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Glycoprotein Interactions Studied Using Electrospray Ionization Mass Spectrometry

  • Author / Creator
    Wang, Yilin
  • This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) based methods for characterizing glycoproteins (GPs) and investigating their interactions with glycan binding proteins (GBPs) and drugs. A multipronged approach was developed to identify and quantify water soluble GBP-GP interactions. First, the catch-and-release (CaR)−ESI−MS assay, implemented with ion mobility separation prior to GBP “release” (i.e., CaRIMS−ESI−MS), was employed to rapidly identify GBP-GP binding in solution. The apparent affinity (Ka,app) of the GBP for the GP was then determined using the competitive proxy ligand-ESI−MS binding assay. Finally, screening of N-glycan libraries enzymatically released from the GPs against the GBP revealed the binding partners. Measurements performed at multiple GBP concentrations allow for affinity ranking of the released N-glycans (grouped as compositional isomers). This approach was demonstrated using the known interactions between a C-terminal domain fragment of human galectin-3 (hGal-3C) and three human serum GPs, α-1-acid glycoprotein (AGP), haptoglobin phenotype 1−1 (Hp1−1) and α-2-macroglobulin (α2M). As an extension of this work, high-resolution MS was employed to characterize the microheterogeneity of AGP, following treatment with glycosidases, a sialidase, an α1,3-4 fucosidase and PNGase F. Using estimated glycoform concentrations, the affinities of warfarin, an anticoagulant drug, were measured by ESI-MS titration experiments. All major unfucosylated glycoforms of asialo-AGP showed similar binding affinities (~ 10^5 M-1) to warfarin, which is consistent with isothermal titration calorimetry (ITC) measurements. Binding properties of warfarin to PNGase F treated AGP, where N-glycans were partially or completely removed, were determined and compared. Unglycosylated AGP showed no binding to warfarin while binding increased with increasing level of AGP glycosylation, indicating the significant roles of N-glycans in promoting AGP recognition of warfarin.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-nz6g-cp60
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.