Comprehensive analysis of protein interactions for determination of protein function in rice

蛋白质芯片是蛋白质组分析中鉴定表达蛋白质和研究蛋白质相互作用的最有前途的工具之一。我们研制了一种新型的蛋白质芯片平板，该平板由类金刚石碳涂层不锈钢(DLC)制成，并在平板表面进行N-羟基琥珀酰亚胺酯的改性。将经十二烷基硫酸钠凝胶电泳法或双向电泳法分离的蛋白质成功地电印迹并共价固定在类金刚石平板上，制备了简便、快速的高密度蛋白质芯片。通过对凝胶和电印迹技术的改进，获得了较高的蛋白质印迹效率(25-70%)。利用DLC板，我们建立了固定在DLC板上的蛋白质的鉴定技术，用于利用基质辅助激光解吸电离/飞行时间质谱仪检测固定在板上的蛋白质或与其相互作用的多肽，并发展了鉴定平板上蛋白质的翻译后修饰的技术。在本研究中，我们应用这些技术对水稻愈伤组织和大豆种子中的一些蛋白质和N-连接的糖蛋白进行了鉴定，这些蛋白质和糖蛋白是用2-DE分离的。所开发的技术为植物高通量蛋白质组分析提供了巨大的潜力。

Protein chip has been one of the most promising tools for identification of expressed proteins and study of protein-protein interaction in proteome analysis. We developed a novel protein chip plate, which is made of diamond-like carbon-coated stainless steel (DLC), and modified with N-hydroxysuccinimide ester on the surface of plate. Proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis (2-DE) were successfully electroblotted and immobilized covalently onto the DLC plate to produce easily and rapidly a high-density protein chip. High blotting efficiency (25-70 %) of proteins from the gels onto the DLC plate was obtained by improvement of electrophoresis and electroblotting techniques. Using the DLC plate, we established the techniques for identification of proteins immobilized on the DLC plate, for detection of proteins or peptides interacted with proteins immobilized on the plate using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and also we developed the technique for identification of post-translational modification of proteins on the plate. In this study, we applied these techniques to identification of a number of proteins and N-linked glycoproteins of the rice calli and soybean seeds, which were separated by 2-DE. The developed techniques have a great potential to make possible the high-throughput proteome analysis in plants.