title: Systematic and quantitative assessment of the ubiquitin-modified proteome.
abstract: 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.
authors: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors: Woong
authors: W
authors: Kim
authors: Eric J
authors: EJ
authors: Bennett
authors: Edward L
authors: EL
authors: Huttlin
authors: Ailan
authors: A
authors: Guo
authors: Jing
authors: J
authors: Li
authors: Anthony
authors: A
authors: Possemato
authors: Mathew E
authors: ME
authors: Sowa
authors: Ramin
authors: R
authors: Rad
authors: John
authors: J
authors: Rush
authors: Michael J
authors: MJ
authors: Comb
authors: J Wade
authors: JW
authors: Harper
authors: Steven P
authors: SP
authors: Gygi
mesh_terms: D002478:Cells, Cultured; D044842:Cullin Proteins / Q000378:metabolism; D006033:Glycylglycine / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D008239:Lysine / Q000235:genetics; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D044842:Cullin Proteins; D020543:Proteome; D025801:Ubiquitin; D006033:Glycylglycine; D008239:Lysine