Abstract
Nitric oxide-derived reactive species have been implicated in many disorders. Protein nitrotyrosine is often used as a stable marker of these reactive species. Using immunohistochemistry, we have previously detected nitrotyrosine in murine Mutatect tumors, where neutrophils are the principal source of nitric oxide. We now report on the identification of several prominent nitrotyrosine-containing proteins. Using Western blot analysis, nitrotyrosine in higher molecular mass proteins (>20 kDa) was detected in tumors containing a high number of neutrophils but not in tumors with fewer neutrophils. Staining for nitrotyrosine was consistently seen in low molecular mass proteins (< or =15 kDa), regardless of the level of neutrophils. Protein nitrotyrosine was not seen in Mutatect cells growing in vitro. Treatment with nitric oxide donors produced nitration of < or =15-kDa proteins, but only after extended periods. These small proteins, both from tumors and cultured cells, were identified by mass spectrometry to be histones. Only a subset of tyrosine residues was nitrated. Selective nitration may reflect differential accessibility of different tyrosine residues and the influence of neighboring residues within the nucleosome. The prominence of histone nitration may reflect its relative stability, making this post-translational modification a potentially useful marker of extended exposure of cells or tissues to nitric oxide-derived reactive species.
Highlights
Nitric oxide (NO1⁄7)1-derived reactive nitrogen oxide species (RNOS) are cytotoxic and mutagenic, and have been implicated in the pathogenesis of several inflammatory disorders [1,2,3,4,5,6,7,8,9]
We report on the identification of several prominent nitrotyrosine-containing proteins
We have recently shown by immunohistochemistry that tumor-infiltrating neutrophils express inducible nitric-oxide synthase and that protein NTyr is present in these experimental tumors [43]
Summary
NO1⁄7, nitric oxide; BSA, bovine serum albumin; CapLC-MS/MS, capillary liquid chromatography-tandem mass spectrometry; hprt, hypoxanthine phosphoribosyltransferase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-offlight mass spectrometry; nESI-MS/MS, nanoelectrospray ionizationtandem mass spectrometry; NTyr, nitrotyrosine; RNOS, reactive nitrogen oxide species; SNP, sodium nitroprusside; MOPS, 4-morpholinepropanesulfonic acid. Mass spectrometry of tryptic peptides is a sensitive and specific technique to identify proteins It has been shown capable of identifying tryptic peptides containing NTyr and specifying which Tyr has been nitrated [14, 40]. One recent report describes a technique of localizing nitrated proteins by Western blotting on a two-dimensional gel, followed by mass spectrometry, to identify putative nitrated proteins in tissue samples [41]. We detect NTyr-containing proteins in Mutatect tumors using West-. This paper is available on line at http://www.jbc.org ern blot analysis, and use mass spectrometric analysis to unambiguously identify some prominent nitrated proteins, including localization of specific Tyr residues that have been nitrated
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