Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay

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 proteinligand 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 ESL-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 Raistonia 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.