Quantitative determination of lysozyme-ligand binding in the solution and gas phases by electrospray ionisation mass spectrometry

Affinity constants for the binding of a range of substrate and non-substrate oligosaccharides to hen egg white lysozyme were determined by direct observation of the protein.ligand complexes using electrospray ionisation mass spectrometry (ESI-MS) with a chip-based nano-ESI source. The values obtained for a series of beta-1,4-N-acetylglucosamine oligomers (NAG(n)) were found to be in good agreement with those determined by fluorescence measurement. Oligomers of alpha-1,4-glucose (Glc(n)), which are believed to bind to lysozyme non-specifically, exhibited a 10(6)- to 10(8)-fold lower affinity for the enzyme. Lysozyme.NAG(n) complexes displayed an increase in K-a from n = 2 to n = 4, but then reached a plateau. In contrast non-specific lysozyme.Glc(n) complexes showed no such trend. Determination of gas-phase complex stability was achieved by quantitative collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) measurements. The collision energy (Ec(50)) or laser power (IRMPD50) required to dissociate precursor ions to 50% of their original intensity was determined for lysozyme.NAG(n) and Glc(n) complexes using the [M+8H](8+) charge state. An excellent correlation between trends in Ka and gas-phase stability was seen for NAG(n) oligomers bound to lysozyme, whereas no such relationship was observed with the non-specific, weaker lysozyme.Glc(n) complexes. These results illustrate that ESI-MS can be used to quantify the interactions between lysozyme and oligosaccharides in both the solution and gas phase and that measurement of gas-phase complex stability by CID or IRMPD can provide information about specific solution binding events. Copyright (C) 2007 John Wiley & Sons, Ltd.