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

J Proteome Res. 2012 Oct 5;11(10):4947-60. doi: 10.1021/pr300465n. Epub 2012 Sep 17.

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-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N 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 (15)N-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 (15)N-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 (15)N-pulse protocols on plants growing in soil under unknown (15)N-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 (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Amino Acid Sequence
  • Animals
  • Bayes Theorem
  • Chromatography, Liquid / standards
  • Herbivory
  • Likelihood Functions
  • Lipoxygenase / chemistry
  • Lipoxygenase / isolation & purification
  • Lipoxygenase / metabolism
  • Molecular Sequence Data
  • Nitrogen / metabolism*
  • Nitrogen Isotopes / metabolism
  • Peptide Fragments / chemistry
  • Peptide Mapping / standards
  • Phosphorylase b / chemistry
  • Plant Extracts / chemistry
  • Plant Extracts / isolation & purification
  • Plant Leaves / chemistry
  • Plant Leaves / metabolism*
  • Plant Proteins / chemistry
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism
  • Rabbits
  • Reference Standards
  • Ribulose-Bisphosphate Carboxylase / chemistry
  • Ribulose-Bisphosphate Carboxylase / isolation & purification
  • Ribulose-Bisphosphate Carboxylase / metabolism*
  • Spectrometry, Mass, Electrospray Ionization / standards
  • Tandem Mass Spectrometry / standards
  • Tobacco / chemistry
  • Tobacco / metabolism*

Substances

  • Nitrogen Isotopes
  • Peptide Fragments
  • Plant Extracts
  • Plant Proteins
  • Lipoxygenase
  • Phosphorylase b
  • Ribulose-Bisphosphate Carboxylase
  • Nitrogen