Targeted Lipid Analysis via MRM-MS

Targeted analysis of lipid species was performed with Thermo Xcalibur 4.0 QF2 (ThermoFisher Scientific) using Multiple Reaction Monitoring (MRM) Mass Spectometry (MS)^{20 (link)}. A Triversa NanoMate (Advion, Ithaca, NY, USA) was used to infuse samples onto a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific, Waltham, USA). MRM-MS was used to identify and quantify lipid species as previously described^{48 (link)}. Data were converted and quantified relative to standard curves of internal standards that were incorporated to the samples before extraction. At least three independent biological replicates were analyzed, each of which was measured twice with three measurement cycles, leading to six technical replicates. Concentration values of lipid species were normalized to the total amount of inorganic phosphate as determined by colorimetric quantification with ammonium molybdate and ascorbic acid of pentachloroacetic acid hydrolyzed total lipid aliquots. To analyze the significance of the fold difference of each lipid species between two conditions, we performed an paired, two-tailed student’s t-test.