Lipid droplets buffer hydrogen sulfide-induced redox imbalance

Summary of Study:
Lipidomic analysis was performed on HT-29 cells cultured under 2% O2 conditions ± 100 ppm H2S for 24 h. The goal was to assess how lipids were impacted with H2S treatment. The experimental design included treating HT-29 cells (approximately 200,000/well) with and without 100 ppm H2S for 24 hours. The treatment media was EBSS, 5% FBS and p/s. There were four biological replicates for the control and treatment cohorts. Total lipid extracts from cell pellets were prepared using a modified MTBE lipid extraction protocol. Total lipid extracts were analyzed by liquid chromatography coupled to targeted tandem mass spectrometry (LC-MS/MS). The LC-MS/MS analyses were performed on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). LC-MS/MS methodology was adapted from the literature (2). The separation was achieved using an ACQUITY Amide BEH column (1.7 µm; 2.1 x 100 mm) column (Waters, Milford, MA) maintained at 45 °C. Mobile phase compositions for solvents A and B consisted of ACN/H2O (95:5, v/v) and (50:50, v/v) respectively, with 10 mM ammonium acetate The gradient profile had a flow rate of 0.6 mL min−1 and ramped from 0.1 to 20% B in 2 min, from 20 to 80% B in 3 min, dropped from 80 to 0.1% B in 0.1 min, and held 0.1% B for 2.9 min. Total chromatographic run time was 8.0 min. The injection volume was 2 μL. The auto-sampler was maintained at 7 °C. Electrospray ionization was achieved using either negative or positive mode. Mass spectrometry detection was done using selective reaction monitoring where predetermined precursor to product ion transitions were used. ESI source parameters were set as follows: voltage 3500 V in positive mode and −2500 V in negative mode, sheath gas (Arb) = 60, aux gas (Arb) = 15, sweep gas (Arb) = 1 and ion transfer tube temperature of 380 °C. Nitrogen was used as the nebulizer and argon as collision gas (1.5 mTor). The vaporizer temperature was set to 350 °C. Collision energies and RF lens voltage were optimized for each lipid class reference standard. LC-MS/MS data was acquired using Thermo’s Xcalibur software and data processing was achieved using Xcalibur 4.2 and TraceFinder 5.1. Additional data analysis was done using Prism 6 (GraphPad, La Jolla, CA) and MetaboAnalyst.