Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry
- PMID: 16841939
- PMCID: PMC3098383
- DOI: 10.1021/ac060525v
Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry
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.
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