Isolation and isotopic analysis of squalenoids:
Cell pellets were freeze-dried overnight, ground with a clean spatula, and extracted three times by sonication in a centrifuge tube filled with 50 mL of 3:1 dichloromethane:methanol (DCM:MeOH). All glassware was combusted overnight at 500°C to remove organics prior to use. After sonication, the extracts were spun in a centrifuge at 125 g for 15 min and the supernatant was decanted to a separate vial. All extracts were combined and the solvent was evaporated to dryness in a rotary evaporator. A maximum of 2 mL of 9:1 DCM:MeOH was added to dissolve the total extract that was then passed over Na2SO4 to remove water. The water-free extract was then separated into different fractions over SepraTM NH2 bulk-packing (P/N 1001711653 572122 – U) silica column by eluting with solvents of increasing polarity (F1 = 5 mL of hexane, F2 = 6 mL of 3:1 hexane:DCM, F3 = 7 mL of 9:1 DCM:acetone, F4 = 8 mL of 4% formic acid in DCM). The apolar fraction (F1) was dried under N2, then re-dissolved in 50 µL of hexane for identification.
The lipids in the apolar fraction were identified and quantified using an Agilent Technologies 5975 inert XL Mass Selective Detector after separation on an Agilent J&W GC HP-5MS UI capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, P/N 19091S – 433UIE) using He as the carrier gas. Samples were injected in pulse splitless mode. The GC oven was from an initial temperature of 70°C, then heated to 150°C at 15°C per min, then to 300°C at 5°C per min. Peaks were quantified by comparison to a 5-point standard curve of a C7-C30 alkane series (P/N 49451 – U, Sigma Aldrich). The isotopic composition of biomarkers in the apolar fraction was determined on a Thermo Scientific Gas Chromatograph-IsolinkII-Isotope Ratio Mass Spectrometer (GC-IsolinkII-IRMS) equipped with an Agilent DB-5 fused silica column (30 m × 0.25 mm i.d., 0.25 µm film thickness) with He as the carrier gas.
Isolation, characterization, and isotopic analysis of amino acids:
Pelleted cells were hydrolyzed with 6 M HCl (Ultrapure grade) with 1% of 11 mM ascorbic acid under N2 at 110°C for 20 h (Henrichs, 1991). After cooling, hydrolyzed amino acids were spiked with internal standard norvaline and derivatized with acidified isopropanol and acetyl chloride for 1 h at 110°C (Silfer et al., 1991). The samples then reacted at 110°C for 1 h on a hot plate. They were then esterified with trifluoroacetic anhydride (TFAA) for 10 min at 110°C for 10 min. The resulting derivatives were dissolved in dichloromethane. The isotopic signatures of derivatized amino acids were determined by GC-IsolinkII-IRMS