INSERM, Laboratoire d'Etude de Dynamique des Protéomes, U880, France.
Mol Cell Proteomics. 2010 Jun;9(6):1063-84. doi: 10.1074/mcp.M900325-MCP200. Epub 2010 Jan 10.
Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.
近年来，蛋白质组学领域的进展使得人们能够对叶绿体样本进行一系列高通量实验，并且这些数据可在几个公共数据库中获得。然而，许多叶绿体蛋白的准确定位通常仍然是假设的。这对于包膜蛋白来说尤其如此。我们通过在同一组实验中关注蛋白质在基质、类囊体和包膜膜中的定位，进一步深入了解叶绿体蛋白质组。基于 LC-MS/MS 的分析首先允许构建 AT_CHLORO 数据库（http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/），该数据库包含通过 10654 个独特肽序列鉴定的 1323 种蛋白质的综合目录，这些蛋白质存在于高度纯化的叶绿体及其从小麦叶中制备的亚部分中。该数据库还提供了广泛的蛋白质组学信息（肽序列和分子量、色谱保留时间、MS/MS 光谱和光谱计数），用于收集具有各自准确质量和时间标记的独特叶绿体蛋白质的肽，这些肽具有各自的精确分析坐标、分子量和保留时间。我们使用半定量蛋白质组学方法（光谱计数）评估每个蛋白质在三个叶绿体区室中的分配情况。这些数据与对文献的深入调查一起被编译，以提供先前已知和新鉴定的蛋白质的准确亚质体定位。因此，获得了一个包含有关包膜部分中鉴定的蛋白质的广泛信息的独特知识库，从而揭示了该膜系统的新见解。总之，我们获得的数据提供了有关一些蛋白质的质体或亚质体定位的意外信息，这些蛋白质以前不被怀疑与该膜系统有关。基于光谱计数的策略进一步得到了验证，因为可以剖析质体中某些途径（例如光合作用和氨基酸、脂肪酸或甘油脂质生物合成）的区室化。它还允许重新审视叶绿体代谢和功能的区室化。
