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. 2006 Jul 15;78(14):5134-42.
doi: 10.1021/ac060525v.

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

Affiliations

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

Cheng Zhao et al. Anal Chem. .

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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Figures

Figure 1
Figure 1
A. (left) ESI spectrum of purified unmodified p21ras treated with DTT. (right) ESI spectrum of purified p21ras treated with peroxynitrite. Inset shows m/z 1185 – 1230. B. ESI spectrum of purified unmodified p21ras that is not treated with DTT.
Figure 2
Figure 2
A. (left) ESI spectrum of peptide 170–185 including the Cys181 – Cys184 disulfide bond (2+). (right) ESI spectrum of this peptide (2+) after DTT treatment. B. CAD MS/MS spectrum of this peptide. Top shows observed cleavages with measured masses and predicted masses in parentheses.
Figure 3
Figure 3
Spectrum of p21ras treated with peroxynitrite and digested with trypsin. The major peaks are labeled with peptide positions. Those labeled with 002A indicate Met-Ox, those labeled with indicate both Met-Os and Cys-SO3H, and those labeled with indicate Tyr-NO2. Insets are the CAD MS/MS spectra of the unmodified peptides 6–16 and 17–42, which were used for internal m/z calibration.
Figure 4
Figure 4
A. Map of oxidative post-translational modifications detected on peroxynitrite-treated p21ras. B: Detected modifications on glutathiolated- p21ras. The most abundant glutathiolation on C118 confirmed by top-down analysis is labeled with *. C: Top down map of the major singly glutathiolated p21ras. The cleavages labeled with & include C118 glutathiolation.
Figure 5
Figure 5
A. m/z 745–775 from Figure 1B. T* = trypsin autodigestion B. CAD MS/MS spectrum from peptide 103–123 with Met111-Ox. C. CAD MS/MS spectrum from peptide 103–123 with both Met111-Ox and Cys118-SO3H modifications. D. ECD MS/MS spectrum from peptide 103–123 with both Met111-Ox and Cys118-SO3H modifications. Peptide sequences (right) show the observed cleavages.
Figure 6
Figure 6
Top down spectra of p21ras. A. ESI spectrum of purified glutathiolated p21ras. B. CAD MS/MS spectrum and C. ECD MS/MS spectrum on isolated +19 singly glutathiolated p21ras. The peak labeled with * indicates electronic noise.
Figure 7
Figure 7
A. CAD MS/MS spectrum of peptide 103–123 with Cys118-SSG modification. The inset is the ESI spectrum of the precursor ion. B. ECD MS/MS spectrum of this modified peptide. The peak labeled with * indicates electronic noise. The sequence at the top shows the observed cleavages.

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