name:
    Johnson_umaryland_0373D_11399.pdf
    Size:
    8.066Mb
    Format:
    PDF
    Download
    Author
    Johnson, Dante cc
    Advisor
    Jones, Lisa M.
    Date
    2022
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein HOS and dynamics requires integrated approaches, including mass spectrometry (MS), which has evolved into an indispensable tool for proteomics research. One approach readily integrated with MS is protein footprinting. In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change. The method can modify thousands of proteins in a single experiment leading to structural information across the proteome. IC-FPOP modifies proteins on the microsecond timescale making the method suitable to study fast biological processes. However, the single cell flow system developed for initial IC-FPOP experiments had temporal limitations motivating the design for a higher throughput platform. My research describes the development of a new platform for IC-FPOP entitled Platform Incubator with XY Movement (PIXY). PIXY permits IC-FPOP to occur in a sterile system using a temperature-controlled stage top incubator, peristaltic pumps for chemical transport, mirrors for laser beam guidance, and a mobile stage for XY movement. Automated communication amongst the entire PIXY system was made possible using LabVIEW software which allows the analysis of one sample in only 20 seconds. Well over 2000 proteins in HEK cells can be oxidatively modified by IC-FPOP in PIXY. This allows for a greater amount of structural information to be obtained. The capabilities of this high throughput platform permit other cell based experimental applications including fluorescent imaging and time-dependent solution transfer. PIXY’s ability to accommodate automated time points and subsequent changes over time make it a powerful tool for probing protein biochemistry in the native cellular environment.
    Description
    University of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 2022.
    Keyword
    fast photochemical oxidation of proteins (FPOP)
    Automation
    Hydroxyl Radical
    Mass Spectrometry
    Molecular Biology
    Protein Footprinting
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/20364
    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.