Dataset Information

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.

SUBMITTER: Zhao C

PROVIDER: S-EPMC3098383 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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

Zhao Cheng C Sethuraman Mahadevan M Clavreul Nicolas N Kaur Parminder P Cohen Richard A RA O'Connor Peter B PB

Analytical chemistry 20060701 14

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 m ...[more]

PMID: 16841939

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Project description:Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.

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Dataset Information

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

Publications

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

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