Abstract
Background: 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 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. Results: In 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. Conclusions: This 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.
| Original language | English (US) |
|---|---|
| Article number | 14 |
| Journal | Clinical Proteomics |
| Volume | 12 |
| Issue number | 1 |
| DOIs | |
| State | Published - 2015 |
| Externally published | Yes |
Bibliographical note
Funding Information:This work was supported in part by the National Institutes of Health under grants and contracts from the National Cancer Institute: Clinical Proteomics Tumor Analysis Consortium (CPTAC, U24CA160036), the Early Detection Research Network (EDRN, U01CA152813 and U24CA115102), and R01CA112314; and from the National Heart, Lung and Blood Institute: Programs of Excellence in Glycosciences (P01HL107153) and Proteomic Center (N01-HV-00240). The authors gratefully acknowledge Sherri Davies and Matthew Ellis of the Washington University Clinical Proteomic Tumor Analysis Consortium (CPTAC) Proteome Characterization Center for providing the patient-derived xenograft tumor tissue.
Funding Information:
This work was supported in part by the National Institutes of Health under grants and contracts from the National Cancer Institute: Clinical Proteomics Tumor Analysis Consortium (CPTAC, U24CA160036), the Early Detection Research Network (EDRN, U01CA152813 and U24CA115102), and R01CA112314; and from the National Heart, Lung and Blood Institute: Programs of Excellence in Glycosciences (P01HL107153) and Proteomic Center (N01-HV-00240).
Publisher Copyright:
© 2015 Thomas et al.
Keywords
- Mass spectrometry
- Proteomics
- Quantification
- Tissue
- Ubiquitylation
- Xenograft
- iTRAQ
