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首页> 外文期刊>Journal of proteome research >Determination of~15N-incorporation into plant proteins and their absolute quantitation: A new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions
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Determination of~15N-incorporation into plant proteins and their absolute quantitation: A new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions

机译：植物蛋白质中〜15N掺入的确定及其绝对定量：研究植物-草食动物相互作用引起的氮通量动态和蛋白质库大小的新工具

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摘要

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS E, combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and 15N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected 15N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate- carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of 15N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using 15N-pulse protocols on plants growing in soil under unknown 15N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and 15N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.
机译：食草导致氮在不同池和组织之间的分配发生变化。但是，缺乏对这些变化的详细定量分析。在这里，我们证明了一种称为LC-MS E的与质谱数据无关的采集方法，并结合了一种新的算法来量化肽中的重原子富集，能够以高通量方式量化引起的蛋白质量和15N通量变化。实现了对掺入叶蛋白提取物中的兔磷酸化酶b蛋白的可靠鉴定/定量。线性动态范围，技术和生物学复制品的可重复性，以及15N掺入到核糖-1,5-双磷酸羧化酶/加氧酶/加氧酶（RuBisCO）和RuBisCO的小（SSU）和大（LSU）亚基中的测定值和预期值之间的差异在不同浓度的15N标记硝酸盐下于水培培养中生长的烟草植物的激活酶2（RCA2）被用于进一步评估该程序。通过在未知的15N掺入水平下在土壤中生长的植物上使用15N脉冲协议，证明了该方法在生态现实环境中进行全植物研究的实用性。此外，我们量化了脂氧合酶2（LOX2）蛋白的含量，该酶在抗草食动物的防御反应中很重要，表明该方法可进行深入的定量蛋白质组学和植物与草食动物相互作用过程中引起的代谢动力学的15N通量分析。

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