Lipidomic raw data of dietary phosphatidylcholine counteracts giant lipid droplet formation in peroxisome-defective Caenorhabditis elegans

From the lipid-rich upper layer, 300 µL of supernatants were aspirated and nitrogen-dried. Preceding the LC-MS analysis, a solvent mixture totaling 150 µL (a mixture of 13.3 µL CH2Cl2 and 6.7 µL MeOH as the solubilizing agent, and 130 µL of a diluent formulated with ACN, IPA, and H2O in the 65:30:5 proportions containing 5mM ammonium acetate) was used to resuspend each sample, followed by 30 seconds of vortexing.

For each sample, 70 µL of the supernatant was subjected to LC-MS analysis. Additionally, a pooled aliquot of supernatant from every sample was combined to form the quality control (QC) samples.(2) Lipidomic data acquisitionLipidomic data were acquired by the Vanquish Flex UPLC system coupled with Thermo Scientific Orbitrap Exploris 240 system equipped with a heated electrospray ionization (H-ESI) source. Samples were separated through a BEH C8 column (2.1 × 100 mm with 1.7 μm particle size, Waters, Milford, MA, USA) with column temperature maintained at 55°C and mobile phases consisting of 2 mM ammonium formate in mobile phase A (40% water and 60% acetonitrile) and mobile phase B (90% isopropanol and 10% acetonitrile).

The gradient started with 90 seconds of isocratic elution with 32% of phase B and 68% of phase A. Then the proportion of phase B was gradually raised to 85% over the next 15.5 minutes, rapidly raised from 85% to 97% within 6 seconds, and maintained at 97% for 144 seconds. Next, the proportion of phase B was rapidly reduced to 32% within 6 seconds and maintained for 5 minutes for column post-equilibration. The flow rate is 0.26 mL/minute.

The injection volume was 2 µL for the positive ions and 5 µL for the negative ions. Mass spectrometer was operated in positive or negative modes using a full scan/data-dependent secondary scan (Full-ddMS2) in the scan range m/z 100–1500 Da. Capillary voltage of 3400 Volts for positive and 3000 Volts for negative.

Ion Transfer Tube Temperature is 320 °C. Vaporizer Temperature is 350 °C. Sheath gas is 40 arb.; auxillary gas is 10 arb.; sweep gas is 10 arb.. Orbitrap resolution was set to 120000 in MS1 and 15000 in MS2, with normalized collision energy type. The internal Mass Calibration remains on the whole time.

The MS data were captured and processed through Xcalibur software (Thermo Fisher Scientific Ltd, San Jose, USA) and LipidSearch5.1 software (Thermo Fisher Scientific Ltd, San Jose, USA).