Simultaneous determination of 15N-incorporation levels in plant proteins and their absolute quantitation to study nitrogen flux and protein turnover
Nitrogen is an essential nutrient for plant growth, reproduction, and defense against herbivores. The metabolic incorporation of 15N into proteins has become a powerful tool to study nitrogen turnover rates in plants, and this approach enables quantitative tracking of isotope flows in whole plants. In the present study we demonstrate that the data-independent acquisition approach known as LC/MSE combined with a novel program MoLE (Molecule Labeling Estimator) that quantifies the degree of heavy atom enrichment, has great potential for simultaneous absolute quantitation of proteins and calculation of their 15N-incorporation levels in high throughput manner. The approach was evaluated using non-sequenced wild tobacco, Nicotiana attenuata, grown in a hydroponic culture at different concentrations of 15N-labeled nitrate. The small (SSU) and large (LSU) subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase (RuBisCO) and RuBisCO activase (RCA2) were used as model proteins for quantitation and isotope enrichment determination. For protein identification and quantitation we used protein sequences of two phylogenetically closely related Nicotiana species (N. tabaccum, N. sylvestris). We show, that the deviation between expected and by MoLE algorithm calculated 15N-ratios did not exceed 1.0 atomic percent (At%). The calculated 15N-abundances were compared to isotope ratios obtained on an elemental analyzer – continuous flow – isotope ratio mass spectrometry (EA–CF–IRMS) system and both values highly correlated with the expected ones (R2=0.9998). The applicability of our approach under greenhouse conditions was demonstrated by applying a 15N-pulse to whole plants grown in soil leading to unknown amounts of 15N-incorporation. Results demonstrate that our approach can be used for ecologically-relevant quantitative proteomics studies and applied to protein turnover and nitrogen flux studies under different natural conditions.