Metabolic labeling of choline PL with deuterium-labeled choline and quantification of lipid molecular species by LC-MS/MS

After culturing the cells for 1 h in choline-deficient DMEM with 10% FBS, 1 mM d9-choline chloride (trimethyl-d9) (Cambridge Isotope Laboratories, Andover, MA) was added to the medium. Brefeldin A (BFA) treatment was performed at a concentration of 20 μg/ml for 2 h as described (16). After culturing for the time indicated, the cells were washed with PBS twice and lysed in distilled water. Lipid was extracted with methanol containing 0.1 nmol of internal standards (d70-PC; Olbracht Serdary Research Laboratories, Toronto, Canada). To eliminate plasmenyl-PC, lipid samples were treated with 0.15 N HCl at room temperature for 2 h as described (18). Choline PL molecular species were analyzed by reverse-phase HPLC using an L-column 2 ODS column (3 μm, 2.0 × 100 mm) (Chemicals Evaluation and Research Institute, Tokyo, Japan) coupled to a 5500 QTRAP mass spectrometer (Sciex, Inc., Framingham, MA). A binary gradient consisting of solvent A (acetonitrile:methanol:water, 1:1:3, v/v/v, containing 5 mM ammonium acetate) and solvent B (2-propanol containing 5 mM ammonium acetate) was used. The gradient profile was 0–1 min, 95% A; 1–9 min, 5–95% B linear gradient; and 9–13 min, 95% B. The flow rate was 0.2 ml/min, and the column temperature was 40°C. Endogenous choline PL was detected in multiple reaction monitoring mode by selecting the m/z of the PL molecular species at Q1 and the precursor ion of m/z 184 at Q3 in positive ion mode. For deuterium-labeled choline PL synthesized via the CDP-choline pathway and PE methylation pathway, the precursor ion of m/z 193 (d9-labeled choline) and 187 (d3-labeled choline) at Q3 was monitored, respectively. Lipids were quantified using MultiQuant version 2.0 (Sciex) and normalized against the internal standards and amount of protein.