A multiplexed work-flow for absolute quantification of Klebsiella oxytoca Nif proteins

Global imbalances of the nitrogen cycle are increasingly recognized as a major challenge to human development, and are exacerbated by the use of synthetic inorganic fertilizers. Biotechnology alternatives to inorganic fertilizers include biofertilisation from nitrogen fixing bacteria (diazotrophs), expressing nitrogenase and auxiliary genes ( genes). In order to directly quantify all twenty Nif proteins through multiple reaction monitoring (MRM) MS, we established a high throughput pipeline to generate a set of Nif protein QconCATs as quantotypic standards. A stringent validation of the pipeline and QconCATs application with regards to isotopic labelling efficiency (100%), limits of detection and quantification, analyte to internal standard concentration boundaries was used for optimisation. Using three QconCATs for the measurement of 20 likely low, middle and high Nif protein abundances enabled detection of all Nif proteins, 19 of which could be accurately quantified and their variation over time monitored. Stoichiometries between Nif proteins and changes between early and late transition into diazotrophy suggest i) a temporal regulation of gene cluster expression that may be linked to nitrogenase expression and maturation; ii) vast disparities in Nif protein abundances and iii) high dependency on the master regulator pair for gene expression.