The successful use of proteasome inhibitors in clinical trials revealed the 
potential of the Ubiquitin Proteasome System for drug development. Protein 
remodeling through ubiquitylation is known to regulate the stability and 
activity of essential cellular factors through largely uncharacterized 
mechanisms. Here, we used Tandem repeated Ubiquitin Binding Entities (TUBEs) 
under non-denaturing conditions followed by mass spectrometry analysis to study 
global ubiquitylation events that may lead to the identification of potential 
drug targets. Using this approach we identified 643 proteins including known and 
unknown ubiquitin targets from human breast adenocarcinoma MCF7 cells treated 
with Adriamycin. Coherent with a global cellular response to this genotoxic 
insult, cellular factors identified are involved in protein synthesis, cellular 
transport, RNA post-transcriptional modification and signaling pathways 
regulating early stress responses. This includes components of large 
macromolecular complexes such as subunits and regulators of the proteasome, 
supporting the use of this method to characterize networks of molecular 
interactions coordinated by ubiquitylation. Further in vitro and in silico 
analysis confirmed that 84% of the total proteins identified here, are 
ubiquitylated. More importantly the enrichment of known biomarkers and targets 
for drug development, underlined the potential of this approach for the 
identification of this clinically relevant information. This article is part of 
a Special Issue entitled: Proteomics: The clinical link.