Methods and Protocols

Cell Extraction and Reconstitution

Phospholipids were extracted using a modified Bligh and Dyer procedure (25). Pellets containing 3 x 10⁶ cells were extracted with 800 mL of 0.1 N HCl: MeOH (1:1) and 400 mL CHCl₃. The samples were vortexed (1 min) and centrifuged (5 min, 18,000 g). The lower phase was then isolated and evaporated (Labconco CentriVap Concentrator, Kansas City, MO), followed by reconstitution with 80 mL MeOH: CHCl₃ (9:1). Prior to analysis, 1 mL of NH₄OH was added to each sample to ensure protonation. Lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL).

Mass Spectrometry Analysis of Phospholipid Cell Extracts

Mass spectral analysis was performed on a Finnigan TSQ Quantum triple quadrupole mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a Harvard Apparatus syringe pump and an electrospray source. Samples were analyzed at an infusion rate of 10 mL/min in both positive and negative modes over the range of m/z 400 to 1200. Instrument parameters were optimized with 1, 2-dioctanoyl-sn-glycero-3-phosphoethanolamine (16:0 PE). Data were collected with the Xcalibur software package (ThermoFinnigan) and analyzed by a software program developed in our research group.