Institute of Molecular Systems Biology at ETH Zurich, Zurich, Switzerland.
Methods Mol Biol. 2023;2706:177-190. doi: 10.1007/978-1-0716-3397-7_13.
Limited proteolysis coupled to mass spectrometry (LiP-MS) is a recent proteomics technique that allows structure-based target engagement profiling on a proteome-wide level. To achieve this, native lysates are first incubated with a compound, followed by a short incubation with a nonspecific protease. Binding of a compound can change accessibility at the binding site or induce other structural changes in the target. This leads to treatment-specific proteolytic fingerprints upon limited proteolysis, which can be analyzed by standard bottom-up MS-based proteomics. Here, we describe a basic LiP-MS protocol using the natural product rapamycin as an example compound. Along with the provided LiP-MS reference data available via ProteomeXchange with identifier PXD035183, this enables the straightforward implementation of the method by scientists with a basic biochemistry and mass spectrometry background. We describe how the procedure can easily be adapted to other protein samples and small molecules.
有限蛋白水解与质谱联用（LiP-MS）是一种新兴的蛋白质组学技术，可在全蛋白质组水平上进行基于结构的靶标结合分析。为实现这一目标，首先将天然裂解物与化合物孵育，然后用非特异性蛋白酶短时间孵育。化合物的结合可以改变结合部位的可及性或诱导靶标发生其他结构变化。这导致在有限蛋白水解时产生特定于处理的蛋白水解指纹图谱，可通过基于标准自上而下的 MS 的蛋白质组学进行分析。在这里，我们使用天然产物雷帕霉素作为示例化合物描述了一个基本的 LiP-MS 方案。与通过 ProteomeXchange 提供的 LiP-MS 参考数据（标识符 PXD035183）一起，这使得具有基本生物化学和质谱背景的科学家能够轻松实现该方法。我们描述了如何轻松地将该程序适应于其他蛋白质样品和小分子。
