Combining Proteome Integral Solubility Alteration (PISA) assay with One-Tip workflow for streamlined drug target deconvolution

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The Proteome Integral Solubility Alteration (PISA) assay is a mass spectrometry (MS)-based method for detecting protein-ligand interactions via changes in thermal stability under treatment conditions. The One-Tip workflow is a streamlined, low-input sample prep method using Evotips, integrating protein digestion, desalting, concentration, and direct injection into the Evosep One LC system. By incorporating the MS-friendly detergent DDM, we combined the two into the high-sensitivity One-Tip-PISA workflow and applied it to EGFR-overexpressing SCC25 squamous carcinoma cells, which depend on EGFR-driven proliferation. To assess One-Tip-PISA’s ability to detect drug-induced stability changes, cells were treated with two concentrations of Erlotinib, Enzastaurin, RMC4550, and Thapsigargin, targeting the EGFR pathway. Using narrow-window DIA on the Orbitrap Astral, we quantified 110,000 peptides and over 7000 proteins. Results included known targets (kinases, phosphatases) and suggested off-target effects for Enzastaurin, RMC4550, and Thapsigargin, consistent with One-Tip-PISA data in HeLa cells. For instance, Thapsigargin disrupted calcium signaling, increased c-fos expression, and induced ER stress via downregulation of a tetrameric transport protein. One-Tip-PISA broadens PISA’s scope, making it suitable for large-scale or low-input studies.

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