Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.
Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.
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DOI：
10.1042/bsr20120032
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发表时间：
2012-12
影响因子：
4
通讯作者：
Yamakura F
中科院分区：
文献类型：
--
作者：
Uda M;Kawasaki H;Shigenaga A;Baba T;Yamakura F
色氨酸的硝化是一种新型的翻译后修饰。在本研究中，我们研究了是否NO2 Trp（硝基色氨酸）-含有蛋白质产生在海马和小脑的成年大鼠在体内生理条件下。用抗6-NO2 Trp特异性抗体进行Western blot分析，我们发现两个区域的蛋白提取物中有许多相似的免疫反应斑点。随后对这些斑点进行胰蛋白酶消化和LC-ESI-MS/MS（LC-电喷雾电离-串联MS）分析。我们确定了几种细胞骨架蛋白和糖酵解酶作为NO2 Trp的含蛋白质，并确定了硝化色氨酸残基的位置与显着的离子得分水平（P<0.05）在两个区域的几个蛋白质。小脑中含NO_2Trp蛋白的总量显著高于海马（P<0.05）。此外，IP（免疫沉淀）测定，使用抗-醛缩酶C抗体表明，NO2 Trp在醛缩酶C的免疫染色的相对强度在小脑比在海马高得多。小脑中神经元型一氧化氮合酶（nNOS）和内皮型一氧化氮合酶（eNOS）的含量明显高于海马。这是首次证明在体内生理条件下蛋白质中存在几个硝化色氨酸的特异位点。
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.