Trace quantification of the oxidative damage products, meta- and ortho-tyrosine, in biological samples by gas chromatography-electron capture negative ionization mass spectrometry.
Oxygen radicals damage biomolecules and may contribute to cellular aging and degenerative disease. We describe a sensitive method for the quantification of two endogenous biomarkers of oxidative damage: meta-tyrosine (m-Tyr) and ortho-tyrosine (o-Tyr). The assay can be applied to direct analysis of free amino acids or protein-bound amino acids following hydrolysis. The assay involves derivatization with pentafluorobenzyl bromide and extraction into n-decane, followed by gas chromatography-mass spectrometry. Stable isotope labeled m- and o-Tyr (2H4) and phenylalanine [i.e., Phe (2H5)] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg of m- and o-Tyr in 100 micrograms protein is possible and the data are expressed as a molar ratio of m- and o-Tyr to native Phe. The assay was used to determine the levels of m- and o-Tyr in freshly isolated human plasma protein (4.05 +/- 0.67 m-Tyr per 10(4) Phe, 0.35 +/- 0.07 o-Tyr per 10(4) Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels of m-Tyr (56.4 +/- 1.1 m-Tyr per 10(4) Phe, P < 0.0001) and o-Tyr (48.9 +/- 1.3 o-Tyr per 10(4) Phe, P < 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of either m- or o-Tyr.
Blount
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Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney, Australia.
Duncan
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D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D010100:Oxygen