Mapping sites of tyrosine nitration by matrix-assisted laser desorption/ionization mass spectrometry.

Protein tyrosine nitration is an important part of nitric oxide biology. This posttranslational modification occurs under normal physiological conditions and is substantially enhanced under various pathological conditions. Studies reveal that protein tyrosine nitration is a dynamic and selective process that influences protein function and turnover and can be considered a diagnostic biomarker of pathology. The identification of nitrated tyrosine residues directly within any given nitrated protein is important for studies on in vivo mechanisms of nitration and for the explanation of functional consequences of nitration. Specific nitrated tyrosines in given proteins may be also more informative as oxidative biomarkers than overall nitrotyrosine levels. However, localization of the sites of nitration remains a methodological challenge. Mass spectrometry (MS) is an ideal method for identifying nitrated tyrosines in proteins because of its sensitivity and specificity. This chapter is not intended to thoroughly discuss the various MS-based approaches for nitrotyrosine identification and merely focuses on the analysis of peptides containing nitrotyrosine by matrix-assisted laser desorption ionization MS (MALDI-MS). The data summarized show that the MALDI-MS pattern of a tyrosine-nitrated peptide includes the unique combination of ions that provides unequivocal evidence for the presence of nitrotyrosine in a given peptide and could be used for mapping sites of tyrosine nitration in proteins.