title: Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.
abstract: 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 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 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 (~10(3) 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.
authors: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Amr
authors: A
authors: El-Hawiet
authors: Glen K
authors: GK
authors: Shoemaker
authors: Rambod
authors: R
authors: Daneshfar
authors: Elena N
authors: EN
authors: Kitova
authors: John S
authors: JS
authors: Klassen
mesh_terms: D002241:Carbohydrates / Q000032:analysis*; D008024:Ligands; D011506:Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002241:Carbohydrates; D008024:Ligands; D011506:Proteins