Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University Nijmegen, Nijmegen, The Netherlands.
Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University Nijmegen, Nijmegen, The Netherlands; Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
J Biol Chem. 2023 Nov;299(11):105279. doi: 10.1016/j.jbc.2023.105279. Epub 2023 Sep 22.
Thermal proteome profiling (TPP) has significantly advanced the field of drug discovery by facilitating proteome-wide identification of drug targets and off-targets. However, TPP has not been widely applied for high-throughput drug screenings, since the method is labor intensive and requires a lot of measurement time on a mass spectrometer. Here, we present Single-tube TPP with Uniform Progression (STPP-UP), which significantly reduces both the amount of required input material and measurement time, while retaining the ability to identify drug targets for compounds of interest. By using incremental heating of a single sample, changes in protein thermal stability across a range of temperatures can be assessed, while alleviating the need to measure multiple samples heated to different temperatures. We demonstrate that STPP-UP is able to identify the direct interactors for anticancer drugs in both human and mice cells. In summary, the STPP-UP methodology represents a useful tool to advance drug discovery and drug repurposing efforts.
热蛋白质组谱分析（TPP）通过促进药物靶点和非靶点的蛋白质组范围鉴定，极大地推动了药物发现领域的发展。然而，由于该方法劳动强度大，需要在质谱仪上花费大量的测量时间，因此尚未广泛应用于高通量药物筛选。在这里，我们提出了单管 TPP 均匀升温法（STPP-UP），它显著减少了所需输入材料的数量和测量时间，同时保留了鉴定目标化合物的药物靶点的能力。通过对单个样本进行增量加热，可以评估蛋白质在一系列温度下的热稳定性变化，同时减轻了测量多个加热到不同温度的样本的需求。我们证明 STPP-UP 能够鉴定人类和小鼠细胞中抗癌药物的直接相互作用物。总之，STPP-UP 方法代表了推进药物发现和药物再利用努力的有用工具。
