Marmagne Anne, Salvi Daniel, Rolland Norbert, Ephritikhine Geneviève, Joyard Jacques, Barbier-Brygoo Hélène
Institut des Sciences du Végétal, CNR, Gif-Sur-Yvette, France.
Methods Mol Biol. 2006;323:403-20. doi: 10.1385/1-59745-003-0:403.
Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.
蛋白质组学是一种非常强大的方法，可将测序基因组（如拟南芥）中包含的信息与植物细胞区室研究提供的功能知识联系起来。然而，膜蛋白质组学仍然是一项挑战。使膜中存在的复杂蛋白质混合物显现出来的一种方法是基于以下两点开展蛋白质组学分析：（1）使用高度纯化的膜组分；（2）对膜蛋白进行分级分离，以尽可能多地检索蛋白质（从疏水性最强到最弱）。为了说明这些策略，我们选择了两种类型的膜，即质膜和叶绿体包膜膜。这两种类型的膜都可以从不同类型的组织中制备到合理的纯度：从培养细胞中制备质膜，从整株植物中制备叶绿体包膜膜。本文仅限于描述制备高度纯化且经过表征的植物膜组分的方法，以及随后根据简单的物理化学标准（即氯仿/甲醇提取、碱性或盐处理）对这些膜蛋白进行分级分离，以便使用现代蛋白质组学方法进行进一步分析。
