Lipid Extraction and Mass Spectrometric Analysis

Lipids were extracted from membrane fractions with chloroform/methanol/HCl (50:100:1.5, vol/vol/vol), as described by Bligh and Dyer 1959. Using a gentle stream of nitrogen, the organic phase was evaporated and the dried lipids were redissolved in chloroform/methanol (1:2, vol/vol). Phosphate determination was performed as described (Rouser et al. 1970), except that the assay volume was reduced by a factor of four. Glassware was used throughout the procedures.
MS was done on a QII triple quadrupole instrument (Micromass) equipped with a nano-ESI source. Nitrogen was used as a drying gas. The source temperature was set to 30°C. A capillary voltage of ± 600–800 V was applied, depending on the ion mode. Argon was used as collision gas at a nominal pressure of 3 × 10⁻³ mbar. Resolution of Q1 and Q3 was set to achieve isotope resolution. A collision energy of 30 eV was used for PC and SM detection in positive PREC mode, selecting a fragment ion of mass/charge (m/z) 184 (phosphocholine ion). Detection of cholesterol as cholesterol sulfate was done in negative PREC mode, selecting for a fragment ion of m/z 97 (sulfate ion) at a collision energy of 62 eV (Sandhoff et al. 1999). The mass range m/z scanned was 600–1,000 for PC and SM detection and 450–480 for cholesterol sulfate detection. For each quantitative measurement, 100 (PC and SM) or 50 (cholesterol) consecutive scans of 4-s duration were averaged.
PC and SM quantification was done as described (Brügger et al. 1997). 1,2-O-dilauroyl-sn-glycero-3-phosphocholine, 1,2-O-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-O-diarachidoyl-sn-glycero-3-phosphocholine, and 1,2-O-dibehenoyl-sn-glycero-3-phosphocholine, as well as N-myristoyl-SM, N-oleoyl-SM, and N-pentacosanoyl-SM were used as standards. In brief, PC and SM standards dissolved in chloroform/methanol (1:2, vol/vol) were added to the extraction solvent before lipid extraction of membrane fractions. Dried lipids were redissolved in chloroform/methanol (1:2, vol/vol). Ammonium acetate (100-mM stock solution in methanol) was added to a final concentration of 5 mM to acidify the solution. Before mass spectrometric analysis, samples were spun at 15,000 g_av for 5 min at 4°C in a microfuge (Eppendorf). Nano flow borosilicate glass tips of type D (Teer Coatings) were used. Instrument parameters were set as described above. For quantitative analysis, mass correction and isotope correction for [M+1], [M+2], and [M+3] isotopes were performed. Quantification of cholesterol was performed as described previously (Sandhoff et al. 1999).