Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum. Issue 6 (14th September 2012)

• Record Type: Journal Article
• Authors: Uda, Munehiro; Kawasaki, Hiroaki; Shigenaga, Ayako; Baba, Takeshi; Yamakura, Fumiyuki
• Is Part Of: Bioscience reports. Volume 32:Issue 6(2012)
• Page Start: 521
• Page End: 530
• Publication Date: 2012-09-14
• Subjects: cerebellum -- hippocampus -- 6-nitrotryptophan -- proteome -- tandem MS analysis -- tryptophan residue; Molecular biology -- Periodicals; Cytology -- Periodicals; 572.8
• DOI: 10.1042/BSR20120032
• Languages: English
• ISSNs: 0144-8463

Abstract

Abstract : Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2 Trp (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-NO2 Trp-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 NO2 Trp-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 NO2 Trp-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 NO2 Trp 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 .