Ligand-protein interaction: CETSA, Kinobeads


Investigation of ligand-protein interactions is fundamental to understand almost all biological processes. Ligand-protein interactions include interactions with other proteins, peptides, natural products or small molecules and involve the investigation of complex formation, drug-protein interaction, determination of binding affinities and kinetics. For example, an unbiased investigation of drug-protein interactions can help to elucidate a drug’s mode of action and to establish a selectivity profiling in order to identify toxic or beneficial off-targets. Different approaches can be useful to gain further insight into ligand-protein interactions, such as chemical proteomic approaches or cellular thermal shift assay (CETSA).

Chemical proteomics offers chemical tools (e.g. affinity matrices) for the investigation of biological questions and enables the determination of binding affinities, selectivity profiling and identification of complex partners.

The cellular thermal shift assay (CETSA) relies on differential protein stability in ligand-free and ligand-bound state, which results in a shift of the proteins melting temperature in a temperature gradient. Target proteins in a complex mixture can be identified simultaneously by a mass spectrometry read-out. After the identification of an optimal temperature, binding affinities can be determined in isothermal dose response (ITDR) experiments. This technique is especially interesting since it is non-invasive and does not require prior immobilization or fluorescence labeling of proteins.


The typical outcome of a ligand-protein interaction experiment based on affinity matrices or ITDRs is a list of identified and quantified target proteins and their binding affinities. CETSA experiments provide information about thermal shift behavior of each identified protein over a pre-defined temperature gradient.