Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
(a–c) LC-MS analysis of synthesized 16:0 LPA and DPPA lipids with or without liposome purification. Lipid DOPG present in the initial composition of the 400-nm vesicles was used as an internal standard to correct for the loss of lipids during purification. Lipid biosynthesis occurred in a one-pot IVTT and acyl transfer reaction starting from 500 μM G3P and 100 μM p-CoA substrates. In some samples SUV membranes were doped with a biotinylated lipid for immobilization of liposomes on streptavidin-coated magnetic beads. Inspection of the amounts of lipids detected for the different experimental conditions allowed us to discriminate between liposome-integrated and free DPPA. Data are mean and s.e.m. of three independent experiments. For each replicate the same sample was injected two times in the MS, their averaged value was calculated and data are reported as the mean and standard error across the three trials. (d) Calculation of the percentage of synthesized DPPA co-localizing with liposome membrane. The use of DOPG as an internal standard enabled the quantification of the fraction of non-immobilized or disrupted vesicles that were washed away during the purification step. Percentage values of recovered DPPA and DOPG were calculated as [counts(purif+|biotin+)–counts(purif+|biotin–)] / counts(purif–|biotin+) × 100. Then, the obtained value for DPPA was divided by that for DOPG to correct for the loss of lipids during purification (Figure C in S1 File), resulting in a value of 28% ± 14% as an estimation of synthesized DPPA that effectively localized in liposomes.