Richards, D. M.; zhai, F.; Li, S.; Yu, Q.

Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of ~9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2 degrees Celsius proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to optimize PISA then apply the optimized conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.