Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry.
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.
Zhao
Cheng
C
Mass Spectrometry Resource, Department of Biochemistry, Cardiovascular Proteomics Center, and Vascular Biology Unit, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Sethuraman
Mahadevan
M
Clavreul
Nicolas
N
Kaur
Parminder
P
Cohen
Richard A
RA
O'Connor
Peter B
PB
D000595:Amino Acid Sequence; D005583:Fourier Analysis; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid; D011499:Protein Processing, Post-Translational; D018631:ras Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D030421:Peroxynitrous Acid; D018631:ras Proteins; D005978:Glutathione