Abstract: | Nitration of tryptophan residues is a novel post-translational modification. In the present
study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in
the hippocampus and cerebellum of the adult rat under physiological conditions in
vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found
many similar immunoreactive spots in the protein extracts from both regions. These spots were
subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS)
analysis. We identified several cytoskeletal proteins and glycolytic enzymes as
NO2Trp-containing proteins and determined the position of nitrated tryptophan residues
with significant ion score levels (P<0.05) in several proteins in both
regions. We also observed that the total amount of NO2Trp-containing proteins in the
cerebellum was significantly greater than that in the hippocampus (P<0.05).
Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative
intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than
in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric
oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of
several specific sites of nitrated tryptophan in proteins under physiological conditions in
vivo. |