Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry
Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry
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DOI：
10.1016/s0003-2697(03)00359-2
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发表时间：
2003-09-15
影响因子：
2.9
通讯作者：
Tannenbaum, SR
中科院分区：
文献类型：
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作者：
Nikov, G;Bhat, V;Tannenbaum, SR
酪氨酸硝化是一种公认的蛋白质修饰，发生在与氧化应激和一氧化氮合酶活性增加相关的疾病状态中。据报道，特定酪氨酸残基的硝化作用会影响蛋白质的结构和功能，这表明3-硝基酪氨酸的形成可能不仅是一种疾病标志物，还可能参与一些疾病的发病机制和正常的调节过程。然而，确定硝化作用的地点一直很困难。我们描述了一种结合特异性分离硝化蛋白质与质谱测定氨基酸序列和单个蛋白质硝化位点的方法。复杂的蛋白质混合物，如血清或细胞裂解液，通过抗酪氨酸抗体免疫沉淀富集含硝基酪氨酸的蛋白质。然后用强还原剂在凝胶和溶液中平行地将硝基酪氨酸还原为氨基酪氨酸。以硝化的人血清白蛋白为模型，我们用二硫苏糖醇还原了二硫键，并用碘乙酰胺烷基化了游离巯基。然后用二亚硝酸钠将硝基酪氨酸还原为氨基酪氨酸，并在pH 5.0时选择性地将可切割的生物素标签附着在氨基酪氨酸上，然后用胰蛋白酶消化白蛋白。用链亲和素亲和柱纯化生物素化胰蛋白酶肽，并用质谱法进行鉴定。我们还从SJL小鼠血浆富集样品中纯化了硝化的人血清白蛋白，并通过肽质量指纹图谱和MASCOT验证了其身份。(C) 2003 Elsevier Science（美国）版权所有。
Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT. (C) 2003 Elsevier Science (USA). All rights reserved.