Vantage triple quadruple mass spectrometer

SR phospholipidomics were conducted as previously described [12 (link), 27 (link)]. Isolated SR fractions were reconstituted in 1.8 mL ddH₂O. Four mL of extraction buffer [2:2 (v/v) chloroform/methanol] was added in the presence of internal standards: 5 μg 14:0-PC ([M+Li]+ m/z 684.58) or 5 μg 14:0-PE ([M-H]- m/z 678.62). After vortex-mixing and centrifugation (800g), the organic (lower) layer was collected, concentrated to dryness under nitrogen, and reconstituted in 200 μl methanol. A 20 μl aliquot was removed, diluted with 180 μL methanol containing 0.6% LiCl, and analyzed by direct injection ESI-MS on a Thermo Vantage triple-quadruple mass spectrometer in positive mode with different neutral loss scans for the analysis of PC (neutral loss of 183) species. Another aliquot of 20 μl was diluted in 200 μl methanol and analyzed in negative mode for PE and phosphatidylserine (PS) species (product ions of 196 and 153). The relative intensity ratio of each individual species to its internal standard was calculated, and then read against a standard curve to obtain its absolute quantity. The final result was reported as nmol/mg protein.

After Bligh-Dyer extraction, organic phases were collected, dried under nitrogen, and reconstituted in 200 μl chloroform/methanol (1:1) with 0.5% sodium acetate. A 50 μl aliquot was directly injected into a Thermo Vantage triple-quadruple mass spectrometer in positive mode for the analysis of PC (including SM) species with neutral loss scan of 183, and for the analysis of PE species with neural loss scan of 141. Each individual species was compared to its internal standard, and absolute quantity was determined using a standard curve, all as described^{33 (link)}.