Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.
Biosci Rep. 2012 Dec;32(6):521-30. doi: 10.1042/BSR20120032.
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.
色氨酸残基的硝化是一种新的翻译后修饰。在本研究中，我们检查了在生理条件下，成年大鼠海马和小脑中是否存在含有 NO2Trp（硝基色氨酸）的蛋白质。使用抗 6-NO2Trp 特异性抗体的 Western blot 分析，我们在两个区域的蛋白提取物中发现了许多相似的免疫反应性斑点。这些斑点随后用胰蛋白酶消化，并进行 LC-ESI-MS/MS（LC-电喷雾电离-串联 MS）分析。我们鉴定了几种细胞骨架蛋白和糖酵解酶作为含有 NO2Trp 的蛋白质，并确定了在两个区域的几种蛋白质中具有显著离子得分水平（P<0.05）的硝化色氨酸残基的位置。我们还观察到小脑中含有 NO2Trp 的蛋白质总量明显大于海马（P<0.05）。此外，使用抗醛缩酶 C 抗体的 IP（免疫沉淀）测定表明，小脑中 NO2Trp 相对于醛缩酶 C 的免疫染色相对强度明显高于海马。小脑中 nNOS（神经元型一氧化氮合酶）和 eNOS（内皮型一氧化氮合酶）的含量明显大于海马。这是体内生理条件下蛋白质中几个特定硝化色氨酸位点的首次证据。
