Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives
Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives
复制标题
DOI：
10.1016/j.freeradbiomed.2008.12.025
复制
发表时间：
2009-05-01
影响因子：
7.4
通讯作者：
Freeman, B. A.
中科院分区：
文献类型：
--
作者：
Schopfer, F. J.;Batthyany, C.;Freeman, B. A.
硝基烯烃脂肪酸衍生物表现出强烈的亲电性质，是临床上可检测的，并诱导多种转录调节的抗炎反应。目前，内源性亲电脂质的表征和定量受到其与蛋白质和小分子亲核靶标的Michael加成的影响。本文报道了硝基脂肪酸与β-巯基乙醇（BME）的反式硝基烷基化反应，并将该反应应用于亲核靶点反应的无偏鉴定和定量。反式硝基烷基化产率在pH 7至8时最大，并且在目标亲核试剂的生理浓度下发生。该反应也适用于蛋白质电泳分离后亲电脂肪酸-蛋白质加合物的基于灵敏质谱的定量。凝胶内反式硝基烷基化反应还允许在没有当前蛋白质组学方法的偏差和缺乏灵敏度的情况下鉴定蛋白质靶。使用这种方法，观察到脂肪酸硝基烯烃在体内通过硝基烯烃还原酶活性和线粒体β-氧化快速代谢，产生各种亲电和非亲电产物，其结构特征在于基于BME的反式硝基烷基化反应。该策略被应用于检测和定量的线粒体中的脂肪酸硝化反应的心肌缺血-再氧合诱导的氧化炎症条件。(C)2008年爱思唯尔公司All rights reserved.
Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation. (C) 2008 Elsevier Inc. All rights reserved.