Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
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分类信号，N末端修饰和叶绿体蛋白质组的丰度。
DOI：
10.1371/journal.pone.0001994
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发表时间：
2008-04-23
期刊：
影响因子：
3.7
通讯作者：
van Wijk, Klaas J.
中科院分区：
文献类型：
--
作者：
Zybailov, Boris;Rutschow, Heidi;Friso, Giulia;Rudella, Andrea;Emanuelsson, Olof;Sun, Qi;van Wijk, Klaas J.
研究叶绿体蛋白质组是了解叶绿体在植物生长发育中的重要作用的基础。本文采用纳米olc - q - tof和纳米olc - ltq - orbitrap质谱（MS）对拟南芥10种独立叶绿体进行了大规模分析，明确鉴定出1325种蛋白质。新型蛋白包括各种激酶和假定的核苷酸结合蛋白。基于重复和独立的MS蛋白鉴定，需要多个匹配的肽序列，以及文献，916个核编码蛋白被高置信度地分配到质体中，其中86%具有预测的叶绿体转运肽（cTP）。可溶性基质蛋白的蛋白质丰度是根据LTQ-Obitrap分析的归一化光谱计数计算的，发现涵盖了四个数量级。与凝胶定量的比较表明，“光谱计数”可以为拟南芥提供大规模的蛋白质定量。这些定量信息用于确定TargetP对蛋白质靶向预测可能存在的偏差，并了解蛋白质污染物的重要性。550个基质蛋白的丰度数据被用来了解代谢途径和叶绿体过程的丰度。我们强调了参与翻译后蛋白质组稳态的48种基质蛋白的丰度（包括氨基肽酶、蛋白酶、去甲酰基酶、伴侣蛋白、蛋白质分选成分），并讨论了其生物学意义。在细胞核和叶绿体编码的蛋白质亚群中发现了n端修饰，并发现了一个新的n端乙酰化基序。对拟南芥叶绿体蛋白ctp及其切割位点的分析，以及对其预测的水稻同源物的分析，发现了新的物种依赖特征，这将有助于改进亚细胞定位预测。没有证据表明通过分泌系统靶向。该研究提供了迄今为止最全面的叶绿体蛋白质组分析和扩展的植物蛋白质组数据库（PPDB），其中所有MS数据都投射到已鉴定的基因模型上。
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.
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