Quantification of oxidative post-translational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags (ICAT) and mass spectrometry

Mahadevan Sethuraman

Nicolas Clavreul

Hua Huang

Mark McComb

Catherine Costello

Richard Cohen

^{Vascular Biology Unit, Boston University School of Medicine, Boston, MA 02118}

^{Cardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA 02118}

^{Mass Spectrometry Resource Center, Boston University School of Medicine, Boston, MA 02118}

Address for correspondence: Richard A. Cohen, MD, Vascular Biology Unit, X720, Boston University School of Medicine, 650 Albany St., Boston, MA 02118, Tel/fax: 617-638-7115/7113, Email: ude.ub@nehocar

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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 (ROS/RNS). Post-translational 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 post-translational thiol modifications of p21ras caused by peroxynitrite (ONOO^{) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased following 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}^{was decreased by 47%, whereas Cys}^{was not significantly affected and was thereby shown to be less reactive. The extent of}S-glutathiolation of Cys^{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 (ICAT), oxidant stress, post-translational modification, p21ras, thiol, S-glutathiolation, mass spectrometry

Abstract

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
TOF: time-of-flight
qTOF: quadrupole TOF
TIC: total ion chromatogram

Abbreviations

Footnotes

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Footnotes

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