Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin γ-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry
Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin γ-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry
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DOI：
10.1038/nprot.2007.298
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Roberts, L. Jackson, II
中科院分区：
文献类型：
--
作者：
Davies, Sean S.;Amarnath, Venkataraman;Roberts, L. Jackson, II
F-2-异前列腺素(F-2-Isoprostanes，F-然而，F-2-Isop的快速清除使得样本收集的时机对于短暂的氧化侮辱至关重要。异酮醛(IsoKs)是通过脂质过氧化的IsoP途径形成的伽马酮醛，它能与蛋白质的赖氨基残基快速反应，形成稳定的蛋白质加合物。氧化应激也可以激活环氧合酶产生前列腺素H-2，它可以形成两种特定的异构体异构体-左旋糖苷(LG)D-2和E-2。由于加合物蛋白不能被迅速清除，IsoK/LG蛋白加合物水平可以作为氧化和炎症损伤的剂量计，在长时间内和短暂的损伤发作。IsoK/LG蛋白加合物的定量始于液相萃取，将蛋白质从脂膜中分离出来，如果需要，可以同时测量同一样品中的IsoK/LG蛋白加合物和F-2-IsoP。提取的蛋白质经蛋白水解液消化、固相萃取、制备后，用液相色谱-串联质谱法测定IsoK/lg-赖氨酰-内酰胺加合物。
Measurement of F-2-isoprostanes (F-2-IsoPs) has been independently verified as one of the most reliable approaches to assess oxidative stress in vivo. However, the rapid clearance of F-2-IsoPs makes the timing of sample collection critical for short-lived oxidative insults. Isoketals (IsoKs) are gamma-ketoaldehydes formed via the IsoP pathway of lipid peroxidation that rapidly react with lysyl residues of proteins to form stable protein adducts. Oxidative stress can also activate cyclooxygenases to produce prostaglandin H-2, which can form two specific isomers of IsoK-levuglandin (LG) D-2 and E-2. Because adducted proteins are not rapidly cleared, IsoK/LG protein adduct levels can serve as a dosimeter of oxidative and inflammatory damage over prolonged periods of time as well as brief episodes of injury. Quantification of IsoK/LG protein adducts begins with liquid-phase extraction to separate proteins from lipid membranes, allowing measurement of both IsoK/LG protein adducts and F-2-IsoP from the same sample if desired. IsoK/LG-lysyl-lactam adducts are measured by liquid chromatography tandem mass spectrometry after proteolytic digestion of extracted proteins, solid-phase extraction and preparative HPLC.