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Evaluation of automated nano‐electrospray mass spectrometry in the determination of non‐covalent protein–ligand complexes
Author(s) -
De Vriendt Kris,
Sandra Koen,
Desmet Tom,
Nerinckx Wim,
Van Beeumen Jozef,
Devreese Bart
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1728
Subject(s) - chemistry , electrospray mass spectrometry , electrospray , mass spectrometry , protein mass spectrometry , nano , chromatography , covalent bond , ligand (biochemistry) , electrospray ionization , organic chemistry , chemical engineering , biochemistry , receptor , engineering
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.