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.
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.
Max Planck Institute for Chemical Ecology, Jena, Germany.
Lynn
L
Ullmann-Zeunert
Alexander
A
Muck
Natalie
N
Wielsch
Franziska
F
Hufsky
Mariana A
MA
Stanton
Stefan
S
Bartram
Sebastian
S
Böcker
Ian T
IT
Baldwin
Karin
K
Groten
Aleš
A
Svatoš
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001499:Bayes Theorem; D002853:Chromatography, Liquid / Q000592:standards; D060434:Herbivory; D016013:Likelihood Functions; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000592:standards; D010763:Phosphorylase b / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012015:Reference Standards; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D014026:Nicotiana / Q000737:chemistry / Q000378:metabolism*
D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D010936:Plant Extracts; D010940:Plant Proteins; D008084:Lipoxygenase; D010763:Phosphorylase b; D012273:Ribulose-Bisphosphate Carboxylase; D009584:Nitrogen