Reckzeh Elena S, Brockmeyer Andreas, Metz Malte, Waldmann Herbert, Janning Petra
Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Methods Mol Biol. 2019;1888:73-98. doi: 10.1007/978-1-4939-8891-4_4.
The identification of protein targets and the elucidation of the molecular mechanism of action (MMoA) of bioactive small molecules are central goals of chemical biology. Many different techniques for target identification and engagement are developed, but none of them is generic. Here we describe one of these techniques-the cellular thermal shift assay (CETSA). The assay works without any labeling of proteins or small molecules, which allows the investigation of the unaltered interaction between the interaction partners. Briefly, the influence of small molecules on the thermal stability of proteins within whole cell lysates is investigated. We describe this approach in two variants: the conventional immunoblot-based approach (CETSA), as well as an unbiased approach based on a proteome-wide mass spectrometric analysis (thermal proteome profiling, TPP). The CETSA approach requires preknowledge about possible target proteins and can only detect a few proteins at once. Although TPP is technically more demanding, it allows for the identification of multiple (off)targets without any preknowledge.
识别蛋白质靶点以及阐明生物活性小分子的分子作用机制是化学生物学的核心目标。人们开发了许多不同的靶点识别和相互作用技术，但没有一种是通用的。在此，我们描述其中一种技术——细胞热位移分析（CETSA）。该分析无需对蛋白质或小分子进行任何标记，从而能够研究相互作用伙伴之间未改变的相互作用。简而言之，研究小分子对全细胞裂解物中蛋白质热稳定性的影响。我们将这种方法描述为两种变体：传统的基于免疫印迹的方法（CETSA），以及基于全蛋白质组质谱分析的无偏差方法（热蛋白质组分析，TPP）。CETSA方法需要预先了解可能的靶蛋白，并且一次只能检测少数几种蛋白质。尽管TPP在技术上要求更高，但它无需任何预先了解就能识别多个（脱）靶标。
