Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry.

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (K) for hen egg white lysozyme (HEWL) and N,N',N″-triacetylchitotriose (NAG) revealed an apparent K value of 313.2 ± 25.9 μM for the ligand titration method. Similar measurements for N,N',N″,N″'-tetraacetylchitotetraose (NAG) revealed an apparent K of 249.3 ± 13.6 μM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a K value of 9.8 ± 0.6 μM. In a second LEMS approach, a calibrated measurement was used to determine a K value of 6.8 ± 1.5 μM for NAG. The capture efficiency of LEMS was measured to be 3.6 ± 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted K value was 11.3 μM for NAG and 9.0 μM for NAG. The calibration method for measuring K developed in this study can be applied to solutions containing unknown analyte concentrations. Graphical Abstract.