Open AccessProtein–Ligand Affinity Determinations Using Covalent Labeling-Mass Spectrometry
Author(s) -
Tianying Liu,
Tyler M. Marcinko,
Richard W. Vachet
Publication year - 2020
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1021/jasms.0c00131
Subject(s) - chemistry , dissociation constant , ligand (biochemistry) , mass spectrometry , protein ligand , covalent bond , dissociation (chemistry) , reagent , target protein , affinity labeling , chromatography , combinatorial chemistry , binding site , biochemistry , organic chemistry , receptor , gene
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 ( K d 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 K d 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.