Applications of a Catch and Release Electrospray Ionization Mass Spectrometry Assay for Carbohydrate Library Screening

Applications of a catch and release electrospray ionization spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M-1 range. Ligands with moderate affinity (10(4) to 10(6) M-1) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 mu M each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (similar to 10(3) M-1) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of similar to 10 mu M each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.