Shotgun Lipidomics

Lipids from primary hepatocytes were extracted by a modified protocol of Folch (Folch et al., 1957) and analyzed by shotgun mass spectrometry as described previously (Schuhmann et al., 2012). Briefly, hepatocytes (an amount equivalent to 10 μg of total protein) were dissolved in 200 μl ammonium bicarbonate solution (150 mM). For the subsequent quantification 10 μl internal standard mixture were added (20 pmol TAG 12:0-12:0-12:0, 20 pmol DAG 17:0- 17:0, 40 pmol diethyl PC 18:0-18:0, 50 pmol diethyl PE 20:0-20-0, 10 pmol PG 17:0-17-0, 40 pmol PS 12:0-12:0, 50 pmol PI 16:0-16-0, 40 pmol LPC 12:0, 40 pmol LPE 14:0, 30 pmol SM d18:1-12:0, 90 pmol CE 12:0, 20 pmol Cer d18:1-12:0, 50 pmol cholesterol d7; Avanti Polar Lipids, Inc., Alabaster, AL, United States). Then, 265 μl of methanol and 730 μl of chloroform were added and the mixture was vortexed for 1 h at 4°C. The lower organic phase was collected, dried in a vacuum centrifuge and the lipid extracts were re-dissolved in 120 μl chloroform:methanol [1:2 (v/v)] mixture. The analysis was performed in both, negative, and positive ion mode. For negative mode analyses, 10 μl extract were mixed with either 12 μl of 13 mM ammonium acetate in isopropanol or 0.1% (v/v) triethylamine in methanol. For positive mode analyses, 10 μl extract were mixed with 90 μl of 6.5 mM ammonium acetate in isopropanol before infusion. The analyses were performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific, Germany) equipped with a robotic nanoflow ion source TriVersa NanoMate (Advion BioSciences, Ithaca, NY, United States). High resolution (140,000 at m/z 200) FT-MS spectra were acquired for 1 min within the range of m/z 420–1000 in negative and 450–1000 in positive mode. Cholesterol was quantified as previously described (Liebisch et al., 2006). Briefly, 30 μl of extract were dried under vacuum, then 75 μl acetyl chloride:chloroform [1:2 (v/v)] were added, incubated for 1 h at room temperature, dried under vacuum and re-dissolved in 60 μl chloroform:methanol [1:2 (v/v)]. 10 μl extract were mixed with 90 μl of 6.5 mM ammonium acetate in propanol before infusion and analyzed in positive ion mode. The following lipid classes were identified and quantified using the LipidXplorer software (Herzog et al., 2011): tri- and diacylglycerides (TAG/DAG), cholesteryl esters (CE), sphingomyelins (SM), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), and cholesterol. The concentrations are plotted in pmol lipid per μg total protein as average of the biological replicates (n = 3) ± standard deviation.