Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.
J Proteome Res. 2020 May 1;19(5):2159-2166. doi: 10.1021/acs.jproteome.0c00063. Epub 2020 Apr 13.
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. ( 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.
热移分析是一种通过测量蛋白质在不同条件下热稳定性变化来发现蛋白质-配体相互作用的强大方法。几种热移分析方法已经被开发出来，并且通过基于串联质量标签的定量蛋白质组学的快速进展，其通量得到了极大的提高。最近 Gaetani 等人的一篇论文（2019 年，18 （11），4027-4037）介绍了蛋白质组整体溶解度变化分析（PISA）测定法，进一步提高了通量并简化了数据分析。ΔSm（熔解曲线下面积差异的代表）和折叠变化（积分样本之间的比率）都是 PISA 测定法的读出值，与 ΔTm（熔点的偏移）呈正相关。在这里，我们表明 PISA 测定法中这些读出值的幅度本质上很小，这对定量分析是一个挑战。模拟和实验结果均表明，选择加热温度子集可以改善微小差异问题并提高 PISA 测定法的灵敏度。
