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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

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Ullmann-Zeunert,  Lynn
Department of Molecular Ecology, Prof. I. T. Baldwin, MPI for Chemical Ecology, Max Planck Society;
IMPRS on Ecological Interactions, MPI for Chemical Ecology, Max Planck Society;

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Muck,  Alexandr
Research Group Mass Spectrometry, MPI for Chemical Ecology, Max Planck Society;

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Wielsch,  Natalie
Research Group Mass Spectrometry, MPI for Chemical Ecology, Max Planck Society;

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Hufsky,  Franziska
IMPRS on Ecological Interactions, MPI for Chemical Ecology, Max Planck Society;

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Stanton,  Mariana
IMPRS on Ecological Interactions, MPI for Chemical Ecology, Max Planck Society;
Department of Molecular Ecology, Prof. I. T. Baldwin, MPI for Chemical Ecology, Max Planck Society;

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Bartram,  Stefan
Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society;

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Baldwin,  Ian Thomas
Department of Molecular Ecology, Prof. I. T. Baldwin, MPI for Chemical Ecology, Max Planck Society;

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Groten,  Karin
IMPRS on Ecological Interactions, MPI for Chemical Ecology, Max Planck Society;

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Svatoš,  Aleš
Research Group Mass Spectrometry, MPI for Chemical Ecology, Max Planck Society;

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MS139s1.pdf
(Supplementary material), 900KB

Citation

Ullmann-Zeunert, L., Muck, A., Wielsch, N., Hufsky, F., Stanton, M., Bartram, S., et al. (2012). 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. Journal of Proteome Research, 11(10), 4947-4960. doi:10.1021/pr300465n.


Cite as: http://hdl.handle.net/11858/00-001M-0000-000F-E606-3
Abstract
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–MSE, 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.