Specific protein-labeling and ligand-binding position analysis with amidopyrene probes as LDI MS tags

To readily analyze the binding mode of protein–ligand interactions, we developed ligand-bound-type and ligand-dissociation-type probes having 6-amidopyrene (apy) as a detecting group. Matrix- and label-assisted laser desorption/ionization mass spectrometry (MALDI and LA-LDI MS) analyses and a covalent docking simulation using these probes precisely determined the binding position of the ligand biotin on the target protein avidin (RMSD = 0.786 and 0.332 A). Our apy-probe-labeling method may be useful for determining the unknown ligand-binding sites of various target proteins.