Identifying small-molecule binders to protein targets remains a daunting task due to the huge diversity in compound structure, activity, and mechanisms of action. Affinity-based target identification techniques are limited by the necessity to modify each drug individually (without losing bioactivity), while non-affinity based approaches are dependent on the drug’s ability to induce specific biochemical/cellular readouts. To overcome these limitations, we have developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) platform coupled to a universally applicable target identification approach that analyzes direct small-molecule binding to its protein target(s). DARTS (drug affinity responsive target stability) relies on a well-known phenomenon in which ligand binding causes thermodynamic stabilization of its target protein’s structure such that the protein becomes resistant to a variety of insults, including proteolysis.

DARTS-MS is performed by screening for proteins that become resistant to proteolysis in the presence of the ligand. Protection from proteolysis is specific to the target protein(s) while proteolysis of non-target proteins is unchanged, allowing for unbiased, proteome-wide screening for small-molecule binders. Samples are proteolyzed and fractionated according to molecular weight (MW). Each molecular weight range is subsequently analyzed by LC-MS/MS (Q-Exactive q-Orbitrap) to identify and quantify the proteins present. Proteins that are differentially digested between a small-molecule-protected sample and the control are determined using Protomap (Protein Topography and Migration Analysis Platform) based on differences in the total amount of each protein and/or differences in sizes of the protein fragments. To date we have utilized our DARTS-MS strategy both to re-discover known protein targets of existing small-molecules as well as discovering novel targets for established drugs and important metabolites.