Batthyány Carlos, Bartesaghi Silvina, Mastrogiovanni Mauricio, Lima Analía, Demicheli Verónica, Radi Rafael
1 Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo , Montevideo, Uruguay .
2 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay .
Antioxid Redox Signal. 2017 Mar 1;26(7):313-328. doi: 10.1089/ars.2016.6787. Epub 2016 Jul 22.
"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function.
Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation.
The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.
“硝基蛋白质组学”正处于积极发展阶段，因为蛋白质中的3 - 硝基酪氨酸构成了一氧化氮衍生氧化剂反应留下的印记，这些反应通常与氧化应激条件相关。此外，蛋白质酪氨酸硝化可导致结构和功能变化，这可能与人类疾病状态的病理生理相关性有关。生物蛋白质酪氨酸硝化是一个自由基过程，涉及酪氨酰自由基的中间体；尽管这是一个非酶促过程，但硝化作用选择性地针对有限的酪氨酸残基子集。蛋白质中3 - 硝基酪氨酸的精确鉴定和定量对研究人员来说是一项“艰巨任务”。
少数蛋白质是硝化的优先靶点（每个蛋白质组通常少于100种蛋白质），这与大量被其他翻译后修饰（如磷酸化、乙酰化，尤其是S - 亚硝基化）修饰的蛋白质形成对比。蛋白质组学方法已经揭示了体内和体外酪氨酸硝化的关键特征，包括选择性、位点特异性以及对蛋白质结构和功能的影响。
由于生物样品中这种氧化修饰的丰度较低，以及其在基于质谱（MS）的技术（即基质辅助激光解吸电离、电喷雾电离和碰撞诱导解离）中的不友好行为，鉴定含3 - 硝基酪氨酸的蛋白质并绘制硝化残基图谱具有挑战性。
将（i）经典的二维电泳与硝化蛋白质的免疫化学检测相结合，随后通过常规串联质谱（MS/MS）进行蛋白质鉴定，与（ii）酪氨酸硝化肽的免疫富集以及（iii）通过MIDAS™实验鉴定硝化肽相结合，正成为一种在体内明确绘制和定量酪氨酸硝化蛋白质的有效方法。《抗氧化与氧化还原信号》26, 313 - 328。
