Proteomic Comparison of Plastids from Developing Embryos and Leaves of Brassica napus

Plastids are highly specialized organelles, responsible for photosynthesis and biosynthesis of various phytochemicals. To better understand plastid diversity and metabolism, a quantitative proteomic study of two plastid forms from Brassica napus (oilseed rape) was performed. Plastids were isolated from leaves (chloroplasts) of two-week-old plants and developing embryos (embryoplasts) three-weeks after flowering, using an approach avoiding protein storage vacuole contamination. Proteins from five different plastid preparations were prefractionated by SDS-PAGE and sectioned into multiple bands, and in-gel proteins were subjected to trypsin digestion. Tryptic peptides from each band were eluted and analyzed by liquid chromatography−tandem mass spectrometry (LC−MS/MS) and spectra were searched against a comprehensive plant database. Proteins were quantified based on MS/MS spectral counting of unique, nonhomologous peptides. Functional classification and quantitative comparison of over 2000 redundant proteins (compiled to 675 nonredundant proteins) determined that light reaction proteins are more prominent in chloroplasts, while many Calvin cycle enzymes are more prominent in embryoplasts. Embryoplasts also contain a diversity of other metabolic enzymes undetected in chloroplasts. Many enzymes involved in de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not chloroplasts. Additionally, protein synthesis-related proteins were prominent in embryoplasts. Collectively, these results indicate that these two plastid types are distinct.质体（Plastids）是一类高度特化的细胞器，负责光合作用与多种植物化学物的生物合成。为深入解析质体的多样性与代谢特征，研究团队针对甘蓝型油菜（Brassica napus，俗称油料油菜）的两种质体形态开展了定量蛋白质组学研究。研究人员从两周龄植株的叶片（叶绿体chloroplasts）以及开花三周后的发育中胚（胚质体embryoplasts）中分离质体，采用的分离策略可避免蛋白质储存液泡的污染。针对5份独立的质体制备物，研究人员通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对其总蛋白进行预分级，将凝胶切割为多条条带，随后对凝胶内的蛋白进行胰蛋白酶酶解。将每条条带中的胰蛋白酶肽段洗脱后，采用液相色谱-串联质谱（LC−MS/MS）进行分析，并将所得质谱谱图与综合植物蛋白质数据库进行比对检索。研究人员基于唯一且非同源肽段的MS/MS谱图计数对蛋白进行定量分析。通过对2000余种冗余蛋白（最终整合为675个非冗余蛋白）进行功能分类与定量比较，研究发现光反应相关蛋白在叶绿体中富集度更高，而诸多卡尔文循环（Calvin cycle）酶类则在胚质体中表达更为显著。胚质体中还存在多种叶绿体中未被检测到的其他代谢酶类。诸多参与脂肪酸与氨基酸从头生物合成的酶类仅在胚质体中被检出，而未在叶绿体中发现。此外，与蛋白质合成相关的功能蛋白在胚质体中富集明显。综上，上述结果表明这两种质体类型具有显著差异。