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

• Author / Creator

  Zhang, Jingjing

• 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.

• Subjects / Keywords

  • Hydrogen/Deuterium exchange
  • Mass-spectrometry
  • Protein-carbohydrate interactions

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3K35MM31

• 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.