Evaluation of automated nano‐electrospray mass spectrometry in the determination of non‐covalent protein–ligand complexes

The use of electrospray ionization mass spectrometry (ESI‐MS) for studying non‐covalent interactions between macromolecules and ligands is well established. ESI‐MS can be a useful tool for the determination of dissociation constants between molecules in the gas phase. We validate this method by studying the binding of the catalytic domain of cellobiohydrolase I (CBH I) from Trichoderma reesei to the disaccharide inhibitor cellobiose. The method was further applied to study two newly synthesized cellobiose derivatives ( m ‐iodobenzyl 2‐deoxy‐2‐azido‐ β ‐cellobioside and p ‐benzyloxybenzyl β ‐cellobioside). In a titration experiment, peak areas of different charge states of the free enzyme and the complex were summed in order to determine the dissociation constant. For cellobiose and m ‐iodobenzyl 2‐deoxy‐2‐azido‐ β ‐cellobioside, the calculated values are in good agreement with those reported from either displacement titration or equilibrium binding experiments in solution. Due to non‐specific binding, the dissociation constant of p ‐benzyloxybenzyl β ‐cellobioside does not correspond with the solution‐based value. Our results indicate the need for careful interpretation of data sets when using nanoESI to study non‐covalent interactions. Copyright © 2004 John Wiley & Sons, Ltd.