Ubi-SCAPE Enables Deep Exploration of the Poly-Ubiquitylome

Abstract Post-translational modification with chains of the 76-amino acid protein ubiquitin (“poly-ubiquitylation”) confers diverse fates to the targeted protein and non-protein substrates, including degradation, intracellular trafficking, and signal transduction. Despite being one of the most frequent modifications, the complexity of poly-ubiquitin chains adds methodological challenges to their characterization. Trypsin-resistant tandem ubiquitin-binding entities (trTUBEs), engineered from natural ubiquitin-binding domains, can capture intact poly-ubiquitylated material with high cumulative avidity. However, such approaches have suffered from considerable co-eluting contaminants in proteomics applications. Here, we introduce an optimized trTUBE-based method for poly-ubiquitylated proteome purification, drastically depleting non-ubiquitylated protein contaminants and mono-ubiquitylated proteins. The method, termed ubiquitylomics by stringent, cleavable, affinity-based proteome extraction (Ubi-SCAPE), offers a streamlined and reproducible (median R2 > 0.98; CV < 8%) means of characterizing the poly-ubiquitylome. Over 7800 poly-ubiquitylated proteins and 8500 ubiquitin-modified peptides (diGly) were quantified with Ubi-SCAPE at a throughput of 40 samples per day, as well as over 6000 proteins from 100 μg of unstressed cell lysate. Upon acute stress by heat-shock, we identified 2700 proteins and 8000 diGly peptides with increased poly-ubiquitylation, offering similar biological insights as far more material-, labor-, and cost-intensive peptide-based ubiquitin enrichment methods would provide. Ubi-SCAPE therefore provides a simple and effective means of comprehensively quantifying a selectively enriched poly-ubiquitylome.