Molecular Cell
Volume 44, Issue 2, 21 October 2011, Pages 325-340
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Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome

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Summary

Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

Highlights

► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates

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4

These authors contributed equally to this work

5

Present address: Division of Biological Sciences, University of California San Diego, La Jolla, CA