Autoflex speed tof tof mass spectrometer

Isolation of lipid A molecules and subsequent analysis by negative-ion matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry was performed as previously described [41, 65, 66] . Briefly, K. pneumoniae strains were grown in LB (Oxoid) and the lipid A was purified from stationary cultures using the ammonium hydroxide/isobutyric acid isolation method described earlier [67] . Mass spectrometry analysis were performed on a Bruker autoflex® speed TOF/TOF mass spectrometer in negative reflective mode with delayed extraction using as matrix an equal volume of dihydroxybenzoic acid matrix (Sigma-Aldrich) dissolved in (1:2) acetonitrile-0.1% trifluoroacetic acid. The ion-accelerating voltage was set at 20 kV. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker) was used to calibrate the MALDI-TOF. Further calibration for lipid A analysis was performed externally using lipid A extracted from Escherichia coli strain MG1655 grown in LB medium at 37°C.