Determination of site-specificity of S-glutathionylated cellular proteins

Biochem Biophys Res Commun. 2005 Jul 1;332(2):362-9. doi: 10.1016/j.bbrc.2005.04.130.


Redox modification by S-glutathionylation is an expanding field within cell signalling research. However, the methods available for analysis of S-glutathionylated proteins in complex mixtures are not sufficiently accurate to specifically and in a high-throughput manner on a structural level establish the effects of S-glutathionylation on the individual proteins. A method has been developed for rapid identification of the S-glutathionylation sites of proteins in diamide-treated ECV304 cells, through tagging of deglutathionylated proteins with a cysteine-reactive biotin-affinity tag, trypsinisation, avidin-affinity purification of tagged peptides, and subsequent analysis by liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. The method has led to identification of the glutathionylation sites of gamma-actin (Cys(217)), heat shock protein 60 (Cys(447)), and elongation factor 1-alpha-1 (Cys(411)). Further developments of accuracy within the field of peptide-affinity capture and mass spectrometry are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Biotinylation / methods
  • Cell Line
  • Chromatography, Liquid / methods*
  • Complex Mixtures / analysis
  • Endothelial Cells / metabolism*
  • Glutathione / metabolism*
  • Humans
  • Isotope Labeling
  • Mass Spectrometry / methods*
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Proteome / metabolism*


  • Complex Mixtures
  • Proteome
  • Glutathione