Lipid A extraction and mass spectrometry analysis.

Triplicate cultures of each strain were grown to an OD₆₀₀ of 1.0 in 25 ml of TYG medium, washed with 1× PBS, pelleted, and frozen. Cell pellets were washed in PBS. Lipid A was extracted using mild acid hydrolysis, as described previously (50, 51). Lipid A samples were subjected to matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). All solvents were high-performance liquid chromatography (HPLC)-grade quality. Lipid A samples were dissolved in a mixture of chloroform and methanol (4:1 [vol/vol]); 5-chloro-2-mercaptobenzothiazole (CMBT) (Sigma) was dissolved in methanol/chloroform/water (4:4:1 [vol/vol/vol]) to a concentration of 25 mg/ml. The matrix was prepared by mixing CMBT with saturated tribasic ammonium citrate (20:1 [vol/vol]). Samples and matrix were mixed (1:1 [vol/vol]), and 1 μl was loaded onto the target plate. MALDI-TOF MS data were calibrated using a peptide calibration standard (Bruker Daltonics), in negative-ion mode. MALDI-TOF MS mass spectra of lipid A extracts were acquired in reflector mode with an Autoflex Speed mass spectrometer (Bruker Daltonics). A total of 500 single laser shots were averaged from each mass spectrum. Data were acquired and processed using FlexControl 3.4 and FlexAnalysis 3.4 software (Bruker Daltonics).