The reported platform for the identification of proteins recruited in artificial granules (IPRAG) combines self-assembling Y15 peptide tag technology and proximity labeling. An intracellular artificial granule containing a bait protein and a biotin ligase TurboID was designed and constructed, and endogenous proteins enriched in the bait-tethered granules were identified by LC-MS/MS analysis.
Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.
