3.3. Monophasic Lipid Extraction and Derivatisation of Aminophospholipids and Plasmalogen-Containing Lipids

From the E. coli culture, 50 mL of EGP culture was transferred to a 50 mL centrifuge tube (Falcon) and spun at 5000× g for 10 min at 4 °C. The supernatant was discarded, and the cells were washed with 40 mL of PBS and pelleted again using the previously mentioned centrifugation conditions. The cell pellet was then washed and pelleted twice more, resuspended in 10 mL PBS and separated into five 1 mL technical replicate aliquots in 1.5 mL microcentrifuge tubes (Eppendorf). The five replicates were pelleted at 8000× g for 5 min at 4 °C, after which the supernatant was removed, then snap-frozen in liquid nitrogen and freeze-dried. Eleven millilitres of SGP culture was diluted to 50 mL to ensure an equal number of cells was harvested for both the EGP and SGP samples, then the cells were washed and pelleted as above. The protein content for each sample replicate was measured with a Pierce™ BCA Protein Assay kit (Thermo Fisher) with the EGP replicates having a mean protein content of 478.4 ± 43.2 µg and the SGP replicates having a mean protein content of 487.4 ± 26.7 µg.

Lipids were subjected to monophasic extraction as reported previously [42,43]. To the freeze-dried cells, a 100 µL scoop of 0.15 mm zirconium oxide beads was added with 200 µL of 60% v/v aqueous MeOH containing 0.01% w/v BHT and 50 µL of the internal lipid standard mix in CHCl₃. The samples were homogenised using a Bullet Blender storm 24 (Next Advance) on Speed 8 for 30 s, which was repeated twice, with the sample being cooled on ice between each homogenisation run. After homogenisation, 120 µL of H₂O, 250 µL of CHCl₃ containing 0.01% w/v BHT and 420 µL of MeOH containing 0.01% w/v BHT was added to the homogenate solution to achieve a 0.74:1:2 H₂O:CHCl₃:MeOH extraction solution. The samples were then vortexed for 1 min and shaken at 1400 rpm for 30 min in a Thermomixer compact mixer (Eppendorf). The samples were pelleted at 10,000 g for 15 min, and the supernatant was transferred to a 2 mL microcentrifuge tube (Eppendorf). The remaining pellet was re-extracted, for which 100 µL of H₂O and 400 µL of 1:2 CHCl₃:MeOH containing 0.01% w/v BHT was added to the pellet, which was homogenised again and then pelleted by centrifugation. The supernatant from the re-extraction was then combined with the supernatant from the first extraction. Both the remaining pellets and the combined supernatant lipid extracts were dried using a miVac centrifugal concentrator (Genevac) until completely dry. Once dry, the supernatants were reconstituted in 500 µL of IPA:MeOH:CHCl₃ (4:2:1) containing 0.01% w/v BHT. For analysis of underivatised samples, 10 µL of each sample was aliquoted onto a 96-well twin.tec PCR plate (Eppendorf), dried and then reconstituted in 40 µL of IPA:MeOH:CHCl₃ (4:2:1) containing 20 mM ammonium formate.

Functional group selective derivatisation of aminophospholipids and plasmalogen lipids to resolve potential isomeric mass overlaps was performed as previously described [43,44,45]. To derivatise the aminophospholipids, a 10 µL aliquot of each sample was aliquoted into a 96-well round-bottom Multi-Chem plate (Whatman) and dried using centrifugal vacuum evaporation for 15 min. The dried samples were reconstituted in 40 µL of CHCl₃ containing 60 µM TEA and 67 µM ¹³C₁-DMBNHS, and the plate was sealed and incubated for 30 min at room temperature on an orbital shaker at 150 rpm. The plate was then unsealed and dried using centrifugal vacuum evaporation. To derivatise the plasmalogens, 40 µL of ice-cold CHCl₃:MeOH (2:1) containing 177 µM I₂ and 667 µM ammonium carbonate was added to each sample then incubated on ice for 5 min. The samples were dried again, washed three times with 40 µL of 10 mM aqueous ammonium carbonate before being dried again, then reconstituted for lipidome analysis in 40 µL of IPA:MeOH:CHCl₃ (4:2:1) containing 20 mM ammonium formate.