Lipid analysis by LCMS2

Bligh and Dyer lipid extracts (200 µL) were dried under vacuum and mixed with an internal standard mix (50 µL) (CDN Isotopes, France and Cayman, France), (14:0)4CL (800 ng), (12:0)2DG (8 µg), and (21:0)2PC (50 ng). Sample (1 µL) was analyzed by LCMSMS using a Zorbax®Eclipse Plus C18 1.8 µm, 2.1 × 100 mm column maintained at 55 °C (Agilent Technologies).

Briefly, digalactosyldiacylglycerols (DGDG), monogalactosyldiacylglycerols (MGDG), and diacylglycerols (DG) were separated on a 1260 Infinity LC system (Agilent Technologies) with a gradient of mobile phase A (acetonitrile/water/1 M ammonium formate (60/39/1 v/v/v) with 0.1% formic acid), and mobile phase B (isopropanol/acetonitrile/1 M ammonium formate (90/9/1 v/v/v) with 0.1% formic acid)⁵⁶ at a flow rate of 0.4 mLmL/min set as follows: 1 min hold at 50% B; 50–60% B in 4 min; 60–85% B in 10 min; 85–99% B in 1 min; 2 min hold at 99%, 99%-50% ramp-down in 0.1 min and maintained at 50% B for 3.9 min. Acquisition was carried out on a 6460 Triple Quadrupole (Agilent Technologies) equipped with an ESI Jet stream source (temperature 250 °C, nebulizer 20 L/min, sheath gas 11 L/min, sheath gas temperature 220 °C, capillary 3500 V, nozzle 1000 V) operating in positive Single Reaction Monitoring (SRM) mode (fragmentor 148 V, collision energy 23 V). Transitions were set as the neutral loss of 359 Da for DGDG or 197 Da for MGDG from their respective [M + NH4] + ions⁵⁷ DG (as NH4 + adducts) were quantified according to the sum of the signal resulting from the neutral loss of either their sn-1 or sn-2 fatty acid. Finally, lipid concentrations were determined by calculating their relative response to (12:0)2DG used as internal standard.

Phosphatidylglycerols (PG) and lysyl-phosphatidylglycerols (lysyl-PG) were separated with the same mobile phases and column as for DGs, and the elution gradient used was set as follows: 2 min hold at 50% B; 50–99% B in 14 min; 2 min hold at 99%, 99%-50% ramp-down in 0.1 min; return to initial conditions in 1.9 min. Acquisition was carried out on a 6460 Triple Quadrupole (Agilent Technologies) equipped with an ESI Jet stream source (temperature 200 °C, nebulizer 20 L/min, sheath gas 11 L/min, sheath gas temperature 220 °C, capillary 3500 V, nozzle 1000 V) operating in positive Single Reaction Monitoring (SRM) mode (fragmentor/collector 116 V/13 V and 300 V/34 V for PG and Lysyl-PG respectively). Transitions were set as the neutral loss of 189 Da or 300 Da for [PG + NH4] + and [Lysyl-PG + H] + ions respectively. Lipid concentrations were determined by calculating relative response ratios with regards to (12:0)2DG used as internal standard.

The analysis of cardiolipins (CL) and phosphatidylcholines (PC) was performed as previously described^58,59 except that a Vanquish LC system, coupled to a triple-stage quadrupole (TSQ) Altis mass spectrometer equipped with a heated electrospray ionization source (Thermo Scientific) was used (sheath gas, 50 arb; auxiliary gas, 10 arb; sweep gas, 1 arb; ion transfer tube temperature, 325 °C; vaporizer tempropylerature, 350 °C and ion spray voltage, 3500 V ( +), and 2500 V (-)). Lipid concentrations for all the quantified lipid classes were expressed in pmol/mg of Bligh and Dyer extract.