Anal. Chem. 2003, 75, 4945-4955
Articles
Influence of Solution and Gas Phase Processes on Protein-Carbohydrate Binding Affinities Determined by Nanoelectrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Weijie Wang, Elena N. Kitova, and John S. Klassen*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
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 (20
µM. The formation of nonspecific complexes leads to an artificially high Kassoc. The influence of nonspecific binding can be minimized; however, by employing an equimolar titration approach. Kassoc was found to decrease with spray duration due to the electrochemical production of the hydronium ion at the nanoES electrode. Limiting the spray duration to
