ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.