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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

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.

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 Creators:
Ullmann-Zeunert, Lynn1, 2, Author              
Muck, Alexandr3, Author              
Wielsch, Natalie3, Author              
Hufsky, Franziska2, Author              
Stanton, Mariana2, 4, Author              
Bartram, Stefan5, Author              
Böcker, S., Author
Baldwin, Ian Thomas4, Author              
Groten, Karin2, Author              
Svatoš, Aleš3, Author              
Affiliations:
1Department of Molecular Ecology, Prof. I. T. Baldwin, MPI for Chemical Ecology, Max Planck Society, ou_24029              
2IMPRS on Ecological Interactions, MPI for Chemical Ecology, Max Planck Society, ou_421900              
3Research Group Mass Spectrometry, MPI for Chemical Ecology, Max Planck Society, ou_421899              
4Department of Molecular Ecology, Prof. I. T. Baldwin, MPI for Chemical Ecology, Max Planck Society, ou_24029              
5Department of Bioorganic Chemistry, Prof. Dr. W. Boland, MPI for Chemical Ecology, Max Planck Society, ou_24028              

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 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.

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 Dates: 2012-082012-09-062012
 Publication Status: Published in print
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 Identifiers: Other: MS139
DOI: 10.1021/pr300465n
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Title: Journal of Proteome Research
  Other : J. Proteome Res.
Source Genre: Journal
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Publ. Info: Washington, D.C. : American Chemical Society
Pages: - Volume / Issue: 11 (10) Sequence Number: - Start / End Page: 4947 - 4960 Identifier: ISSN: 1535-3893
CoNE: https://pure.mpg.de/cone/journals/resource/111019664290000