Protein-Ligand Affinity Determinations Using Covalent Labeling-Mass Spectrometry.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chemical Biology

Background:

• Characterizing protein-ligand interactions is crucial in molecular biology.
• Accurate determination of binding affinity (Kd values) is essential for understanding these interactions.
• Existing methods for measuring binding affinity can be challenging for certain protein-ligand complexes.

Purpose of the Study:

• To develop and validate a novel covalent labeling (CL)-mass spectrometry (MS) approach for determining protein-ligand dissociation constants (Kd).
• To utilize diethylpyrocarbonate (DEPC) as a labeling reagent within the CL-MS framework.
• To establish CL-MS as a viable alternative for characterizing challenging protein-ligand interactions and potentially identifying binding sites.

Main Methods:

• Employed covalent labeling (CL) with diethylpyrocarbonate (DEPC) followed by mass spectrometry (MS).
• Maintained protein labeling extent below 30% to ensure accurate Kd determination.
• Validated the method using two established and one poorly characterized protein-ligand systems.

Main Results:

• Demonstrated that CL-MS can accurately determine protein-ligand dissociation constants (Kd values).
• Achieved Kd values comparable to those obtained through traditional methods across different systems.
• Showcased the method's effectiveness even when labeling reaction time scales approach protein-ligand dissociation/reassociation rates.

Conclusions:

• The developed CL-MS strategy offers a robust alternative for measuring protein-ligand binding affinities.
• This approach is particularly valuable for systems that are difficult to analyze with conventional techniques.
• The CL-MS method holds potential for simultaneously providing both binding affinity and binding site information.