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.