Printz Bruno, Dos Santos Morais Raphaël, Wienkoop Stefanie, Sergeant Kjell, Lutts Stanley, Hausman Jean-Francois, Renaut Jenny
Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology Belvaux, Luxembourg ; Groupe de Recherche en Physiologie Végétale, Earth and Life Institute Agronomy, Universiteì catholique de Louvain Louvain-la-Neuve, Belgium.
Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology Belvaux, Luxembourg.
Front Plant Sci. 2015 Apr 10;6:237. doi: 10.3389/fpls.2015.00237. eCollection 2015.
Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.
采用三步提取程序，依次使用氯化钙、乙二醇双四乙酸（EGTA）和氯化锂补充缓冲液，从苜蓿茎中提取细胞壁蛋白。将该提取细胞壁蛋白的方案的效率与之前发表的两种针对苜蓿茎细胞壁蛋白分析进行优化的方法进行了比较。经过液相色谱-串联质谱（LC-MS/MS）分析，三步提取程序鉴定出的细胞壁蛋白数量最多（242个NCBInr标识符），非细胞壁蛋白的比例最低（约30%）。然而，这三种方案并非相互替代，而是相互补充，因为43%的鉴定蛋白是某一种方案所特有的。因此，选择该三步方案，使用二维凝胶电泳进行更详细的蛋白质组学表征。通过该技术，75%的鉴定蛋白显示为组分特异性，72.7%的蛋白预计属于细胞壁组分。虽然基于凝胶的方法在检测和鉴定低丰度蛋白方面不如LC-MS/MS方法灵敏，但它是区分和相对定量蛋白质异构体和/或修饰蛋白的有价值工具。特别是检测和表征了氨基酸序列有变异和/或携带不同N-连接聚糖链的异构体。这项研究强调了用于植物细胞壁蛋白质组研究的提取方案和分析技术是如何相互补充的，以及它们如何结合起来以阐明生物学研究中植物细胞壁蛋白质组的动态变化。数据可通过ProteomeXchange获得，标识符为PXD001927。
