The proteome is constantly remodeled to meet the changing environmental challenges of the cell. Protein degradation facilitated by the ubiquitin-proteasome system (UPS) is a major contributor to proteome remodeling. In this pathway, ubiquitin is activated and transferred to substrates via an E1-E2-E3 cascade (Ye and Rape, 2009
). The chemical properties of the isopeptide bond formed between the C-terminal glycine in ubiquitin and the ε-amino group of lysine (Lys) residues in substrates provides a route for detection of ubiquitylated targets by mass spectrometry, as trypsinolysis of ubiquitin conjugates yields a characteristic “diGly remnant” due to cleavage of the C-terminal Arg-Gly-Gly sequence of ubiquitin (Peng et al., 2003
It is convenient to consider two major classes of UPS targets: 1) regulatory substrates that undergo programmed ubiquitylation during a physiological process, and 2) quality control substrates that undergo ubiquitylation in response to misfolding, inappropriate complex formation, or aggregation. Signal-dependent regulatory ubiquitylation often results in the complete degradation of the target protein, eliminating its function. In contrast, quality control proteolysis generally affects only the fraction of protein that is defective. Errors in co-translational folding of proteins or translation may account for a significant fraction of the flux through the UPS (Schubert et al., 2000
; Vabulas and Hartl, 2005
It is currently thought that a wide cross-section of the proteome is subject to ubiquitin modification at some point during its lifetime. As such, there are two central challenges facing the field. First, a complete and quantitative description of the ubiquitinome – the array of proteins that are modified by the ubiquitin system as well as the actual site of modification – requires a means by which to detect, catalog, and quantify individual ubiquitylation events on proteins. Previous studies have focused on either the use of ubiquitin binding domains/antibodies or overexpression of epitope-tagged ubiquitin in an attempt to capture ubiquitylated proteins for identification by mass spectrometry (Danielsen et al., 2011
; Matsumoto et al., 2005
; Meierhofer et al., 2008
; Peng et al., 2003
; Tagwerker et al., 2006
). However, the low occupancy of ubiquitylation challenges detection of endogenously modified proteins in the absence of overexpression of either ubiquitin or substrate. Advances in mass spectrometry and enrichment strategies, including affinity-capture of the diGly remnant, have assisted in the identification of a greater number of sites, with recent reports identifying 753 and 374 ubiquitylation sites (Danielsen et al., 2011
; Xu et al., 2010
). However, these studies relied upon exogenous expression of epitope-tagged ubiquitin, possibly subverting endogenous ubiquitin modification pathways. Despite these advances, the overall number of modification sites is small in comparison to the extent of acetylation and phosphorylation (Choudhary et al., 2009
; Huttlin et al., 2010
; Olsen et al., 2010
), and it is unclear the extent to which ubiquitin overexpression affects the occupancy and specificity of ubiquitylation.
The second central challenge for the field is matching ubiquitylation targets with the vast array of ubiquitylation machinery encoded by eukaryotic genomes. The majority of substrates for E3s have been identified based on a physical interaction between the E3 and the substrate. While mutational analysis is most often used to identify candidate ubiquitylation sites in targets, this approach does not always provide a direct route to the actual sites of endogenous ubiquitylation in vivo, due to unmasking of cryptic sites and effects of overexpression.
Here, we employ an improved method for antibody-based capture of endogenous diGly-containing peptides to identify ~19,000 ubiquitylation sites in ~5000 proteins, and to quantitatively monitor temporal changes in the ubiquitinome in response to proteasome inhibition. This analysis reveals both increased ubiquitylation of proteasome targets and a loss of ubiquitin from a cohort of putatively monoubiquitylated proteins, presumably as a response to ubiquitin depletion. Surprisingly, detection of a large cross-section of the ubiquitinome upon proteasome inhibition requires on-going translation. We also demonstrate the utility of diGly capture for identification of cullin-RING ubiquitin ligase (CRL) substrates, and demonstrate that ubiquitin depletion promotes charging and transfer of the ubiquitin-like (UBL) protein NEDD8 by the ubiquitin conjugating machinery, thereby altering the targeting specificity of this UBL. Using a multi-classifier approach on merged quantitative data from multiple experiments, we categorize individual modified lysine residues into putative classes representing distinct functional outcomes. This dataset represents a powerful resource for the identification and classification of ubiquitin-modified lysine residues in both known UPS substrates and newly identified substrates allowing for facile future interrogation of individual site utilization.