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Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by 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.

Pubmed ID: 17370265

Authors

  • Denis NJ
  • Vasilescu J
  • Lambert JP
  • Smith JC
  • Figeys D

Journal

Proteomics

Publication Data

March 4, 2007

Associated Grants

None

Mesh Terms

  • Animals
  • Binding Sites
  • Breast Neoplasms
  • Cell Line, Tumor
  • Female
  • Humans
  • Mass Spectrometry
  • Mice
  • Reference Standards
  • Trypsin
  • Ubiquitin