Phosphoproteomic Analysis of Seed Maturation in Arabidopsis, Rapeseed, and Soybean
Phosphoproteomic Analysis of Seed Maturation in Arabidopsis, Rapeseed, and Soybean
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DOI：
10.1104/pp.111.191700
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发表时间：
2012-05-01
期刊：
影响因子：
7.4
通讯作者：
Thelen, Jay J.
中科院分区：
文献类型：
--
作者：
Meyer, Louis J.;Gao, Jianjiong;Thelen, Jay J.
为了表征发育种子中的蛋白磷酸化，对大豆（Glycine max）、油菜（Brassica napus）和拟南芥（Arabidopsis thaliana）发育的五个连续阶段的整个种子进行了大规模的基于质谱的磷酸化蛋白质组学研究。使用固定化金属亲和和金属氧化物亲和色谱的组合策略从0.5 mg总肽中富集磷酸肽。通过Orbitrap串联质谱法分析富集的磷酸肽，并以正向和随机方向针对同源基因组或cDNA数据库进行质谱挖掘，后者用于计算错误发现率。我们从956种蛋白质中鉴定出总共2,001种磷酸肽，其中包含1,026个明确的磷酸化位点，整个研究的平均错误发现率为0.78%。将整个数据集上传到植物蛋白磷酸化数据库（www.p3db.org）中，包括所有元数据和注释的光谱。植物蛋白磷酸化数据库是所有植物磷酸化数据的门户，并允许基于同源性的查询实验确定的磷酸化位点。与其他大规模的磷酸化蛋白质组学研究的比较确定，652磷蛋白是新的这项研究。独特的蛋白质分为几个基因本体论类别，其中一些在我们的研究以及其他大规模磷酸蛋白质组学研究中被过度代表，包括代谢过程和RNA结合;其他类别在我们的研究中只被过度代表，如胚胎发育。这项研究表明，分析多个植物和植物器官的重要性，以全面绘制完整的植物磷酸蛋白质组。
To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg of total peptides using a combined strategy of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and mass spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate. We identified a total of 2,001 phosphopeptides containing 1,026 unambiguous phosphorylation sites from 956 proteins, with an average false discovery rate of 0.78% for the entire study. The entire data set was uploaded into the Plant Protein Phosphorylation Database (www.p3db.org), including all meta-data and annotated spectra. The Plant Protein Phosphorylation Database is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally determined phosphosites. Comparisons with other large-scale phosphoproteomic studies determined that 652 of the phosphoproteins are novel to this study. The unique proteins fall into several Gene Ontology categories, some of which are overrepresented in our study as well as other large-scale phosphoproteomic studies, including metabolic process and RNA binding; other categories are only overrepresented in our study, like embryonic development. This investigation shows the importance of analyzing multiple plants and plant organs to comprehensively map the complete plant phosphoproteome.