National Institute of Crop Science, Tsukuba 305-8518, Japan.
J Proteome Res. 2009 Oct;8(10):4487-99. doi: 10.1021/pr9002883.
The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.
质膜作为细胞细胞质与细胞外环境的主要界面。为了研究质膜在应对水淹胁迫中的功能，我们使用双水相分配法从大豆幼苗的根和下胚轴中纯化质膜。使用二维聚丙烯酰胺凝胶电泳结合质谱和蛋白质测序（2-DE MS/测序仪）的蛋白质组学或纳流液相色谱结合质谱（nanoLC-MS/MS）的蛋白质组学分析纯度为 81%的纯化质膜蛋白。通过 nanoLC-MS/MS 蛋白质组学鉴定的疏水性蛋白质数量与通过 2-DE MS/测序仪蛋白质组学鉴定的疏水性蛋白质数量进行了比较。这些技术被应用于鉴定对水淹胁迫有反应的大豆中的蛋白质。结果表明，质膜深入了解了大豆对水淹胁迫的反应：（i）位于细胞壁中的蛋白质在质膜中上调；（ii）与抗氧化系统相关的蛋白质在保护细胞免受氧化损伤方面发挥关键作用；（iii）热休克同源蛋白在水淹胁迫期间发挥作用，防止蛋白质变性和降解；（iv）信号相关蛋白可能调节离子稳态。
