Simultaneous determination of 3-nitrotyrosine and tyrosine in plasma proteins of rats and assessment of artifactual tyrosine nitration

Thierry Delatour, Janique Richoz, Paul Vouros, Robert J. Turesky

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26 Scopus citations

Abstract

A sensitive and specific isotope dilution liquid chromatography-electrospray tandem mass spectrometry method was developed for the determination of the 3-nitrotyrosine residue levels in rat plasma proteins. The assay is based on the cleavage of proteins with concentrated hydrochloric acid to release both 3-nitrotyrosine and tyrosine. To control the potential artifactual nitration of tyrosine residues during the proteolysis, samples are spiked with 13C9-labeled tyrosine and the level of 13C9-labeled 3-nitrotyrosine is measured. The clean-up process entails hydrolysate fortification with 2,5,6-d3-3-nitrotyrosine, followed by solid-phase extraction on octadecylsilyl (to isolate tyrosine) and aminopropylsilyl (to isolate 3-nitrotyrosine) cartridges. Tyrosine and 3-nitrotyrosine fractions are mixed in an appropriate ratio prior to the analysis. The method was applied to animals exposed to ferric nitrilotriacetate to induce oxidative stress.

Original languageEnglish (US)
Pages (from-to)189-199
Number of pages11
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume779
Issue number2
DOIs
StatePublished - Nov 5 2002

Keywords

  • Nitration
  • Nitrotyrosine
  • Proteins
  • Tyrosine

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