Prototeomics of the Thylakoids from Arabidopsis Thaliana; Protein Identification and Interactions, Post-Translational Modifications and Differential Expression

据预测，绿藻和高等植物的叶绿体中含有大约2500-3000种不同的蛋白质，占植物蛋白质组总数的10-12%。叶绿体具有四种多亚基蛋白质复合物的类囊体膜，由至少66种不同的蛋白质组成，并进行光合反应。这些复合物的组装涉及辅因子的靶向、插入、加工和连接，并需要转录、翻译和翻译后水平的调控。在类囊体膜中至少存在100种参与生物发生相关过程的蛋白质，但迄今为止只发现了少数几种。随着拟南芥基因组的完成以及二维电泳和质谱（MS）技术的改进，对拟南芥类囊体蛋白质组的系统表征已成为可能。本项目的目的是系统地表征拟南芥类囊体蛋白质组。这一特性包括大多数类囊体蛋白及其蛋白相互作用的全球鉴定。高重复性的二维电泳，非变性生化分离技术，以及最先进的质谱仪将被用来以一种有效的方式获得这种表征。在线质谱将用于小蛋白质或非常疏水的整体膜蛋白的众所周知的困难鉴定。总之，这将给出一个独特的概述类囊体蛋白质组。此外，这也将为类囊体生物发生和维持的研究提供更合理的遗传设计（反向）策略。该项目的成功实现也有助于加深我们对细胞器生物发生以及原核生物内膜生物发生的一般认识。总之，这个项目的目标是：1。用质谱法从一维和二维凝胶分离蛋白中系统鉴定可溶性和外周类囊体蛋白。2。有机溶剂分离和在线串联质谱法（反相高效液相色谱）鉴定整体类囊体膜蛋白。3。（低丰度）类囊体蛋白复合物和蛋白相互作用的非变性纯化方法和质谱选择鉴定。4 .叶绿体靶向信号的分析和对预测程序的反馈，以及预测蛋白质序列（来自cDNA或基因组DNA）与实验确定的蛋白质序列之间的推定差异分析。五、开发专业定制的蛋白质组学实验室信息管理系统，包括web界面。

The chloroplasts in green algae and higher plants are predicted to contain around 2500-3000 different proteins, corresponding to 10-12% of the total plant proteome. Chloroplasts possess thylakoid membranes with four multi-subunit protein complexes, comprising together at least 66 different proteins and carrying out the photosynthetic reactions. Assembly of these complexes involves targeting, insertion, processing as well as ligation of cofactors, and requires regulation at transcriptional, translational and post-translational levels. At least 100 proteins involved in such biogenesis related processes should be present in the thylakoid membrane, but only a few have been identified so far. With the completion of the Arabidopsis thaliana genome and the improvement of 2-dimensional electrophoresis and mass spectrometry (MS), a systematic characterization of the thylakoid proteome has become feasible. The objective of this project is to systematically characterize the thylakoid proteome of Arabidopsis thaliana. This characterization includes a global identification of most thylakoid proteins and their protein-protein interactions. Highly reproducible 2-dimensional electrophoresis, non-denaturing biochemical separation techniques, and state of the art mass spectrometers will be employed to obtain this characterization in a time-effective manner. On-line MS will be carried out for the notoriously difficult identification of small proteins or very hydrophobic integral membrane proteins. Together this will give a unique overview of the thylakoid proteome. In addition, it will also enable more rational design (reverse) genetic strategies for studies of thylakoid biogenesis and maintenance. A successful realization of the project could serve also to deepen our general understanding of organelle biogenesis, as well as inner membrane biogenesis of prokaryotes. In summary, the objectives of this project are:I. Systematic identification of soluble and peripheral thylakoid proteins by mass spectrometry from gel-separated proteins from 1-dimensional and 2-dimensional gels. II. Identification of integral thylakoid membrane proteins by fractionation in organic solvents and on-line tandem mass spectrometry (reverse phase HPLC). III. Selected identification of (low abundant) thylakoid protein complexes and protein-protein interactions by non-denaturing purification methods and mass spectrometry. IV. Analysis of chloroplast targeting signals and feedback to prediction programs and analysis of putative discrepancies between predicted protein sequences (from cDNA or genomic DNA) and experimental determined protein sequences. V. Development of a professional custom built proteomics LIM (laboratory information management) system, including a web interface.