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鈥揌erbivore Interac

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• 作者：Lynn Ullmann-Zeunert ; Alexander Muck ; Natalie Wielsch ; Franziska Hufsky ; Mariana A. Stanton ; Stefan Bartram ; Sebastian B枚cker ; Ian T. Baldwin ; Karin Groten ; Ale拧 Svato拧
• 刊名：Journal of Proteome Research
• 出版年：2012
• 出版时间：October 5, 2012
• 年：2012
• 卷：11
• 期：10
• 页码：4947-4960
• 全文大小：568K
• 年卷期：v.11,no.10(October 5, 2012)
• ISSN：1535-3907

文摘

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鈥揗S^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鈥揾erbivore interactions.

Keywords:

LC鈭扢S^E; ¹⁵N-incorporation; absolute protein quantification; Nicotiana attenuata; herbivory; soil-grown plants