A "fluorescence switch" technique increases the sensitivity of proteomic detection and identification of S-nitrosylated proteins
A "fluorescence switch" technique increases the sensitivity of proteomic detection and identification of S-nitrosylated proteins
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DOI：
10.1002/pmic.200900070
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发表时间：
2009-12-01
期刊：
影响因子：
3.4
通讯作者：
Martinez-Ruiz, Antonio
中科院分区：
文献类型：
--
作者：
Tello, Daniel;Tarin, Carlos;Martinez-Ruiz, Antonio
蛋白质s -亚硝基化是蛋白质半胱氨酸的可逆翻译后修饰，越来越被认为是一种信号转导机制。“生物素开关”技术标志着s -亚硝基蛋白质组研究的开始，该技术基于更稳定的生物素化特异性取代不稳定的s -亚硝基化，从而允许进一步的检测和纯化。然而，由于其相对较低的灵敏度，限制了其在蛋白质组学研究中的应用。因此，典型的蛋白质组学实验需要大量的蛋白质提取物，这就排除了在许多生物学环境中使用这种方法。我们开发了一种“荧光开关”技术，当与2-DE蛋白质组学方法相结合时，可以通过使用有限数量的起始材料来检测和鉴定s -亚硝基化蛋白质，从而显着提高灵敏度。我们已经应用这种方法来检测暴露于s -亚硝基- l -半胱氨酸（一种生理s -亚硝基硫醇）时内皮细胞中变成s -亚硝基化的蛋白质，识别已知的s -亚硝基化靶点，以及新靶点的蛋白质。这种“荧光开关”方法还使我们能够在细胞因子激活的RAW264.7（小鼠巨噬细胞）细胞中鉴定出几种被硫氧还蛋白脱氮的蛋白质。我们认为这种方法代表了一种进步，以便在生理条件下接近s -亚硝基化蛋白的鉴定。
Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting Material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.