Protocol for identification and validation of 2′3′-cGAMP-binding proteins by photoaffinity probes

Mammalian cyclic dinucleotide 2′3′-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2′3′-cGAMP photoaffinity probes to capture 2′3′-cGAMP-binding or 2′3′-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC–based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2′3′-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells. For complete details on the use and execution of this profile, please refer to Hou et al. (2021).