title: Influence of solution and gas phase processes on protein-carbohydrate binding affinities determined by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry.
abstract: The influence of solution pH, analyte concentration and in-source dissociation on the measurement of the association constant for a single chain variable fragment of a monoclonal antibody (scFv) and its native trisaccharide ligand by nanoelectrospray-Fourier transform ion cyclotron resonance mass spectrometery has been systematically investigated. From the results of this study, experimental conditions that preserve the original distribution of bound and unbound protein in solution into the gas phase, such that the nanoES mass spectrum provides a quantitative measure of the solution composition, were identified. These include the use of short spray durations (<10 min) to minimize pH changes, equimolar concentrations of protein and ligand to minimize the formation of nonspecific complexes, and short accumulation times (<2 s) in the hexapole of the ion source to avoid collisional heating and dissociation of the gaseous complex. Application of this methodology to the scFv and a series of carbohydrate ligands yields results that are in agreement with values previously determined by isothermal titration calorimetry. Competitive binding experiments performed on solutions containing the scFv and a mixture of carbohydrate ligands were also found to yield accurate association constants.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Weijie
authors.initials: W
authors.lastname: Wang
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D005583:Fourier Analysis; D036103:Nanotechnology / Q000379:methods*; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012996:Solutions / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D009844:Oligosaccharides; D011506:Proteins; D012996:Solutions