Mass spectrometry-based thermal shift assay for protein-ligand binding analysis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chemical Biology

Background:

• Protein-ligand interactions are crucial in biological processes.
• Screening these interactions in complex mixtures presents analytical challenges.
• Existing methods may lack sensitivity or require purified proteins.

Purpose of the Study:

• To develop a mass spectrometry-based assay for detecting protein-ligand binding in multicomponent mixtures.
• To utilize an oxidation labeling protocol coupled with thermal denaturation analysis.
• To demonstrate the assay's efficacy in a proof-of-principle study.

Main Methods:

• A mass spectrometry assay employing an oxidation labeling protocol with hydrogen peroxide.
• Selective oxidation of methionine residues to probe solvent accessibility as a function of temperature.
• Analysis of intact proteins (MALDI) and tryptic peptide fragments (LC-MS) after oxidation.
• Construction of thermal denaturation curves from mass spectral data.

Main Results:

• The assay successfully generated thermal denaturation curves for proteins in a four-protein mixture.
• Protein-ligand binding interactions were detected by comparing denaturation curves with and without ligand.
• The known binding of cyclosporin A (CsA) to cyclophilin A (CypA) was confirmed using both MALDI- and LC-MS readouts.

Conclusions:

• The developed mass spectrometry assay is effective for identifying protein-ligand binding in complex biological samples.
• Oxidation labeling coupled with thermal denaturation provides a sensitive method for probing binding interactions.
• This assay offers a valuable tool for drug discovery and chemical biology research.