Isolation of nitrotyrosine-containing peptides by using an insoluble-antibody column

Antibodies to nitrotyrosine were prepared in goats or rabbits by injecting a nitrotyrosine-protein conjugate. The antibodies were purified by using a protein that had been nitrated with tetranitromethane. These antibodies were used to isolate nitrotyrosine-containing peptides from nitrated lysozyme. The nitrotyrosine-containing peptides were thus purified (55% yield) in one step and the positions of nitration in lysozyme were found to be at tyrosine-20 and tyrosine-23. This method is of general applicability for the determination of the position of nitrotyrosine in proteins.