Abstract
SIGNIFICANCE: The conversion of protein-bound Tyr residues to 3-nitrotyrosine (3NY) can occur during nitrative stress and has been correlated to aging and many disease states. Proteomic analysis of this post-translational modification, using mass spectrometry-based techniques, is crucial for understanding its potential role in pathological and physiological processes. RECENT ADVANCES: To overcome some of the disadvantages inherent to well-established nitroproteomic methods using anti-3NY antibodies and gel-based separations, methods involving multidimensional chromatography, precursor ion scanning, and/or chemical derivatization have emerged for both identification and quantitation of protein nitration sites. A few of these methods have successfully detected endogenous 3NY modifications from biological samples. CRITICAL ISSUES: While model systems often show promising results, identification of endogenous 3NY modifications remains largely elusive. The frequently low abundance of nitrated proteins in vivo, even under inflammatory conditions, is especially challenging, and sample loss due to derivatization and cleaning may become significant. FUTURE DIRECTIONS: Continued efforts to avoid interference from non-nitrated peptides without sacrificing recovery of nitrated peptides are needed. Quantitative methods are emerging and are crucial for identifying endogenous modifications that may have significant biological impacts.