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

Zhu Liang; Andreas Damianou; Athina Grigoriou; Hannah B.L. Jones; Vassilena Sharlandijeva; Frederik Lassen; Iolanda Vendrell; Elena Di Daniel; Benedikt M. Kessler

STAR Protocols (Dec 2024)

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

Zhu Liang,
Andreas Damianou,
Athina Grigoriou,
Hannah B.L. Jones,
Vassilena Sharlandijeva,
Frederik Lassen,
Iolanda Vendrell,
Elena Di Daniel,
Benedikt M. Kessler

Affiliations

Zhu Liang: 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; Corresponding author
Andreas Damianou: 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; Corresponding author
Athina Grigoriou: Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK
Hannah B.L. Jones: Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK
Vassilena Sharlandijeva: Weatherall Institute for Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK
Frederik Lassen: Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford OX3 7LF, UK
Iolanda Vendrell: 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
Elena Di Daniel: Alzheimer’s Research UK Oxford Drug Discovery Institute, University of Oxford, Oxford OX3 7FZ, UK
Benedikt M. Kessler: 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; Corresponding author

Journal volume & issue: Vol. 5, no. 4
p. 103417

Abstract

Read online

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

About the journal

Abstract

Keywords