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Anal Chem. 2006 Jul 15;78(14):5134-42.

Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry.

Author information

1
Mass Spectrometry Resource, Department of Biochemistry, Cardiovascular Proteomics Center, and Vascular Biology Unit, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Abstract

P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high-resolution and high mass accuracy Fourier transform mass spectrometry was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by "bottom-up" analysis, and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, "top-down" analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications, especially using top-down analysis.

PMID:
16841939
PMCID:
PMC3098383
DOI:
10.1021/ac060525v
[Indexed for MEDLINE]
Free PMC Article

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