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.
Journal article
2024-12-20T00:00:00+00:00
5
immunology, mass spectrometry, molecular biology, proteomics, NLR Family, Pyrin Domain-Containing 3 Protein, Inflammasomes, Humans, Tandem Mass Spectrometry, Chromatography, Liquid, DNA-(Apurinic or Apyrimidinic Site) Lyase, Ascorbate Peroxidases, HEK293 Cells, Endonucleases, Multifunctional Enzymes