MALDI-TOF Analysis of Lipid A

For structural analysis, lipid A was extracted by an ammonium hydroxide-isobutyric acid method as previously described^{23 (link)}, and subjected to MALDI-TOF MS analysis. Briefly, freshly washed cells (10 mg) were suspended in 400 μL of isobutyric acid-1M ammonium hydroxide mixture (5:3, vol/vol), and incubated for 2 h at 100 °C in a screw-cap test tube, with occasional vortexing. The mixture was then cooled on ice and centrifuged at 8000 g for 15 min. The supernatant was transferred to a new tube, mixed with an equal volume of water, and lyophilized overnight at −70 °C. The lyophilized sample was then washed twice with 400 μL of methanol, and centrifuged at 5000 rpm for 15 min. Finally, the insoluble lipid A was solubilized in 100 μL chloroform-methanol-water mixture (3:1.5:0.25, v/v/v). The lipid A structure was analyzed using MALDI-TOF mass spectrometry in the negative-ion mode^{8 (link),24 (link)}. All MALDI-TOF analyses were performed on a Bruker Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics, Coventry, UK) using the FlexControl 3.0 acquisition software. The matrix used for lipid A analysis was 2,5-dihydroxybenzoic acid (DHB; Sigma Chemical Co., St. Louis, MO, USA). The DHB solution (10 mg/mL) was prepared using a mixture of water and acetonitrile (1:4, vol/vol). All lipid A samples were premixed with the DHB solution (1:1, vol/vol) before MALDI measurements, and 1 μL of the resulting mixture was spotted on the MALDI metallic target. Lipid A from E. coli F583 was used as an external standard and mass calibrant.