Nuriel Tal, Deeb Ruba S, Hajjar David P, Gross Steven S
Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA.
Methods Enzymol. 2008;441:1-17. doi: 10.1016/S0076-6879(08)01201-9.
Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues.
一氧化氮（NO）衍生的物质对酪氨酸残基进行硝化作用，会导致蛋白质中3-硝基酪氨酸的积累，这是细胞和组织中亚硝化应激的一个标志。酪氨酸硝化被认为是NO衍生的物质在健康和疾病状态下用于调节蛋白质结构和功能的多种信号传导方式之一。已经描述了多种用于定量蛋白质3-硝基酪氨酸残基的方法，并且为了在全蛋白质组范围内鉴定蛋白质酪氨酸修饰位点这一目标，也提出了几种策略。本章详细介绍了一种使用高压液相色谱-电化学检测法定量细胞和组织中3-硝基酪氨酸的实用方案。此外，本章还描述了一种用于特异性富集含3-硝基酪氨酸肽段的新型生物素标记策略。预计将该策略与基于高通量MS/MS的肽段测序相结合，有助于编制细胞和组织中亚硝化应激诱导的蛋白质酪氨酸修饰位点的综合清单。
