Label-free target identification using in-gel fluorescence difference via thermal stability shift.

Target engagement is a prerequisite for the therapeutic effects of bioactive small molecules, and unbiased identification of their target proteins can facilitate drug discovery and chemical biology research. Structural modifications of bioactive natural products for target identification exhibit potential limitations such as synthetic difficulties, limited supplies from natural sources, and loss of original efficacy. Herein, we developed a label-free method for proteome-wide target identification using in-gel fluorescence difference caused by thermal stability shift, namely TS-FITGE. Quantitative intra-gel image analysis of each protein spot revealed target proteins with shifted thermal stability upon drug engagement, and plotting of melting curves by inter-gel analysis confirmed the positive targets. We demonstrated the robustness and applicability of the TS-FITGE method by identifying target proteins, including membrane-anchored proteins, of complex bioactive compounds. Furthermore, we identified and functionally validated nucleophosmin as a novel target protein of hordenine, a natural product upregulator of in vitro translation.