Both these authors have contributed equally to this work.
Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry
Article first published online: 16 MAR 2007
Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: SYSTEMS BIOLOGY
Volume 7, Issue 6, pages 868–874, No. 6 March 2007
How to Cite
Denis, N. J., Vasilescu, J., Lambert, J.-P., Smith, J. C. and Figeys, D. (2007), Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry. Proteomics, 7: 868–874. doi: 10.1002/pmic.200600410
- Issue published online: 16 MAR 2007
- Article first published online: 16 MAR 2007
- Manuscript Received: 4 JUN 2006
- Canada Research Chair
- In-gel digestion;
- Mass spectrometry;
Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.