Lipid Extraction and Mass Spectrometric Analysis

Lipid extraction was performed according to the procedure of Bligh and Dyer³⁷ in the presence of non-naturally occurring internal lipid standards. Stable isotope-labelled and unlabelled PL species were quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a Quattro Ultima™ Triple Quadruple mass spectrometer (Micromass, Wilmslow, UK) as described previously³⁸. Briefly, a precursor ion scan of m/z 184 was used for phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) detection. [D9]-Choline-labelled lipids were analysed using a precursor ion scan of m/z 193. A neutral loss scan of 141 was used for phosphatidylethanolamine (PE) detection. [D4]-Ethanolamine-labelled lipids were analysed using a neutral loss scan of 145. Fragment ions of m/z 364, 390 and 392 were used for detecting phosphatidylethanolamine-based plasmalogens (PE P) PE P-16:0, PE P-18:1 and PE P-18:0. The isotopic overlap of lipid species was corrected and data analysis was performed using self-programmed Excel macros³⁹. Lipid species were annotated according to a standard methodology for reporting lipid species identified by mass spectrometry⁴⁰. Glycerophospholipid annotation is based on the assumption of even numbered carbon chains only. SM species annotation is based on the assumption that a sphingoid base with two hydroxyl groups is present. The quantitative values were normalized with respect to the cellular protein content and are expressed as nmol/mg or pmol/mg protein. Only PL species with concentrations higher than 1% of the corresponding PL class, and more than three times higher than the internal standard blank, were taken into account.