A systematic and quantitative assessment of the ubiquitin modified proteome was conducted using a monoclonal antibody that recognizes diglycine (diGly) containing isopeptides following trypsin digestion. The study identified approximately 19,000 diGly modified lysine residues in ~5000 proteins. Quantitative proteomics was used to monitor temporal changes in diGly site abundance in response to proteasomal and translational inhibition, revealing both a dependence on ongoing translation for site abundance changes and distinct dynamics of individual modified lysines in response to proteasome inhibition. The study also demonstrated the utility of diGly proteomics for identifying substrates for cullin-RING ubiquitin ligases. The ubiquitinome analysis allowed for a quantitative assessment of protein homeostasis fidelity and identification of substrates for individual ubiquitin pathway enzymes. The study found that proteasome inhibition leads to increased ubiquitylation of proteasome targets and a loss of ubiquitin from a cohort of putatively monoubiquitylated proteins. The ubiquitinome was found to be largely composed of proteins that are modified by ubiquitin, with NEDD8 and ISG15 contributing minimally. The study also showed that proteasome inhibition requires ongoing translation for the accumulation of a large cross-section of the ubiquitinome. The diGly proteome was found to represent a newly synthesized population of the proteome, with many highly abundant proteins being ubiquitylated. The study also identified CRL substrates using diGly proteomics and found that altering free UBL levels can lead to inappropriate E1 utilization. The study provided a comprehensive resource for the identification and classification of ubiquitin-modified lysine residues in both known and newly identified substrates. The results suggest that the ubiquitinome is a distinct and important component of the proteome, with a significant portion of proteins being ubiquitylated. The study also highlighted the importance of diGly modification in protein quality control and the potential for diGly-modified sites to serve as sensitive endogenous biomarkers for protein quality control fidelity. The findings have implications for understanding protein homeostasis and the role of ubiquitin in various diseases.A systematic and quantitative assessment of the ubiquitin modified proteome was conducted using a monoclonal antibody that recognizes diglycine (diGly) containing isopeptides following trypsin digestion. The study identified approximately 19,000 diGly modified lysine residues in ~5000 proteins. Quantitative proteomics was used to monitor temporal changes in diGly site abundance in response to proteasomal and translational inhibition, revealing both a dependence on ongoing translation for site abundance changes and distinct dynamics of individual modified lysines in response to proteasome inhibition. The study also demonstrated the utility of diGly proteomics for identifying substrates for cullin-RING ubiquitin ligases. The ubiquitinome analysis allowed for a quantitative assessment of protein homeostasis fidelity and identification of substrates for individual ubiquitin pathway enzymes. The study found that proteasome inhibition leads to increased ubiquitylation of proteasome targets and a loss of ubiquitin from a cohort of putatively monoubiquitylated proteins. The ubiquitinome was found to be largely composed of proteins that are modified by ubiquitin, with NEDD8 and ISG15 contributing minimally. The study also showed that proteasome inhibition requires ongoing translation for the accumulation of a large cross-section of the ubiquitinome. The diGly proteome was found to represent a newly synthesized population of the proteome, with many highly abundant proteins being ubiquitylated. The study also identified CRL substrates using diGly proteomics and found that altering free UBL levels can lead to inappropriate E1 utilization. The study provided a comprehensive resource for the identification and classification of ubiquitin-modified lysine residues in both known and newly identified substrates. The results suggest that the ubiquitinome is a distinct and important component of the proteome, with a significant portion of proteins being ubiquitylated. The study also highlighted the importance of diGly modification in protein quality control and the potential for diGly-modified sites to serve as sensitive endogenous biomarkers for protein quality control fidelity. The findings have implications for understanding protein homeostasis and the role of ubiquitin in various diseases.