Yamaguchi Kenichi
Division of Biochemistry, Nagasaki University, Nagasaki, Japan.
Methods Mol Biol. 2011;775:241-64. doi: 10.1007/978-1-61779-237-3_13.
Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.
菠菜和衣藻叶绿体核糖体的蛋白质组学揭示了质体核糖体独特的蛋白质组成和结构。这些研究表明存在一些质体核糖体特有的核糖体蛋白，它们可能参与质体特有的翻译调控。鉴于拟南芥在遗传分析和分子生物学方面有强大的背景，对拟南芥质体核糖体进行深入的蛋白质组学表征将有助于进一步了解高等植物中的质体翻译。在此，我描述了使用蔗糖梯度从衣藻和拟南芥中制备质体核糖体的简单快速方法。我还描述了纯化质体核糖体及其亚基的纯度标准和产量估算方法、质体核糖体蛋白的制备方法，以及通过基质辅助激光解吸/电离质谱法鉴定一些拟南芥质体核糖体蛋白的方法。
