Systematic and quantitative assessment of the ubiquitin modified proteome

By Woong Kim, E. Bennett, Edward L. Huttlin, A. Guo, Jing Li, A. Possemato, M. Sowa, R. Rad, John Rush, M. Comb and 2 more. Molecules and Cells, 2011

1,651 citations recorded by the source corpus.

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 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 of on-going 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.

Subfields: Molecular Biology, Oncology

View Paper: https://www.semanticscholar.org/paper/983d9fa69f1f441c98cfccb1bddbe13b8de13160

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Citations in the corpus

Insights in Post-Translational Modifications: Ubiquitin and SUMO (2022, background): “Immunopurification methods using monoclonal antibodies against selective diGly residues have been exploited for the MS identification of ubiquitination sites [155,156].”
Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics (2012, positive): “To confirm that the MG132 treatment effectively blocked the proteasome, we analyzed our total data set for the abundance of K48-linked ubiquitin and histone H2A ubiquitin conjugates, which were previously shown to change following proteasome inhibition (37).”
Deubiquitylating enzymes and drug discovery: emerging opportunities (2017, methodology): “For example, Altun et al. (2011), used ABPs to demonstrate the selectivity of P22077 for USP7 in cells, in contrast to PR-619 which inhibited a broad range of DUBs(239).”
Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization (2013, methodology): “12,13 To overcome inherent stochastic sampling for low abundance diGLY peptides (see Figure S1a,b), 12,13 we performed 73 control and QdiGLY profiling experiments in 4 cell lines with and without elevated PARKIN levels (~10-fold) (Figure S2a) in a 3-tiered manner, varying the length of…”
Quantitative proteomics reveal a feed-forward model for mitochondrial PARKIN translocation and UB chain synthesis (2014, neutral): “Ubiquitylation site identification by mass spectrometry was performed as described (Kim et al., 2011; Sarraf et al., 2013).”
The ubiquitin code. (2012, background): “Alternatively, ubiquitylation can occur at endosomal membranes to control localization after internalization.”