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Quantification of phosphorylated metabolites, organic acids, and intermediates of the TCA cycle using capillary ion chromatography tandem mass spectrometry (capIC-MS/MS) following treatment of Escherichia coli with ciprofloxacin

<p>Capillary ion chromatography tandem mass spectrometry (capIC-MS/MS)&nbsp;was used to quantify phosphorylated metabolites, organic acids, and intermediates of the TCA cycle of Escherichia coli treated with ciprofloxacin, BTP-001 (a novel antimicrobial peptide), and a combination of the two . Metabolite extracts were analyzed with a Xevo TQ-XS triple quadrupole mass spectrometer (Waters, USA).</p><p>Samples were gathered from E. coli cultures grown in batch cultivations using 1 liter bioreactors. Briefly, intracellular metabolites were extracted by cycling samples between −20 °C EtOH and N2 (<i>l</i>) in three consecutive freeze–thaw cycles, with vortexing every 10 min during the thawing phase. Filters were removed and the cell debris was pelleted (4500 rcf, 10 min, -9 °C). The supernatants were transferred to a new tube, snap frozen in N2 (<i>l</i>), and lyophilized. Lyophilized extracts were reconstituted in 500 µL cold Milli-Q H2O and cleared by spin-filtration with a 10 kDa molecular cutoff (20817 rcf, 10 min, 0 °C). A mix of 80 µL centrifuged sample and 20 µL 13C-labeled ISTD extract from yeast was sent to analysis.&nbsp;</p><p>Data processing and absolute quantification was performed as earlier described using the TargetLynx application manager of MassLynx v 4.1 (Waters) to interpolate calibration curves made with appropriate dilutions of analytical grade standards (Sigma-Aldrich). The response factor of the corresponding U13C-isotopologues were used to correct the standard and sample extract response factors. Extract concentrations were normalized to the CDW, which was calculated from interpolation of the OD600 vs. CDW (g/L) curve.&nbsp;</p><p>Further statistical analysis in MetaboAnalyst v 5.0&nbsp;replaced missing values with 1/5 of the minimum value of the respective metabolite. An unpaired T-test with unequal variance determined differential enriched metabolites with a false discovery rate (FDR) &lt; 0.05 which are presented as log2 fold-change compared to control.&nbsp;</p>