Introduction
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has driven plant biotechnologists for decades. The potential benefits are manifold: reduction in the negative environmental effects from chemical fertilizer use; availability of reactive nitrogen; potential economic benefits; and, opportunities to provide affordable crops to farmers in developing countries to increase yield. The combination of recent advances in bioengineering and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made this vision more possible. Of critical importance is the mitochondrial/chloroplast targeting and processing of the nitrogenase proteins in eukaryotes if the goal of nitrogen fixation in plants is to be a reality.
Methods
Liquid chromatography-mass spectrometry (LC-MS) offers many advantages over traditional methods for protein detection and quantitation, especially when dealing with transgenic membrane proteins that are often difficult to express or generate antibodies against. In this study, proteomics aimed to identify, characterise and quantify 16 nitrogenase (Nif) proteins that had been engineered into tobacco (Nicotiana benthamiana) and Escherichia coli. Samples were analysed with data-dependent micro-flow LC-MS/MS acquisition (SCIEX TripleTOF 6600) and multiple-reaction-monitoring LC-MRM-MS (SCIEX 6500 QTRAP).
Results
A panel of 168 peptides were tested for their suitability as Nif-specific MRM peptide markers. MRM methods have been developed for 14 nitrogenase proteins. A targeted LC-MS/MS method was employed to study the N-terminal region of the expressed NifK protein after engineering into tobacco to assess the mitochondrial processing and putative cleavage site. We have identified a mitochondrial processing peptidase (MPP) putative cleavage site and developed an assay capable of monitoring the efficiency of cleavage.
Conclusion
MRM technology is a valuable strategy to provide insights into the generation of modified plants where the N2 fixation machinery is introduced and highlights the usefulness of interdisciplinary collaboration to progress the engineering of plants to express nitrogenase.