LC-MS/MS analysis of SMALP extracted membrane lipids. (a) TIC of SMALP extracted E. coli BL21(DE3) membrane lipids on the Luna® C8(2) column under a THF/MeOH/H2O gradient. Dotted lines (red) indicate the change in gradient as solvent B percentage and typical elution time windows for the three membrane phospholipids of E. coli (PG, PE, and CL) are indicated with dotted lines (black). (b–d) Combined negative ion mass spectra across these regions showing the variety of PG (b), PE (c), and CL (d) phospholipids. The phospholipids are labelled as C:n, whereby C = number of carbons in the fatty acyl chains; n = double bond equivalents. Theoretical masses for the lipids can be found in Supplementary Table S2. (e) Product ion (MS/MS) scan in negative ion mode of PE 34:1 (m/z 716) showing the fragmentation of this species to yield different fatty acyl chains (insert, m/z 250–290). (f) The potential structures of molecular species of PE(34:1), of which 16:0_18:1 was the most abundant species.