Selection of Heating Temperatures Improves the Sensitivity of the Proteome Integral Solubility Alteration Assay

The thermal shift assay is a robust method of discovering protein–ligand interactions by measuring the alterations in protein thermal stability under various conditions. Several thermal shift assays have been developed and their throughput has been advanced greatly by the rapid progress in tandem mass tag-based quantitative proteomics. A recent paper by Gaetani et al. ( J. Proteome Res. 2019, 18 (11), 4027−4037) introduced the proteome integral solubility alteration (PISA) assay, further increasing throughput and simplifying the data analysis. Both ΔSm (a proxy of the difference between areas under the melting curves) and fold changes (ratios between integral samples) are readouts of the PISA assay and positively related to ΔTm (shift in melting temperatures). Here, we show that the magnitudes of these readouts are inherently small in PISA assay, which is a challenge for quantitation. Both simulation and experimental results show that the selection of a subset of heating temperatures ameliorates the small difference problem and improves the sensitivity of the PISA assay.