期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 31, 期 7, 页码 1544-1553出版社
AMER CHEMICAL SOC
DOI: 10.1021/jasms.0c00131
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资金
- National Institutes of Health (NIH) [R01 GM075092]
- NIH [1S10OD010645-01A1]
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.
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