title: Quantifying protein interactions with isomeric carbohydrate ligands using a catch and release electrospray ionization-mass spectrometry assay.
abstract: The application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to quantify interactions between proteins and isomeric carbohydrate ligands is described. Absolute affinities for each ligand are determined from the abundance ratio of ligand-bound to free protein measured directly by ESI-MS and the relative abundances of the individual isomeric ligands, which are established by releasing the ligands, in their deprotonated form, from the protein using collision-induced dissociation (CID) and subjecting them to ion mobility separation (IMS) or another stage of CID to fragment the ions. Using Gaussian functions to represent the contributions of individual ligands to the arrival time distributions (ATDs) measured by IMS, the relative abundance of each ligand bound to the protein can be established. A modification of this method, suitable for cases where nonspecific ligand-protein binding occurs during the ESI process, is also described. In cases where the ATDs are not sufficiently different to distinguish the isomeric ligands, CID can establish the relative abundance of each ligand bound to the protein from the relative abundance of the resulting fragment ions. The implementation and reliability of the CaR-ESI-MS assay for the analysis of isomeric carbohydrate ligands is demonstrated using three carbohydrate-binding proteins, a single chain antibody, an antigen binding fragment, and a fragment of a bacterial toxin, and their interactions with isomeric carbohydrate ligands with affinities ranging from 10(3) to 10(5) M(-1).
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Amr
authors.initials: A
authors.lastname: El-Hawiet
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D002241:Carbohydrates / Q000737:chemistry*; D007536:Isomerism; D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002241:Carbohydrates; D008024:Ligands; D011506:Proteins