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题名:Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry
作者:Mahadevan Sethuraman, Nicolas Clavreul, Hua Huang, Mark E. McComb, Catherine E. Costello and Richard A. Cohen
来源:Free Radical Biology and Medicine[IF=4.971], Volume 42, Issue 6, 15 March 2007, Pages 823-829
URL :http://dx.doi.org/10.1016/j.freeradbiomed.2006.12.012
日期:070415
摘要: Mahadevan Sethuramana, b, Nicolas Clavreula, b, Hua Huangb, Mark E. McCombb, Catherine E. Costellob, c and Richard A. Cohena, b, ,

aVascular Biology Unit, Boston University School of Medicine, Boston, MA 02118, USA
bCardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA 02118, USA
cMass Spectrometry Resource Center, Boston University School of Medicine, Boston, MA 02118, USA

Received 17 August 2006; revised 11 December 2006; accepted 12 December 2006. Available online 16 December 2006.

Summary

p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO?) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO?. The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO? showed that ICAT labeling of Cys118 was decreased by 47%, whereas Cys80 was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys118 by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras. 3.

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