The Alberta Glycomics Centre Screening Centre collaborates with academic investigators to employ advanced electrospray ionization mass spectrometry (ESI-MS) instrumentation and assays, which are alternatives to conventional glycan microarrays to screen carbohydrate libraries (as well as other classes of compounds) against target proteins to identify novel inhibitors and natural receptors that may be exploited for the development of new and effective treatments for a variety of bacterial and viral infections as well as other diseases.
Types of Screening Assays:
- Direct ESI-MS Assay
The direct ESI-MS assay strictly relies on the direct and accurate detection of gas phase ions of the free and ligand-bound protein. The binding constant (Ka) for a given protein-ligand complex is determined from the ratio of abundances of the bound and unbound protein ions, as measured by ESI-MS for solutions of known initial concentrations of protein and ligand. The technique boasts a number of strengths, including its simplicity (no labeling or immobilization required), speed (measurements can usually be completed within a few seconds), and the unique ability to provide direct insight into stoichiometry and to study multiple binding equilibria simultaneously.
Typical number of replicates is 3-4 for individual binding constant measurements using the direct ESI-MS assay. Typical amount of protein and ligands required for assay is ~ 0.1-0.5 mg.
- Km Shams-Ud-Doha, Elena N. Kitova, Pavel I. Kitov, Yves St-Pierre, and John S. Klassen “Human Milk Oligosaccharide Specificities of Human Galectins. Comparison of Electrospray Ionization Mass Spectrometry and Glycan Microarray Screening Results”. Anal. Chem. 2017 89(9):4914-4921
- Hong Lin, Elena N. Kitova, John S. Klassen “Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide”. Journal of The American Society for Mass Spectrometry. 2014, 25(1), 104-110.
- El-Hawiet, A., Kitova, E.N.,and Klassen, J.S. “Quantifying Carbohydrate-Protein Interactions by Electrospray Ionization Mass Spectrometry Analysis”. Biochemistry, 2012, 51(21), 4244-4253.
- Deng L, Sun N, Kitova EN, Klassen JS. “Direct Quantification of Protein-Metal Ion Affinities by Electrospray Ionization Mass Spectrometry”. Anal. Chem. 2010, 82, 2170-2174.
- Catch & Release (CaR)-ESI-MS assay
CaR-ESI-MS assay is a novel and powerful assay widely used in the screening center. It can be used towards both water-soluble glycan libraries and model membrane solubilized glycolipid libraries screening against different target proteins, ranging from small proteins or much larger ones such as bacteriophage and viruses. In the CaR-ESI-MS assay, ligands are identified after release from collisionally activated protein-ligand complex based on their molecular weights (MWs), ion mobility (IMS) separation arrival times and fragmentation fingerprints. With the assistance of IMS, isomers presented in the binding hits can be distinguished.
The beauty of the CaR-ESI-MS helps to achieve a high-throughput and label- and immobilization-free ESI-MS assay for screening a library of free human milk oligosaccharides (HMOs, reducing sugars) against lectins in vitro in the screening center. For this fully characterized in-house library, it takes < 1 h to complete each screening for each lectin towards the library, and consume < 5 ng of each HMO and < 0.5 µg of protein.
- Amr El-Hawiet, Yajie Chen, Km Shams-Ud-Doha, Elena N Kitova, Yves St-Pierre, and John S. Klassen “High Throughput, Label- and Immobilization-Free Screening of Human Milk Oligosaccharides Against Lectins”. Anal. Chem. 2017 89 (17): 8713–8722.
- Ling Han, Elena N. Kitova and John S. Klassen”Detecting Protein-Glycolipid Interactions using Glycomicelles and CaR-ESI-MS”. J Am Soc Mass Spectrom. 2016, 27:1878.
- Jun Li, Xuxin Fan, Elena N Kitova, Chunxia Zou, Christopher W. Cario, Luiz Eugenio, Kenneth KS Ng, Zi Jiang Xiong, Gilber G. Prive and John S. Klassen “Screening Glycolipids Against Proteins in vitro using Picodiscs and Catch-and-Release Electrospray Ionization Mass Spectrometry”. Anal. Chem. 2016, 88(9):4742-4750.
- Aneika C. Leney, Reza Rezaei Darestani, Jun Li, Sanaz Nikjah, Elena N. Kitova, Chunxia Zou, Christopher W. Cairo, Zi Jian Xiong, Gilbert G. Privé , and John S. Klassen “Picodiscs for Facile Protein-Glycolipid Interaction Analysis”. Anal Chem. 2015, 87, 4402-4408.
- Yixuan Zhang, Lan Liu, Rambod Daneshfar, Elena N. Kitova, Caishun Li, Feng Jia, Christopher W. Cairo and John S. Klassen “Protein-Glycosphingolipid Interactions Revealed using Catch-and-Release Mass Spectrometry”. Anal. Chem. 2012, 84(18), 7618-7621.