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Study of Non-Covalent Protein-Carbohydrate Interactions using Electrospray Ionization Mass Spectrometry

  • Author / Creator
    El-Hawiet, Amr Mostafa
  • This thesis describes the development and application of electrospray ionization mass spectrometry (ESI-MS) methods to study protein-carbohydrate interactions in vitro. The affinities (Ka) of two recombinant fragments of Clostridium difficile toxins (TcdA & TcdB) and a library of the most abundant human milk oligosaccharides (HMOs) were quantified using the direct ESI-MS assay. The results of the study revealed that both of the toxin fragments recognize, albeit weakly, a variety of HMOs ranging in size from tri- to heptasaccharides. The results of molecular docking simulations suggest that a disaccharide moiety (lactose or lactosamine) is the core HMO recognition element for both toxin fragments. The protective effects of HMOs fractions, extracted from human milk, were tested using the verocytotoxicity neutralization assay. However, the results revealed that the HMOs fractions do not significantly inhibit the cytotoxic effects of TcdA or TcdB. Combining the direct ESI-MS assay and competitive binding, two new ESI-MS assays were developed. The reference ligand ESI-MS method allows for the quantification of protein-ligand complexes that are prone to dissociation in the gas phase while the proxy protein ESI-MS method allows for the quantification of carbohydrate binding to large protein complexes that cannot be directly detected by ESI-MS. Using the reference ligand ESI-MS method, affinities of two carbohydrate-binding proteins for monosaccharide ligands were quantified, while the proxy protein ESI-MS method was used to quantify the interactions of tail spike protein of bacteriophage P22 (180 kDa) together with its mutant to their natural receptors. The results of binding measurements performed using these new methods were in excellent agreement with the reported values. A catch-and-release (CaR) ESI-MS assay for screening carbohydrate libraries against target proteins was also developed. Ligands with moderate affinity (104 - 106 M-1) were successfully detected from mixtures containing >200 carbohydrates. Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~103 M-1) were successfully detected in mixtures containing >20 carbohydrates. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

  • Subjects / Keywords
  • Graduation date
    2012-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3V03R
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Klassen, John (Department of chemistry)
  • Examining committee members and their departments
    • Lowary,Todd (Department of Chemistry)
    • Irvin, Randall (Department of Medical Microbiology & Immunology)
    • Cairo, Christopher (Department of Chemistry)
    • Vachet, Richard (University of Massachusetts, Amherst, MA)
    • Brown, Alexander (Department of Chemistry)