title: Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay.
abstract: An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay.
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada , T6G 2G2.
authors.forename: Ling
authors.initials: L
authors.lastname: Han
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada , T6G 2G2.
authors.forename: Km
authors.initials: K
authors.lastname: Shams-Ud-Doha
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada , T6G 2G2.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada , T6G 2G2.
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D029322:Norovirus / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D043368:Ralstonia solanacearum / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D036022:Capsid Proteins; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; C027112:fucose-binding lectin