A proteomics approach to understanding protein ubiquitination

Nat Biotechnol. 2003 Aug;21(8):921-6. doi: 10.1038/nbt849. Epub 2003 Jul 20.


There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Gene Expression Regulation, Fungal / physiology
  • Molecular Sequence Data
  • Protein Interaction Mapping / methods*
  • Proteome / chemistry
  • Proteome / metabolism
  • Proteomics / methods*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment / methods*
  • Sequence Analysis, Protein / methods*
  • Sequence Homology, Amino Acid
  • Ubiquitin / chemistry*
  • Ubiquitin / metabolism*


  • Proteome
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin