Quantitation of Protein-Bound 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine by High-Performance Liquid Chromatography with Electrochemical Array Detection

Abstract

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in myriad disease etiologies and may represent an obligate pathologic sequelus of inflammation. Unfortunately, few sensitive and specific analytical techniques exist for the routine assay of biomarkers indicative of ROS and RNS elaboration. In this study, high-performance liquid chromatography is used in conjunction with coulometric electrochemical array (HPLC-EC) detection to allow ultrasensitive determination of protein-bound 3-nitrotyrosine and 3, 4-dihydroxyphenylalanine (3-hydroxytyrosine) as specific in situ biomarkers of protein exposure to reactive nitrating and oxidizing species. Tyrosine and derivatives can be analyzed simultaneously with practical detection limits for tyrosine, 3-NT, and 3,4-Dopa being 10, 50, and 2 pmol, respectively, in as little as 20 microL of sample. HPLC-EC array detection allows two-dimensional resolution of chromatograms, greatly facilitating peak detection and confidence assignment. A method of sample preparation wherein tyrosine analogs are enzymatically hydrolyzed from protein without the need for sample extraction, concentration, or derivatization is reported.