Discovering Protein-Glycolipid Interactions Using Electrospray Ionization Mass Spectrometry

• Author / Creator

  Li, Jianing

• This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) based techniques to detect and quantify proteins interactions with carbohydrates or glycolipids in model membranes.
  In Chapter 2, the native ESI-MS based direct/competitive binding assay and catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay were employed to investigate the ganglioside specificities of a series of anti-GD2 monoclonal antibodies (mAbs) and their antigen binding fragments (Fabs). First, the affinities of anti-GD2 antibodies including hu3F8 and its double mutant E1K/D32H Fabs and 14G2a mAb to a library of fourteen ganglioside oligosaccharides were quantified using the direct ESI-MS assay. The binding data revealed that the anti-GD2 antibodies were ranked in order of affinity: hu3F8 E1K/D32H Fab (Ka,GD2os = (22 ± 1.5) x 105 M-1) > hu3F8 Fab (Ka,GD2os = (4.3 ± 0.1) x 105 M-1) > 14G2a mAb (Ka1,app = (1.2 ± 0.9) x 105 M-1, Ka2,app = (4.1 ± 0.4) x 104 M-1 and Ka,int,GD2os = (7.0 ± 0.2) x 104 M-1 (per binding site). Measurements performed on other ganglioside oligosaccharides indicated that all of the ganglioside oligosaccharides tested were recognized by these antibodies, although with lower affinities, in the 2.0 x 102 M-1 - 5.8 x 103 M-1 range. In addition, the hu3F8 Fab exhibits better specificity to GD2os than the hu3F8 E1K/D32H Fab and 14G2a mAb. The CaR-ESI-MS assay implemented with model membrane nanodiscs (NDs) to solubilize gangliosides, was then used to screen mixtures of gangliosides against different types of anti-GD2 antibodies including hu3F8 and its double mutant E1K/D32H and 14G2a mAbs. As expected, GD2 was found to be the dominant ligand of these anti-GD2 antibodies. However, CaR-ESI-MS also revealed 14G2a mAb has measurable binding to GM3 and GM4. Finally, the competitive binding ESI-MS assays were applied to quantify affinities of the hu3F8 and its double mutant E1K/D32H Fabs and 14G2a mAb for GD2 incorporated into NDs. The binding data revealed the extent of GD2 binding to these antibodies was found to be sensitive to ganglioside content of the NDs.
  In Chapter 3, the membrane anchor-assisted electrospray ionization mass spectrometry approach was introduced for detecting low affinity interactions between glycan binding proteins (GBPs) and glycospingolipids (GSLs) presented in model membranes. The method involves covalent cross-linking the GBP to the model membrane through a modified lipid. The resulting membrane anchor serves to enhance the local concentration of GBP on the surface of the membrane and enhance binding to GSL ligands. The implementation and reliability of this new approach was demonstrated using three human galectins, C-terminal fragment of human galectin-3, recombinant human galectin-1 and recombinant human galectin-7, and their interactions with gangliosides presented in nanodiscs. The results of this study were validated using binding data measured for the corresponding ganglioside oligosaccharides.

• Subjects / Keywords

  • Protein-Glycolipid Interactions

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-s63b-hf44

• License

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