STUPPIT is a proximity labeling tool for labeling intermediary proteins that bridge two non-interacting proteins.

Decoding the complexities of signaling pathways is fundamental for deciphering the mechanisms underlying tissue development, homeostasis, and disease pathogenesis. Proximity labeling tools have been instrumental in identifying upstream or downstream effectors of specific proteins within signaling pathways. However, currently, there are no tools available to directly label and capture intermediary proteins that bridge two non-interacting proteins. Here, we developed Split-TurboID and PUP-IT based Protein Identification Tool (STUPPIT), a novel method combining split-TurboID and PUP-IT to biotinylate intermediary proteins of two non-interacting proteins through a two-step enzymatic reaction. STUPPIT was validated using three well-characterized protein triads, including YAP1/AMOT/β-actin, YAP1/LATS1/MOB1A, and β-catenin/α-catenin/β-actin using HEK293T human cell lines. Combining STUPPIT and proteomics, we identified novel intermediary proteins including ERC1 and USP7, which interacted both with β-catenin and SMAD4, key components of the Wnt and BMP signaling pathways. In conclusion, STUPPIT represents a powerful tool for labeling and capturing intermediary proteins between non-interacting partners, offering new insights into protein-protein interactions and advancing signal transduction research.