Development of Mass Spectrometry Methods to Study Protein-Ligand Interactions

  • Author / Creator
    Yao, Yuyu
  • This thesis describes the development of mass spectrometry methods to study protein-ligand interactions in vitro. Liquid sample desorption electrospray ionization mass spectrometry (liquid sample DESI-MS) was first applied to quantify protein-carbohydrate interactions in aqueous ammonium acetate solutions. Protein-carbohydrate interactions were measured using liquid sample DESI-MS were found to be in good agreement with values measured by isothermal titration calorimetry (ITC) and the direct ESI-MS assay. The suitability of liquid sample DESI-MS for quantitative binding measurements carried out using solutions containing high concentrations of phosphate buffered saline (PBS) was also first explored. Binding of lysozyme to b-D-GlcNAc-(1→4)-b-D-GlcNAc-(1→4)-D-GlcNAc in aqueous solutions containing up to 1x PBS was successfully monitored using liquid sample DESI-MS; with ESI-MS the binding measurements were limited to concentrations less than ~0.02x PBS. The influence of sulfolane on ESI-MS measurements of protein-ligand affinities was investigated. Having found evidence that sulfolane generally reduces the apparent affinity, a detailed study of the origin of the reduced affinity was carried out using ITC, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to establish how sulfolane affects the structure and stability of the protein-ligand complex in solution. Finally, binding measurements performed using liquid sample DESI-MS revealed that the introduction of sulfolane into the ESI solution results in protein supercharging without any loss in affinity.

  • Subjects / Keywords
  • Graduation date
    Fall 2016
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • 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
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • McDermott, Mark (Chemistry)
    • Klassen, John (Chemistry)
    • Campbell, Robert (Chemistry)