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Investigating Protein-Carbohydrate Interactions with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) Open Access


Other title
Mass spectrometry
Hydrogen/Deuterium exchange
Protein-carbohydrate interactions
Type of item
Degree grantor
University of Alberta
Author or creator
Zhang, Jingjing
Supervisor and department
Klassen, John (Chemistry)
Examining committee member and department
Li, Liang (Chemistry)
Cairo, Christopher (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Master of Science
Degree level
The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to investigating protein-carbohydrate interactions is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin (CTB5) and Shiga toxin type 1 (Stx1B5) and a fragment of Clostridium difficile toxin A (TcdA-A2), and their interactions with native carbohydrate receptors, GM1 pentasaccharide (GM1-os), Pk trisaccharide and CD-grease, respectively, were first served as model systems for this study. The results suggested that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. Following this, HDX-MS measurements were applied to explore the existence of distinct HMOs binding sites on toxins. Altogether, two toxins were studied, CTB5 and TcdA-A2, and their interactions with HMOs, 2ʹ-fucosyllactose (2ʹ-FL) and lacto-N-tetraose (LNT), respectively. For CTB5 and its interaction with 2ʹ-FL, a novel binding site was localized for 2ʹ-FL, different from the one for native receptor GM1-os. For TcdA-A2 and its interaction with LNT, however, the localized binding site was the same as its native carbohydrate receptor CD-grease. A HDX-MS based titration method Protein-Ligand Interactions in solution by Mass Spectrometry, Titration and hydrogen/deuterium Exchange (PLIMSTEX), was also applied to CTB5 and its interactions GM1-os, to test the reliability of using peptides as indicators to obtain the protein-carbohydrate binding affinities. The average apparent association constant measured for the addition of GM1-os to CTB at pH 7.0 and 20 °C was found to be (1.6 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (3.2 ± 0.2) × 106 M-1, which was measured using direct ESI-MS assay at pH 6.9 and room temperature.
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