A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress
A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress
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DOI：
10.1016/j.freeradbiomed.2005.08.046
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发表时间：
2006-02-01
影响因子：
7.4
通讯作者：
Darley-Usmar, VM
中科院分区：
文献类型：
--
作者：
Landar, A;Oh, JY;Darley-Usmar, VM
蛋白质组学与高度特异性和灵敏的亲和技术的结合对于识别活性氧和氮物种 (ROS/RNS) 的翻译后修饰非常重要。这种方法最紧迫的问题之一是准确确定复杂蛋白质样品中特定氨基酸（例如半胱氨酸残基）的修饰程度。许多与自由基生物学相关的技术使用生物素标记作为高灵敏度和特异性跟踪蛋白质修饰的方法。为了实现这种方法提供定量数据的潜力，我们通过赖氨酸残基的修饰制备了一系列生物素化蛋白质。然后将这些蛋白质用作电泳分离用生物素缀合的碘乙酰胺标记的蛋白质样品的定量标准品。通过测量硫醇暴露于硫醇氧化剂时的修饰以及与生物素标记的亲电脂质形成的蛋白质加合物的量来评估该方法的实用性。此外，使用天然和生物素标记的细胞色素 c 的组合，该方法用于定量二维凝胶上给定点中硫醇的量相对于蛋白质的量。因此，我们开发了一种多功能、经济高效的标准品，可用于蛋白质组学方法来定量响应氧化应激的生物素标签。 (c) 2005 Elsevier Inc. 保留所有权利。
The combination of proteomics with highly specific and sensitive affinity techniques is important for the identification of posttranslational modifications by reactive oxygen and nitrogen species (ROS/RNS). One of the most pressing problems with this approach is to determine accurately the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample. A number of techniques relevant to free radical biology use biotin tagging as a method to follow protein modification with high sensitivity and specificity. To realize the potential of this approach to provide quantitative data, we have prepared a series of biotinylated proteins through the modification of lysine residues. These proteins were then used as quantitative standards in electrophoretic separation of protein samples labeled with biotin-conjugated iodoacetamide. The utility of the approach was assessed by measuring modification of thiols in response to exposure to thiol oxidants, as well as the amount of protein adduct formation with a biotin-tagged electrophilic lipid. Furthermore, using a combination of native and biotin-tagged cytochrome c, this method was used to quantitate the amount of thiol relative to the amount of protein in a given spot on a two-dimensional gel. Thus, we have developed a versatile, cost-effective standard that can be used in proteomic methods to quantitate biotin tags in response to oxidative stress. (c) 2005 Elsevier Inc. All rights reserved.