Optimized Automated Workflow for BioID Improves Reproducibility and Identification of Protein-Protein Interactions.

Proximity-dependent biotinylation is an important method to study protein-protein interactions in cells, for which an expanding number of applications has been proposed. The laborious and time-consuming sample processing has limited project sizes so far. Here, we introduce an automated workflow on a liquid handler to process up to 96 samples at a time. The automation not only allows higher sample numbers to be processed in parallel but also improves reproducibility and lowers the minimal sample input. Furthermore, we combined automated sample processing with shorter liquid chromatography gradients and data-independent acquisition to increase the analysis throughput and enable reproducible protein quantitation across a large number of samples. We successfully applied this workflow to optimize the detection of proteasome substrates by proximity-dependent labeling.

Quantitative mapping of proteasome interactomes and substrates using ProteasomeID.

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.