Lipid Profiling by High-Resolution LC-MS

Lipid samples were analyzed at the Harvard Center for Mass Spectrometry. The LC–MS analyses were modified from Miraldi^{81 (link)} and were performed on an Orbitrap Exactive plus (Thermo Scientific) in line with an Ultimate 3000 LC (Thermo Scientific). Each sample was analyzed in positive and negative modes, in top 5 automatic data-dependent MSMS mode. Column hardware consisted of a Biobond C4 column (4.6 × 50 mm, 5 μm, Dikma Technologies). Flow rate was set 100 μl min⁻¹ for 5 min with 0% mobile phase B (MB), then switch to 400 μl min⁻¹ for 50 min, with a linear gradient of MB from 20 to 100%. The column was then washed at 500 μl min⁻¹ for 8 min at 100% MB before being reequilibrated for 7 min at 0% MB and 500 μl min⁻¹. For positive mode runs, buffers consisted for mobile phase A (MA) of 5 mM ammonium formate, 0.1% formic acid and 5% methanol in water, and for mobile phase B (MB) of 5 mM ammonium formate, 0.1% formic acid, 5% water, 35% methanol in isopropanol. For negative runs, buffers consisted for MA of 0.03% ammonium hydroxide, 5% methanol in water, and for MB of 0.03% ammonium hydroxide, 5% water, 35% methanol in isopropanol. Lipids were identified and quantified using the Lipidsearch© software (version 4.2.27, Mitsui Knowledge Industry, University of Tokyo). Integrations and peak quality were curated manually before exporting and analyzing the data in Microsoft Excel.