Abstract
BackgroundPost-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.ResultsIn this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.ConclusionsThis is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9086-5) contains supplementary material, which is available to authorized users.
Highlights
Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth
Following protein extraction and trypsin digestion, peptides from each breast cancer patientderived xenograft tumor tissue sample were subjected to di-Gly ubiquitin remnant motif (K-ε-GG) immunoaffinity enrichment using an antibody that recognizes Lys residues that are modified with a di-Gly ubiquitin remnant (K-ε-GG)
In summary, we conducted an integrated isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis that enabled the interrogation of the relationship between global protein and endogenous ubiquitylation relative abundance using basal and luminal breast cancer patient-derived xenograft tissue
Summary
Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. Covalent post-translational modification (PTM) by ubiquitin entails the formation of an isopeptide bond between the C-terminus of ubiquitin and the ε-amino group of the targeted Lys residue on the substrate protein, it is possible, but rare, for N-terminal. The peptide-based affinity enrichment of the ubiquitylated proteome entails the use of a K-ε-GG antibody that recognizes the di-Gly ubiquitin remnant that remains conjugated to Lys residues on substrate proteins following trypsin digestion [16, 22, 23]. For the current study, the di-Gly-modified proteome and K-ε-GG-containing peptides will be referred to as the ubiquitylated proteome and ubiquitylated peptides, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
