Detection of 3-nitrotyrosine in human platelets exposed to peroxynitrite by a new gas chromatography/mass spectrometry assay.
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 at m/z 595 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 microM. Decomposed peroxynitrite was without the effect. Nitrogen dioxide (43 microM) was also a potent tyrosine nitrating molecule, increasing the levels of 3-nitrotyrosine 153-fold. HOCl (50 microM) in the presence of nitrite (50 microM) 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.
Jiang
H
H
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
Balazy
M
M
D001792:Blood Platelets / Q000378:metabolism*; D005464:Fluorobenzenes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D009566:Nitrates; D009585:Nitrogen Dioxide / Q000737:chemistry; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D005466:Fluorocarbons
D005464:Fluorobenzenes; D009566:Nitrates; D009585:Nitrogen Dioxide; D014443:Tyrosine; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons; C039642:peroxynitric acid; C002744:3-nitrotyrosine