期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 332, 期 2, 页码 362-369出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2005.04.130
关键词
protein thiols; deglutathionylation; biotin-tagged peptides; affinity chromatography; mass spectrometry
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. (C) 2005 Elsevier Inc. All rights reserved.
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