doi:10.1016/j.freeradbiomed.2006.12.012 
Copyright © 2006 Elsevier Inc. All rights reserved.
Original Contribution
Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry
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.
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Abstract
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.
Keywords: Isotope-coded affinity tag; Oxidant stress; Posttranslational modification; p21ras; Thiol; S-Glutathiolation; Mass spectrometry; Free radicals
Abbreviations: ONOO−, peroxynitrite; GSSG, glutathione disulfide; IAM, iodoacetamide; ICAT, isotope-coded affinity tag; ACN, acetonitrile; TFA, trifluoroacetic acid; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl) phosphine; LC, liquid chromatography; HPLC, high-performance LC; capLC, capillary LC; MS, mass spectrometry; MS/MS, tandem MS; ESI, electrospray ionization
Fig. 1. Amino acid sequence of human p21ras. Six cysteines, Cys51, Cys80, Cys118, Cys181, Cys184, and Cys186 of p21ras are highlighted in bold font and underlined.
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Fig. 2. ICAT approach to quantitatively evaluate reversible and irreversible oxidative posttranslational thiol modification. Protein samples with free cysteine thiols ■ (reactive) and less reactive cysteine thiols □ are exposed to peroxynitrite or GSSG and normal conditions or control buffer before being labeled with ICAT reagent. Some of the reactive cysteine thiols are oxidized depending upon the level of the oxidant, and oxidized thiols are designated as ▲. After oxidation, labeling of the free thiols is performed, with light 
and heavy 
ICAT reagent. ICAT-labeled samples are mixed and digested with trypsin, followed by purification through HPLC cation exchange and avidin-affinity cartridges as outlined under Experimental methods. Affinity-captured peptides are analyzed by LC-MS and MS/MS. As shown for the reactive cysteines, the oxidized cysteines are not susceptible to labeling by ICAT reagent, and hence there is a decreased intensity in the signal from the heavy-labeled peptide in MS of the reactive cysteine-containing peptide. For less reactive cysteines, the peptides in samples prepared under normal and oxidant stress conditions exhibit equivalent signal intensity in MS. From the relative peak intensities of the MS of light and heavy ICAT-labeled peptides, the ratio of oxidized thiols in the samples can be estimated. The identity of the peptide sequences can be derived from MS/MS analysis of these peptides. In order to evaluate whether the oxidation caused by peroxynitrite or GSSG is reversible or irreversible, the oxidized protein samples are treated with TCEP (for disulfide reduction of S-glutathiolation modification) or ascorbate (for reducing S-nitrosation) before being labeled with heavy ICAT. From the LC-MS analysis, reversibility of the modifications caused by peroxynitrite or GSSG can be quantitated.
Fig. 3. S-Glutathiolation, but not ONOO−, promotes p21ras guanine nucleotide exchange activity. The guanine nucleotide exchange activity of recombinant p21ras was measured by monitoring the fluorescence of p21ras-bound Mant-GDP complexes. The protein was either untreated (control) or treated with peroxynitrite (ONOO−, 10 or 100 μM) or GSSG (10 or 100 μM). The bar graph shows the percentage of the guanine nucleotide exchange activity of p21ras (i.e., the unbound Mant-GDP expressed as a percentage of the total bound) in control and peroxynitrite- and GSSG-treated samples 5 min after treatment. *p < 0.05; n = 3.
Fig. 4. LC-MS/MS of ICAT-labeled tryptic peptides of p21ras. (a) Total ion chromatogram for LC-ESI-MS/MS of ICAT-labeled tryptic peptides of p21ras. (b) The m/z 400–1800 region of the mass spectrum of the peptides eluting between 46.9 and 47.9 min. (c) Expanded view of the mass spectrum around m/z 920 showing the pair of light (12C) and heavy (13C) ICAT-labeled p21ras peptides, Thr74–Lys88.
Fig. 5. Effect of ONOO− on ICAT labeling of cysteines of p21ras. (a) MS of the ICAT-labeled peptide pair (Thr74–Lys88) of p21ras containing less reactive Cys80, (b) MS of the ICAT-labeled peptide pair Val103–Arg123 of p21ras containing reactive Cys118, and (c) MS of the ICAT-labeled peptide pair Lys170–Lys185 of p21ras containing the terminal Cys181 and Cys184. (d) Summary of ICAT labeling experiments quantitating individual cysteine modifications of p21ras treated with peroxynitrite.
Fig. 6. Effect of GSSG on ICAT labeling of cysteines of p21ras. (a) MS of ICAT-labeled peptide pair (Val103–Arg123) containing reactive Cys118 in which the methionine is in oxidized form, (b) MS of the ICAT-labeled peptide pair (Thr74–Lys88) of p21ras containing less reactive Cys80, and (c) MS of the ICAT-labeled peptide pair (Lys170–Lys185) of p21ras containing terminal cysteines Cys181 and Cys184 in which both cysteines were ICAT labeled and the methionine is in oxidized form. (d) Summary of ICAT labeling of p21ras to quantitate cysteine modifications caused by GSSG.
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