Recent advances in proteome‐wide label‐free target deconvolution for bioactive small molecules

Abstract Small‐molecule drugs modulate biological processes and disease states through engagement of target proteins in cells. Assessing drug–target engagement on a proteome‐wide scale is of utmost importance in better understanding the molecular mechanisms of action of observed beneficial and adverse effects, as well as in developing next generation tool compounds and drugs with better efficacies and specificities. However, systematic assessment of drug–target engagement has been an arduous task. With the continuous development of mass spectrometry‐based proteomics instruments and techniques, various chemical proteomics approaches for drug target deconvolution (i.e., the identification of molecular target for drugs) have emerged. Among these, the label‐free target deconvolution approaches that do not involve the chemical modification of compounds of interest, have gained increased attention in the community. Here we provide an overview of the basic principles and recent biological applications of the most important label‐free methods including the cellular thermal shift assay, pulse proteolysis, chemical denaturant and protein precipitation, stability of proteins from rates of oxidation, drug affinity responsive target stability, limited proteolysis, and solvent‐induced protein precipitation. The state‐of‐the‐art technical implications and future outlook for the label‐free approaches are also discussed.