Determination of site-specificity of S-glutathionylated cellular proteins.
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.
Hamnell-Pamment
Ylva
Y
Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm SE-17177, Sweden.
Lind
Christina
C
Palmberg
Carina
C
Bergman
Tomas
T
Cotgreave
Ian A
IA
D001665:Binding Sites; D019921:Biotinylation / Q000379:methods; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D045424:Complex Mixtures / Q000032:analysis; D042783:Endothelial Cells / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000378:metabolism*
D045424:Complex Mixtures; D020543:Proteome; D005978:Glutathione