热蛋白组学分析（Thermal proteome profiling，TPP）通过实现蛋白组学范围内的靶点和非靶点鉴定，显著推动了药物发现领域的进展。然而，由于该方法涉及大量实验操作和在质谱仪上较长的测量时间，TPP在高通量药物筛选方面的应用尚不广泛。本文介绍了单个管道统一进展（Single-tube TPP with Uniform Progression，STPP-UP），该方法显著减少所需输入材料的数量和测量时间，并保留了鉴定感兴趣化合物的药物靶点的能力。通过对单个样品进行渐进加热，可以评估一系列温度下蛋白质热稳定性的变化，同时减轻了对加热到不同温度的多个样品测量的需求。我们证明了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质。总之，STPP-UP方法是推进药物发现和药物再利用工作的有用工具。版权所有©2023作者。由Elsevier Inc.出版，保留全部权利。

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 anti-cancer 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.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.