| The presence of 3-nitrotyrosine (nTyr) in proteins and peptides serves as a marker for the production of reactive nitrogen species. Nitrotyrosine occurs in cells in vivo at normal, basal levels, and in increasing amounts during states involving oxidant stress, such as inflammation. Tyrosine nitration can change the activity of many enzymes and is believed to occur in a site-specific manner. Tandem mass spectrometry provides the definitive method for identifying the presence and site of nitration, but the amounts of nTyr in vivo are often below detectable levels. To address this need we developed methods to specifically concentrate proteins and peptides containing nTyr to facilitate the identification of the site of modification.; The first series of methods focused on specific, solution-phase labeling of dithionite-reduced nitrotyrosine with an amine-reactive biotin label (sulfo-NHS-S-S-biotin). After protein digestion, biotinylated peptides were affinity-captured with streptavidin-bound Sepharose and eluted by reducing the disulfide linker on the biotin tag. Although nitrated peptides from tetranitromethane-treated bovine serum albumin could be isolated with several variants of the method, all required very large amounts of sample to yield suitable data. A solid-phase capture approach was developed that used similar chemistry and also took advantage of the lower pKa of aminotyrosine to increase selectivity of binding. This yielded a 10-fold increase in sensitivity. The best implementation of the method, however still suffered from inadequate sensitivity and reproducibility. Of the two methods presented, however, it holds greater potential. Given the amount of non-specific reactivity of amines with the matrices that were used, it may he necessary to develop a more specific capture chemistry. The substrates also may have difficulty accessing reactive sites on the resin. Further optimization of binding conditions might yield a more robust and reproducible capture reaction. Given the methods developed, and with potential improvements to the reaction conditions, solid-phase capture appears to be a promising method to isolate nTyr-containing peptides and proteins. |
