Detection of 3-Nitrotyrosine in Human Platelets Exposed to Peroxynitrite by a New Gas Chromatography/Mass Spectrometry Assay

Abstract

A new sensitive and specific assay was developed and applied for the quantitative determination of 3-nitrotyrosine in proteins of human platelets. 3-Nitrotyrosine was quantitatively converted into a new pentafluorobenzyl derivative in a single step and detected as an abundant carboxylate anion atm/z595 using negative ion chemical ionization gas chromatography/mass spectrometry. The internal standard, [13C6]-3-nitrotyrosine, was prepared via a new and efficient method using nitronium borofluorate dissolved in hydrochloric acid. The assay showed excellent linearity and sensitivity. Intact human platelets contained 1.4 ± 0.6 ng of 3-nitrotyrosine per milligram of protein. Peroxynitrite increased 3-nitrotyrosine levels 4- to 535-fold at the concentration range of 10 to 300 μM. Decomposed peroxynitrite was without the effect. Nitrogen dioxide (43 μM) was also a potent tyrosine nitrating molecule, increasing the levels of 3-nitrotyrosine 153-fold. HOCl (50 μM) in the presence of nitrite (50 μM) increased the 3-nitrotyrosine levels 3-fold. Exposure of platelets to nitric oxide, nitrite, thrombin, adenosine diphosphate, platelet activating factor, and arachidonic acid had no effect on platelet 3-nitrotyrosine levels.