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

Applications of a catch and release electrospray ionization mass 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 103 to 106 M–1 range. Ligands with moderate affinity (104 to 106 M–1) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM 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 (∼103 M–1) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ∼10 μM 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.