Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide.
Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide.
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DOI：
10.1016/j.redox.2015.04.006
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发表时间：
2015-08
期刊：
影响因子：
11.4
通讯作者：
Fedorova, Maria
中科院分区：
文献类型：
--
作者：
Vemula, Venukumar;Ni, Zhixu;Fedorova, Maria
关键词：
羰基化是一个通用术语，是指由于活性氧物质诱导的氧化反应而存在于生物分子中的活性羰基。羰基化蛋白质、脂质和核酸已被深入研究，并且通常与氧化应激相关病症的发作或进展相关。为了揭示潜在的羰基化途径和生物学相关性，研究它们的细胞内形成和空间分布是至关重要的。通常使用特定化学探针衍生化后，在细胞溶解物和体液中识别和定量羰基化物质。然而，空间细胞和组织分布很少被研究。在这里，我们报告香豆素酰肼，荧光化学探针的时间和成本效益的标记细胞羰基随后通过荧光显微镜，以评估其在时间和空间的细胞内形成。香豆素酰肼的特异性在时间和剂量依赖性实验中得到证实，使用人原代成纤维细胞与百草枯强调，并与传统的DNPH为基础的免疫细胞化学。这两种技术染色羰基化物种积累在细胞质中具有强大的核周聚类。使用互补阵列的分析方法的特异性香豆素酰肼探针对蛋白质和脂质结合的羰基已被证明。此外，证明了羰基化物质和氧化磷脂的共分布。香豆素-酰肼（CHH）化学探针用于标记细胞羰基。CHH荧光显微镜允许监测蛋白质和脂质羰基分布。CHH对蛋白质和脂质结合羰基的特异性被证明。比较CHH标记和DNPH免疫细胞化学用于显微镜成像。
Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated. Coumarin-hydrazide (CHH) chemical probe was used to label cellular carbonyls. CHH fluorescence microscopy allowed to monitor protein and lipid carbonyl distribution. CHH specificity towards protein- and lipid-bound carbonyls was demonstrated. CHH labeling and DNPH immunocytochemistry for microscopy imaging were compared.
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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作者：
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作者：
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Chedin, Stephane
DOI：
10.1111/j.1582-4934.2006.tb00407.x
发表时间：
2006-04
影响因子：
5.3
作者：
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通讯作者：
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