Ubi-SCAPE enables deep exploration of the poly-ubiquitylome

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 proteins 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 7,800 poly-ubiquitylated proteins and 8,500 ubiquitin-modified peptides (diGly) were quantified with Ubi-SCAPE at a throughput of 40 samples per day, as well as over 6,000 from an equivalent of 33 g unstressed cell lysate. Upon acute stress by heat-shock, we identified 2,700 proteins and 8,000 diGly peptides with increased poly-ubiquitylation--offering similar biological insight as with far more material-, labor-, and cost-intensive peptide-based ubiquitin enrichment methods. Ubi-SCAPE therefore provides a simple and effective means of comprehensively quantifying a selective-enriched poly-ubiquitylome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/703496v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@a28120org.highwire.dtl.DTLVardef@cb801aorg.highwire.dtl.DTLVardef@47577aorg.highwire.dtl.DTLVardef@1c030af_HPS_FORMAT_FIGEXP M_FIG C_FIG