Axima confidence maldi tof ms

Purified lipid A samples were dissolved in a small amount (30–100 μL) of 2:1 chloroform/methanol. A sample of this solution was mixed in situ on a MALDI target slide with 20 mg/mL 6-aza-2-thiothymine in 9:9:2 water/acetonitrile/10% ammonium citrate in ratios ranging from 4:1 to 1:4, with a maximum total load volume of 2 μL. Spectral data were collected by Shimadzu Axima Confidence MALDI-TOF MS in the negative or positive linear mode (pulsed extraction 2000 Da) as the average of up to 2000 shots at moderate power (see individual figure legends for details). Shimadzu MALDI-MS software was used to perform the following processing of the raw data: (1) Calibration of all spectra to mode-specific standard datasets built from spectral data for wild-type E. coli hexa-acyl bis-phosphate lipid A (Figure 1) and E. coli tetra-acyl bis-phosphate (data not shown); (2) Averaging of all profiles collected for each sample; (3) Processing with a baseline filter width of 100 channels, threshold-apex peak detection, and an averaged peak smoothing width of 30–100 channels to resolve molecular weights from the clusters of individual isotopic masses.

Purified lipid A samples were dissolved in a small amount (30–100 μL) of 2:1 chloroform/methanol. A sample of this solution was mixed in situ on a MALDI target slide with 20 mg/mL 6-aza-2-thiothymine in 9:9:2 water/acetonitrile/10% ammonium citrate in ratios ranging from 4:1 to 1:4, with a maximum total load volume of 2 μL. Spectral data were collected by Shimadzu Axima Confidence MALDI-TOF MS in the negative or positive linear mode (pulsed extraction 2000 Da) as the average of up to 2000 shots at moderate power (75–90 arbitrary units; see the individual figure legends for details) calibrated with standard mass controls (ProteoMass, Sigma-Aldrich, St. Louis, MO, USA). Shimadzu MALDI-MS software was used to perform the following processing: (1) fine mass calibration to mode-specific standard datasets built from spectral data for E. coli W3110 lipid A (per the manufacturer instructions); (2) averaging of all profiles collected for each sample; and (3) processing using manufacturer-recommended settings for display of averaged molecular weight peaks from data collected with isotopic resolution: baseline filter width of 100 channels, threshold-apex peak detection and an averaged peak smoothing width of 50–100 channels.