• About
    • Login
    View Item 
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of Institutional RepositoryCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartment

    My Account

    LoginRegister

    High Mass Accuracy Coupled to Spatially-Directed Proteomics for Improved Protein Identifications in Imaging Mass Spectrometry Experiments

    Rizzo, David Geoffrey
    : https://etd.library.vanderbilt.edu/etd-06072016-091330
    http://hdl.handle.net/1803/12499
    : 2016-06-08

    Abstract

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the study of protein distributions in biological tissue specimens. This has traditionally been performed using time-of-flight (TOF) systems due to their large practical mass range, high dynamic range, and high throughput of the TOF analyzer. While many proteins are detected with this technology, the unambiguous identification of these analytes remains challenging. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. This project incorporates high mass resolving power and high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) into protein IMS experiments. With sample preparation and instrument methodologies tailored for improved protein signal detection, proteins up to 17 kDa were detected with resolving powers of 75,000 and mass accuracies less than 5 ppm. Additionally, in situ digestions were utilized to investigate proteins above current observable mass ranges of the FTICR. Incubation times for enzymatic digestions were compared using IMS approaches to evaluate maximum peptide signal while minimizing delocalization. In order to improve confidence in protein and peptide identifications, identification strategies that preserve some form of spatial information were employed. Initially, liquid microextractions were used to selectively interrogate regions of tissue after in situ digestion. Localization of extracted peptides provided an additional level if information to correlate back to the IMS data. To further decrease the extraction diameter, hydrogel technologies for spatially-localized protein digestion/extraction were modified. Parameters such as percent polyacrylamide used in hydrogel construction as well as concentration of trypsin with which the hydrogel is loaded were investigated to maximize the number of protein identifications from LC-MS/MS analysis of hydrogel extracts. Increased polyacrylamide concentrations led to more rigid polymers, which were more amenable to perforating hydrogels using small punch biopsies. Hydrogels were fabricated with diameters as small as 260 µm, while still providing over 600 protein identifications. These improved methods to the hydrogel process allow researchers to target smaller biological features for robust spatially-localized proteomic analyses. Integrating high mass accuracy instrumentation with regio-specific proteomics experiments allows for confident identifications of proteins, providing insight into underlying biology of heterogeneous samples.
    Show full item record

    Files in this item

    Icon
    Name:
    Rizzo_Dissertation.pdf
    Size:
    4.302Mb
    Format:
    PDF
    View/Open

    This item appears in the following collection(s):

    • Electronic Theses and Dissertations

    Connect with Vanderbilt Libraries

    Your Vanderbilt

    • Alumni
    • Current Students
    • Faculty & Staff
    • International Students
    • Media
    • Parents & Family
    • Prospective Students
    • Researchers
    • Sports Fans
    • Visitors & Neighbors

    Support the Jean and Alexander Heard Libraries

    Support the Library...Give Now

    Gifts to the Libraries support the learning and research needs of the entire Vanderbilt community. Learn more about giving to the Libraries.

    Become a Friend of the Libraries

    Quick Links

    • Hours
    • About
    • Employment
    • Staff Directory
    • Accessibility Services
    • Contact
    • Vanderbilt Home
    • Privacy Policy