Absolute quantification of the Kdp subunits of Escherichia coli by multiple reaction monitoring.

The sensor kinase/response regulator system KdpD/KdpE of Escherichia coli regulates the expression of the kdpFABC operon, encoding the high-affinity KdpFABC potassium (K(+) )-transport complex. Additionally, it has been suggested that the kdpDE operon itself is subjected to autoregulation by its gene products KdpD and KdpE. However, since kdpFABC and kdpDE expression has mainly been studied on the transcriptional level, accurate information on absolute amounts and the stoichiometric subunit composition of KdpFABC and KdpD/KdpE under K(+) -limiting and K(+) -nonlimiting growth conditions are lacking. In this study, we used highly sensitive mass spectrometric methods to quantify the amount of subunits of the Kdp(F)ABC complex and KdpD/KdpE. Data-dependent shotgun MS was used to assess protein coverage and accessible peptides. Absolute amounts of Kdp(F)ABC and KdpD/KdpE were quantified by targeted MRM analysis in the presence of corresponding heavy labeled standard peptides. Baseline synthesis of Kdp(F)ABC and KdpD/KdpE was found to be in the attomolar range under K(+) -nonlimiting conditions. Under K(+) -limitation, synthesis of Kdp(F)ABC (KdpA:KdpB:KdpC ratio of 1:1:1) was amplified more than 100-fold, whereas only a tenfold amplification of KdpD/KdpE (KdpD:KdpE ratio of 1:4) was observed. The results obtained provide a solid basis for follow-up studies on the dynamic regulation of the Kdp system.