Rapid and reproducible phosphopeptide enrichment by tandem metal oxide affinity chromatography: application to boron deficiency induced phosphoproteomics
Rapid and reproducible phosphopeptide enrichment by tandem metal oxide affinity chromatography: application to boron deficiency induced phosphoproteomics
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通过串联 MOAC 快速、可重复地富集磷酸肽：在缺硼诱导的磷酸蛋白质组学中的应用
DOI：
10.1111/tpj.14215
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发表时间：
2019-04-01
期刊：
影响因子：
7.2
通讯作者：
Hoehenwarter, Wolfgang
中科院分区：
文献类型：
--
作者：
Chen, Yanmei;Hoehenwarter, Wolfgang
质谱法已经有助于通过蛋白质翻译后修饰（特别是磷酸化）的位点特异性定量在细胞尺度上研究分子信号传导。在这里，我们描述了一个更新的串联金属氧化物亲和色谱（MOAC）相结合的磷蛋白/磷酸肽富集策略，一个可扩展的磷酸蛋白质组学的方法，可以快速识别成千上万的磷酸肽在植物材料。我们对原始串联MOAC程序的几个步骤进行了修改，以增加定量磷酸肽的量，从而增加样品中蛋白质的位点特异性磷酸化，从少量组织和大量提取的蛋白质开始。我们应用这项技术，以产生时间分辨的地图硼信号在拟南芥根。我们表明，连续富集磷蛋白在第一和磷酸肽提取在第二步，使用我们的优化程序强烈富集根磷酸蛋白质组。我们的研究结果表明，缺硼影响超过20%的测得的根磷酸化蛋白质组和许多磷酸化位点与已知的生物学功能，甚至更大数量的以前未描述的网站，在缺硼的时间过程中进行修改。我们确定转录因子作为激素信号通路的关键调节因子，调节缺硼植物的基因表达。此外，我们的磷酸化动力学数据表明，有丝分裂原活化蛋白激酶（MAPK）级联介导的极化运输硼在拟南芥根。总之，我们建立和验证了一个强大的方法，在植物生物学研究的蛋白质组范围内的磷酸化分析。
Mass spectrometry has been instrumental in enabling the study of molecular signaling on a cellular scale by way of site-specific quantification of protein post-translational modifications, in particular phosphorylation. Here we describe an updated tandem metal oxide affinity chromatography (MOAC) combined phosphoprotein/phosphopeptide enrichment strategy, a scalable phosphoproteomics approach that allows rapid identification of thousands of phosphopeptides in plant materials. We implemented modifications to several steps of the original tandem MOAC procedure to increase the amount of quantified phosphopeptides and hence site-specific phosphorylation of proteins in a sample beginning with the less amounts of tissue and a substantially smaller amount of extracted protein. We applied this technology to generate time-resolved maps of boron signaling in Arabidopsis roots. We show that the successive enrichment of phosphoproteins in a first and phosphopeptide extraction in a second step using our optimized procedure strongly enriched the root phosphoproteome. Our results reveal that boron deficiency affects over 20% of the measured root phosphoproteome and that many phosphorylation sites with known biological function, and an even larger number of previously undescribed sites, are modified during the time course of boron deficiency. We identify transcription factors as key regulators of hormone signaling pathways that modulate gene expression in boron deprived plants. Furthermore, our phosphorylation kinetics data demonstrate that mitogen-activated protein kinase (MAPK) cascades mediate polarized transport of boron in Arabidopsis roots. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in plant biology research.