Protocols for identifying endogenous interactors of RNA-binding proteins in mammalian cells using the peroxidase APEX2 biotin-labeling method

Engineered ascorbate peroxidase, APEX2, is widely applied for the identification of intracellular molecule-molecule interaction analyses. Here, we present a protocol for identifying interactors of RNA-binding proteins (RBPs) in living HeLa cells using the APEX2 fusion construct. We describe steps for generation of RBP-APEX2, proximity biotin labeling, and preparation of labeled molecules for mass spectrometry analysis. This protocol may be applicable to other cell cultures and RBPs of interest. For complete details on the use and execution of this protocol, please refer to Uozumi et al.1.