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

Ryota Uozumi; Kohji Mori; Shoshin Akamine; Manabu Ikeda

doi:10.1016/j.xpro.2024.103368

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

STAR Protoc. 2024 Oct 9;5(4):103368. doi: 10.1016/j.xpro.2024.103368. Online ahead of print.

Authors

Ryota Uozumi¹, Kohji Mori², Shoshin Akamine¹, Manabu Ikeda¹

Affiliations

¹ Department of Psychiatry, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
² Department of Psychiatry, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: kmori@psy.med.osaka-u.ac.jp.

PMID: 39392747
DOI: 10.1016/j.xpro.2024.103368

Abstract

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.

Keywords: Bioinformatics; Cell Biology; Protein expression and purification; Proteomics.