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IMMOBILIZATION OF OLIGOSACCHARIDES IN AFFINITY CHROMATOGRAPHY AND APPLICATIONS TO SCREENING HUMAN MILK OLIGOSACCHARIDE RECEPTORS
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- Author / Creator
- Hernandez Armada, Daniel
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A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are available, in many cases there is a lack of satisfactory chemical characterization. In this thesis we describe work towards improved characterization of chemistries with improved efficiencies which may be essential for work with glycans available only in limited quantity. Several chemical approaches were compared to divinyl sulfone based on immobilization efficiency, attachment to the solid support, and binding capacity to a protein receptor. We observed that immobilizations using p-Toluene sulfonyl hydrazide offered higher immobilization efficiencies and comparable binding capacities to the DVS method. We also generated a series of affinity matrices containing individual human milk oligosaccharides (HMO) species. To test the utility of the affinity matrix for identifying glycan receptors, we used the lysate from human cells. Proteins which specifically bound to the affinity matrix were captured, and then identified using LC-MS/MS-based proteomics analysis. This method has successfully identified a number of HMO-receptors, including members if the galectin family.
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- Subjects / Keywords
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- Graduation date
- Spring 2015
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- 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.