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 (NAGn) were found to be in good agreement with those determined by fluorescence measurement. Oligomers of alpha-1,4-glucose (Glcn), which are believed to bind to lysozyme non-specifically, exhibited a 10(6)- to 10(8)-fold lower affinity for the enzyme. Lysozyme.NAGn complexes displayed an increase in Ka from n=2 to n=4, but then reached a plateau. In contrast non-specific lysozyme.Glcn 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 (Ec50) or laser power (IRMPD50) required to dissociate precursor ions to 50% of their original intensity was determined for lysozyme.NAGn and Glcn complexes using the [M+8H]8+ charge state. An excellent correlation between trends in Ka and gas-phase stability was seen for NAGn oligomers bound to lysozyme, whereas no such relationship was observed with the non-specific, weaker lysozyme.Glcn 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.
Veros
Christopher T
CT
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Oldham
Neil J
NJ
D000117:Acetylglucosamine / Q000737:chemistry; D000818:Animals; D002645:Chickens; D005260:Female; D005740:Gases; D008024:Ligands; D008832:Microchemistry / Q000379:methods; D009113:Muramidase / Q000737:chemistry*; D036103:Nanotechnology; D011485:Protein Binding; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005740:Gases; D008024:Ligands; D012996:Solutions; D009113:Muramidase; D000117:Acetylglucosamine