Label-free target protein characterization for small molecule drugs: recent advances in methods and applications

Target protein identification is the key to identification of the mechanisms, side effects, and evaluating druglikeness of small-molecule drugs. The commonly used "labeled" target-characterization methods, including activity-based proteome profiling (ABPP), require the synthesis of a derivatized probe, which are time-consuming and may affect the active drug conformation. Label-free target identification methods do not involve any chemical modification of small-molecules drugs and have received increasing attention in recent years. We reviewed the basic principles, workflow, applications, advantages, and disadvantages of the promising label-free target identification methods, including cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), pulse proteolysis (PP), stability of proteins from rates of oxidation (SPROX), drug affinity responsive target stability (DARTS), limited proteolysis-coupled mass spectrometry (LiP-MS) and solvent-induced protein precipitation (SIP). We also reviewed the prospective applications of these label-free methods for efficient target identification. The approaches based on peptide mapping using high-resolution mass spectrometry (MS) may provide more information regarding comprehensive target proteins and binding sites, which may be useful for target identification in multi-target or complex drug systems.