Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry.
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 Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) 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.
Sethuraman
Mahadevan
M
Vascular Biology Unit, Boston University School of Medicine, Boston, MA 02118, USA.
Clavreul
Nicolas
N
Huang
Hua
H
McComb
Mark E
ME
Costello
Catherine E
CE
Cohen
Richard A
RA
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D005609:Free Radicals; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D005609:Free Radicals; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; D016283:Proto-Oncogene Proteins p21(ras); D005978:Glutathione; D003545:Cysteine; D010100:Oxygen