Development and Application of ESI-MS Based Techniques for Quantitative Determination of Protein-Carbohydrate Interactions

  • Author / Creator
    Shams-Ud-Doha, Km
  • This thesis describes the development and application of mass spectrometry methods to study protein-ligand interactions in vitro. Liquid sample desorption electrospray ionization mass spectrometry (liquid sample DESI-MS) was employed to quantify protein-carbohydrate interactions in aqueous buffer solutions. Protein-carbohydrate interactions were quantified using liquid sample DESI-MS and it was found to be in agreement with values obtained by the direct ESI-MS assay. A rapid and quantitative method was developed to screen oligosaccharides libraries against lectins by the proxy protein ESI-MS assay. In this present work, the proxy protein ESI-MS method combined with direct ESI-MS protein-ligand binding assay and competitive protein binding assay was developed to detect and quantify interactions of protein target e.g. pathogenic protein, with glycan library. When direct detection of target protein by ESI-MS is not possible this method will facilitate the quantification of protein against ligand libraries. This method was applied to screen small oligosaccharide libraries against the N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding bacterial lectin from Ralstonia solanacearum and the P particle of human norovirus, to demonstrate the reliability and versatility of the assay. Finally, comparative studies of human milk oligosaccharides (HMOs) specificities of human galectin-1, 3, 9 were described by using direct ESI-MS assay as compared to glycan array method. Human milk oligosaccharides (HMOs) have a variety of biological functions against pathogens and serve as prebiotic. To quantify the interaction between three human galectins and 32 HMOs, electrospray ionization mass spectrometry (ESI-MS) direct binding assay method was used. The data show that galectins have broad HMO specificity with association constants ranging from 103 to 105 M-1 along with no affinity for several specific HMOs. Binding affinity of galectins was found to be similar to binding affinity to histo-blood type oligosaccharides. Weak or no binding was established for galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). These values were compared to values obtained from glycan array analysis. Significant difference was observed which depicts the reliability of the direct ESI-MS assay for quantifying protein-ligand interaction.

  • 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.