Direct aliquoting from a single heated well enables fully automated high-throughput thermal proteome profiling

Thermal proteome profiling (TPP) measures drug target engagement across the proteome by detecting ligand-induced changes in protein thermal stability, but conventional workflows are laborious and costly, which has limited their use in compound screening. We present DASH-PISA, a single-well thermal fractionation approach in which a lysate is heated through a series of defined temperatures in one well and sampled at each step, and the aliquots are pooled into another well. Because sampling and pooling both take place in plate format, the thermal treatment runs on a standard liquid-handling robot with an integrated thermocycler and a 96-channel pipette. Combined with filter-based separation of soluble protein, data-independent acquisition (DIA), and a spike-in SILAC reference, DASH-PISA forms a fully automated, high-throughput TPP workflow. As a proof of concept, stepwise single-well fractionation reproduced the melting curves of traditional TPP, and the pooled DASH-PISA workflow detected known kinase targets of staurosporine; a spike-in SILAC reference further improved their recovery. By running on commercially available automation, this workflow makes proteome-wide target identification practical at the scale required for compound-library screening.