Sorting Signals, N-Terminal Modifications and Abundance of the Chloroplast Proteome

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that ��spectral counting�� can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.要阐明叶绿体在植物生长与发育过程中的核心贡献，需先对叶绿体蛋白质组开展定性表征。本研究采用纳升液相色谱-四极杆-飞行时间质谱（nanoLC-Q-TOF）与纳升液相色谱-线性离子阱-轨道阱质谱（nanoLC-LTQ-Orbitrap）两种质谱（MS）技术，对十份独立制备的拟南芥（Arabidopsis thaliana）叶绿体样品进行大规模蛋白质组分析，明确鉴定出1325种蛋白质。新鉴定的蛋白质涵盖多种激酶及推定的核苷酸结合蛋白。基于需匹配多条肽段的重复独立质谱蛋白质鉴定结果，并结合已有文献，我们以高置信度将916种核编码蛋白质定位于质体，其中86%带有预测的叶绿体转运肽（chloroplast transit peptide, cTP）。通过LTQ-轨道阱质谱分析得到的归一化光谱计数，我们计算得到可溶性叶绿体基质蛋白质的丰度范围跨越四个数量级。与基于凝胶的定量方法对比后证实，"光谱计数"可用于拟南芥的大规模蛋白质定量分析。该定量信息被用于评估TargetP蛋白质靶向预测工具可能存在的偏倚，同时也用于解析蛋白质污染物的生物学意义。我们利用550种基质蛋白质的丰度数据，分析了代谢通路与叶绿体相关进程的蛋白质丰度特征。我们重点关注了48种参与蛋白质组翻译后稳态调控的基质蛋白质，包括氨肽酶、蛋白酶、脱甲酰基酶、分子伴侣及蛋白质分选组分，并探讨了其生物学意义。我们在部分核编码及叶绿体编码蛋白质中鉴定到了N末端修饰，并发现了一种全新的N末端乙酰化基序。通过分析拟南芥叶绿体蛋白质的叶绿体转运肽（cTP）及其切割位点，以及其预测的水稻同源蛋白，我们鉴定出了新的物种依赖性特征，这将有助于优化亚细胞定位预测算法。未发现有证据支持蛋白质通过分泌系统进行叶绿体靶向的假说。本研究提供了迄今为止最为全面的叶绿体蛋白质组分析结果，并构建了扩展版的植物蛋白质组数据库（Plant Proteome Database, PPDB），所有质谱数据均被映射至已鉴定的基因模型上。