Abstract
Determining the binding affinity is an important aspect of characterizing protein-ligand complexes. Here, we describe an approach based on covalent labeling (CL)-mass spectrometry (MS) that can accurately provide protein-ligand dissociation constants (Kd values) using diethylpyrocarbonate (DEPC) as the labeling reagent. Even though DEPC labeling reactions occur on a time scale that is similar to the dissociation/reassociation rates of many protein-ligand complexes, we demonstrate that relatively accurate binding constants can still be obtained as long as the extent of protein labeling is kept below 30%. Using two well-established model systems and one insufficiently characterized system, we find that Kd values can be determined that are close to values obtained in previous measurements. The CL-MS-based strategy that is described here should serve as an alternative for characterizing protein-ligand complexes that are challenging to measure by other methods. Moreover, this method has the potential to provide, simultaneously, the affinity and binding site information.