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Study of Non-Covalent Protein-Carbohydrate Interactions using Electrospray Ionization Mass Spectrometry Open Access


Other title
Human milk oligosaccharide
Carbohydrate-Protein interactions
Insource dissociation
Electrospray ionization mass spectrometry
library screening
Type of item
Degree grantor
University of Alberta
Author or creator
El-Hawiet, Amr Mostafa
Supervisor and department
Klassen, John (Department of chemistry)
Examining committee member and department
Brown, Alexander (Department of Chemistry)
Lowary,Todd (Department of Chemistry)
Irvin, Randall (Department of Medical Microbiology & Immunology)
Cairo, Christopher (Department of Chemistry)
Vachet, Richard (University of Massachusetts, Amherst, MA)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
This thesis describes the development and application of electrospray ionization mass spectrometry (ESI-MS) methods to study protein-carbohydrate interactions in vitro. The affinities (Ka) of two recombinant fragments of Clostridium difficile toxins (TcdA & TcdB) and a library of the most abundant human milk oligosaccharides (HMOs) were quantified using the direct ESI-MS assay. The results of the study revealed that both of the toxin fragments recognize, albeit weakly, a variety of HMOs ranging in size from tri- to heptasaccharides. The results of molecular docking simulations suggest that a disaccharide moiety (lactose or lactosamine) is the core HMO recognition element for both toxin fragments. The protective effects of HMOs fractions, extracted from human milk, were tested using the verocytotoxicity neutralization assay. However, the results revealed that the HMOs fractions do not significantly inhibit the cytotoxic effects of TcdA or TcdB. Combining the direct ESI-MS assay and competitive binding, two new ESI-MS assays were developed. The reference ligand ESI-MS method allows for the quantification of protein-ligand complexes that are prone to dissociation in the gas phase while the proxy protein ESI-MS method allows for the quantification of carbohydrate binding to large protein complexes that cannot be directly detected by ESI-MS. Using the reference ligand ESI-MS method, affinities of two carbohydrate-binding proteins for monosaccharide ligands were quantified, while the proxy protein ESI-MS method was used to quantify the interactions of tail spike protein of bacteriophage P22 (180 kDa) together with its mutant to their natural receptors. The results of binding measurements performed using these new methods were in excellent agreement with the reported values. A catch-and-release (CaR) ESI-MS assay for screening carbohydrate libraries against target proteins was also developed. Ligands with moderate affinity (104 - 106 M-1) were successfully detected from mixtures containing >200 carbohydrates. Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~103 M-1) were successfully detected in mixtures containing >20 carbohydrates. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.
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.
Citation for previous publication
El-Hawiet, A.; Kitova, E. N.; Klassen, J. S. Biochemistry 2012, 51, 4244-4253.El-Hawiet, A.; Shoemaker, G. K.; Daneshfar, R.; Kitova, E. N.; Klassen, J. S. Anal. Chem., 2012, 84, 50-58.El-Hawiet, A.; Kitova, E. N.; Arutyunov, D.; Simpson, D. J.; Szymanski, C. M.; Klassen, J. S. Anal. Chem. 2012, 84, 3867–3870.El-Hawiet, A.; Kitova, E. N.; Liu, L.; Klassen, J. S. J. Am. Soc. Mass Spectrom. 2010, 21, 1893-1899.El-Hawiet, A.; Kitova, E. N.; Kitov, P.; Eugenio, L.; Ng, K. K. S.; Mulvey, G. L.; Dingle, T. C.; Szpacenko, A.; Armstrong, G. D.; Klassen, J. S. Glycobiology 2011, 21, 1217-1227.

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File title: Binding of Clostridium difficile Toxins to Human Milk Oligosaccharides
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