Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling

• DOI: 10.1016/j.xpro.2024.103417
• Publication Date: 2024-10-25T17:57:53Z
• Authors: Zhu Liang; Andreas Damianou; Athina Grigoriou; Hannah B.L. Jones; Vassilena Sharlan...; Frederik Lassen; Iolanda Vendrell; Elena Di Daniel; Benedikt M. Kessler
• Keywords: immunology; Q1-390; 0303 health sciences; 03 medical and health sciences; proteomics; Science (General); Protocol; molecular biology; mass spectrometry

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.1.