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Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Published on: November 11, 2016
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A quantitative, high-throughput method identifies protein-glycan interactions via mass spectrometry
Pavel I Kitov1, Elena N Kitova1, Ling Han1
11Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2 Canada.
Communications Biology
|July 26, 2019
Summary
We developed a new assay, the competitive universal proxy receptor assay (CUPRA), for high-throughput screening of glycan interactions. This method quantifies binding affinities and enzyme activities, advancing glycan-mediated biology research.
Area of Science:
- Glycobiology
- Biochemistry
- Analytical Chemistry
Background:
- Glycan interactions mediated by glycan-binding proteins and carbohydrate-active enzymes are crucial in biological processes.
- Understanding these interactions is key for developing new diagnostics and therapeutics.
- Current methods for mapping glycan interactions can be limited in scope or throughput.
Purpose of the Study:
- To introduce a versatile, quantitative, high-throughput method for screening glycan libraries.
- To enable comprehensive mapping of glycan-protein interactions.
- To characterize the activity and specificity of carbohydrate-active enzymes.
Main Methods:
- Development of the competitive universal proxy receptor assay (CUPRA).
- Integration of electrospray ionization mass spectrometry, competitive binding, and heterobifunctional glycan-based ligands.
- Application to diverse proteins including human, plant, and bacterial examples.
Main Results:
- CUPRA successfully identified ligands with affinities (Kd) ≤ 1 mM for various proteins.
- Demonstrated versatility across different protein types and glycan libraries.
- Showcased potential for time-resolved application to measure enzyme activity and substrate specificity.
Conclusions:
- CUPRA is a powerful, quantitative, high-throughput assay for glycan-binding and glycan-processing proteins.
- The assay significantly advances the study of glycan-mediated biology.
- CUPRA offers a versatile platform for discovering novel glycan interactions and characterizing enzyme functions.
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