The ubiquitin machinery, including E3 ligases and deubiquitinases, are critical regulators of cellular processes and can be chemically targeted with therapeutic potential. For example, PROteolysis TArgeting Chimeras (PROTACs) are bifunctional compounds that recruit an E3 ligase to a disease-relevant neo-substrate for targeted protein degradation. To uncover the intricacies of this system and identify new ubiquitin pathways for disease intervention, comprehensive profiling using mass spectrometry-based proteomics is essential.

In this study, we present an extensive proteomic profiling of the ubiquitination machinery and investigate cellular responses to their targeted modulation. Using proximity-dependent interactomics and quantitative proteomics, we profiled deubiquitinases and E3 ligase substrate receptors. Our analyses, conducted in the presence and absence of selective and potent ligands, revealed a broad spectrum of non-degradative and degradative substrates. These findings offer insights into the dynamic regulatory roles these enzymes play beyond their well-known function in protein degradation. Moreover, we developed a novel serial enrichment strategy for ubiquitin proteomics, which enables the identification of ubiquitin sites induced by PROTAC treatment on a neo-substrate. This innovative approach demonstrates high specificity and sensitivity, and can be easily extended to other post-translational modifications and applications.

Our study highlights the versatility and complexity of the ubiquitination machinery and underscores its potential as a therapeutic target. The methodologies we have established provide robust tools for dissecting ubiquitin-related pathways and their implications in health and disease.