Determination of Archaeal Lipids by Mass Spectrometry.

Lipids of P. furiosus were extracted from the cells according to a methyl tert-butyl ether (MTBE) protocol (58). The lipid extract was injected on a Waters BEH C8 100 × 1-mm 1.7-μm high performance liquid chromatography (HPLC) column used with an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Solvent A was water with 1% ammonium acetate and 0.1% formic acid, and solvent B was acetonitrile/2-propanol 5:2 with 1% ammonium acetate and 0.1% formic acid. Gradient elution started at 50% mobile phase B, rising to 100% B over 40 min; 100% B was held for 10 min, and the column was reequilibrated with 50% B for 8 min before the next injection. The flow rate was 150 μL⋅min⁻¹.

Data acquisition was performed according to a previously reported protocol (59) by means of an Orbitrap mass spectrometer (LTQ-Orbitrap; Thermo Fisher Scientific) full scan in preview mode at a resolution of 100,000 and <2 ppm mass accuracy with external calibration. The spray voltage was set to 4,500 V, and the capillary temperature was at 300 °C. From the Fourier transform mass spectrometry preview scan, the 10 most abundant m/z values were picked in data-dependent acquisition mode, fragmented in the linear ion trap analyzer, and ejected at nominal mass resolution. Normalized collision energy was set to 50%, the repeat count was 2, and the exclusion duration was set at 10 s. Data analysis was performed using Lipid Data Analyzer, a custom-developed software tool (60, 61).