期刊
ANALYTICAL CHEMISTRY
卷 78, 期 14, 页码 5134-5142出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac060525v
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资金
- NCRR NIH HHS [P41 RR010888, P41-RR10888] Funding Source: Medline
- NHLBI NIH HHS [T32 HL07224, P01 HL81587, T32 HL007224, P01 HL081587, N01HV28178, N01-HV28178] Funding Source: Medline
- NIA NIH HHS [R01 AG027080, R01 AG27080] Funding Source: Medline
- NIGMS NIH HHS [R01 GM078293-02] Funding Source: Medline
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, Cys(118)-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 topdown analysis.
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