Identification of dimedone-trapped sulfenylated proteins in plants under stress.
Identification of dimedone-trapped sulfenylated proteins in plants under stress.
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DOI：
10.1016/j.bbrep.2016.11.014
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发表时间：
2017-03
影响因子：
2.7
通讯作者：
Messens J
中科院分区：
文献类型：
--
作者：
Akter S;Carpentier S;Van Breusegem F;Messens J
在受到胁迫的植物中，活性氧（ROS）水平上升。ROS信号转导研究的关键是检测和鉴定蛋白质半胱氨酸亚磺酰化（-SOH），ROS介导的巯基（-SH）氧化产物。用过氧化氢（H2 O2）胁迫拟南芥幼苗，并用二甲酮标记亚磺酰化蛋白。双甲酮标记的次磺酸蛋白可视化的二维电泳（2DE）免疫印迹与抗半胱氨酸次磺酸抗体，随后通过质谱检测。优化了拟南芥蛋白质亚磺酰化的检测方法。我们的结论是，双甲酮可以穿透细胞壁，不胁迫植物，并可以“阅读”氧化胁迫下的蛋白质磺酰化模式的变化。我们观察到，亚磺酰化的蛋白质在植物中处理10 mM H2 O2的数量高于未处理的植物。在2DE免疫印迹上共发现39个亚磺酰化蛋白点。通过质谱分析，发现了11个参与初级代谢、氧化还原调节、翻译和信号转导途径的亚磺酰化蛋白。因此，通过结合免疫化学的2DE策略与质谱，我们能够确定亚磺酰化蛋白在过氧化氢胁迫拟南芥幼苗。亚磺酰化的蛋白质可以被考虑作为植物中的氧化还原调节剂用于进一步验证。提出了一种植物蛋白质亚磺酰化的化学检测方法。双甲酮是一种无毒的，植物细胞可渗透的，硫烯类“阅读器”。H2 O2响应亚磺酰化蛋白质鉴定相结合的免疫化学2DE策略与质谱。
In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H2O2) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis. We conclude that dimedone can penetrate the cell wall, does not stress plants, and can “read” the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H2O2 was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H2O2-stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants. A chemical detection method for protein sulfenylation in plants is proposed. Dimedone is a non-toxic, plant cell permeable, sulfenome ‘reader’. H2O2 responsive sulfenylated proteins identified by combining an immunochemical 2DE strategy with mass spectrometry.