Screening and identification of protein-protein interaction using proximity labeling

Cell death is a fundamental biological process with critical roles in both normal physiology and pathological conditions, especially programmed cell death, such as apoptosis, necroptosis and pyroptosis. Programmed cell death is mediated by cascade signaling transduction rely on protein-protein interaction. Necroptosis mediator RIPK1, RIPK3 and MLKL have been shown to be regulated by different types of post-translational modifications (PTMs), suggesting that additional factors must associate with them during necroptosis. Proximity labeling (PL) has been used to tag and identify proteins, RNAs, or other biomolecules in close proximity (∼10-20 nm) to a target protein of interest. Therefore, utilizing proximity labeling coupled mass spectrometry to identify weak and transient interactors of necroptosis mediators, will be helpful for the further understanding of cell death mechanisms and functions. Here, in this chapter, we provide a step-by-step protocol for using TurboID-based proximity labeling to map interactors and regulators of key necroptotic proteins (RIPK1, RIPK3, MLKL and ZBP1).