High-throughput proteome integral solubility alteration assay for low cell input using One-Tip.

Mass spectrometry-based versions of the cellular thermal shift assay (CETSA), like proteome integral solubility alteration (PISA), enable simultaneous monitoring of thousands of proteins for drug-target engagement. These methods are constrained in throughput and scalability, while the sample requirement limits the applicability to widely available material. Here, we combine PISA with the One-Tip method to simplify and streamline sample preparation. Using the mass spectrometry-compatible n-Dodecyl-β-D-Maltoside (DDM) non-ionic detergent for cell lysis in PISA sample preparation enables direct transfer to One-Tip with decreasing cell requirements down to 200 cells per µL. One-Tip provides similar depth and higher reproducibility, with lower material and solvent usage and a faster proteolytic digestion compared to a conventional sample cleaning and digestion protocol, making it a cost-effective, fast, and user-friendly option. To demonstrate its scalability, we applied One-Tip-PISA in a 96-well plate format, profiling a kinase inhibitor panel, allowing cell treatment to injection within 12 h, enhancing workflow efficiency and accessibility for a wide range of laboratories.