title: Evaluation of automated nano-electrospray mass spectrometry in the determination of non-covalent protein-ligand complexes.
abstract: 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-beta-cellobioside and p-benzyloxybenzyl beta-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-beta-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 beta-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.
authors.affiliation: Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: De Vriendt
authors.forename: Koen
authors.initials: K
authors.lastname: Sandra
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
authors.forename: Wim
authors.initials: W
authors.lastname: Nerinckx
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
mesh_terms: D001331:Automation; D001665:Binding Sites; D001667:Binding, Competitive; D002475:Cellobiose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014242:Trichoderma / Q000201:enzymology
chemical_list: D008024:Ligands; D011506:Proteins; D002475:Cellobiose; D043366:Cellulose 1,4-beta-Cellobiosidase