Liang Zhu, Damianou Andreas, Grigoriou Athina, Jones Hannah B L, Sharlandijeva Vassilena, Lassen Frederik, Vendrell Iolanda, Di Daniel Elena, Kessler Benedikt M
Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK; Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
STAR Protoc. 2024 Dec 20;5(4):103417. doi: 10.1016/j.xpro.2024.103417. Epub 2024 Oct 25.
The NLRP3 inflammasome is a key multi-protein complex controlling inflammation, particularly interleukin-1β (IL-1β) production. Here, we present a protocol to profile spatially resolved NLRP3 inflammasome complexes using ascorbic peroxidase 2 (APEX2)-based proximity labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for design and generation of the fusion construct, characterization of the stable FLAG-NLRP3-APEX2 expression cell line by western blotting/imaging, biotinylated proteome enrichment, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Liang et al..
NLRP3炎性小体是一种控制炎症的关键多蛋白复合物，尤其是白细胞介素-1β（IL-1β）的产生。在此，我们展示了一种使用基于抗坏血酸过氧化物酶2（APEX2）的邻近标记结合液相色谱-串联质谱（LC-MS/MS）对空间分辨的NLRP3炎性小体复合物进行分析的方案。我们描述了融合构建体的设计和生成步骤、通过蛋白质印迹/成像对稳定的FLAG-NLRP3-APEX2表达细胞系进行表征、生物素化蛋白质组富集以及质谱分析。有关此方案使用和执行的完整详细信息，请参考Liang等人的研究。
