LX Reference Saito 2009

Materials and Methods

Lipid extracts were analyzed as described previously (48, 49). Briefly, aliquots were diluted 100-fold with chloroform/methanol/2-propanol 1:2:4 (vol/vol/vol) containing 3.5 mM ammonium acetate, vortexed thoroughly, and centrifuged for 2 min at 13,000 × g on a Minispin centrifuge (Eppendorf). Analyses were performed on an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source, NanoMate HD (Advion BioSciences). Mass spectrometry survey scans were acquired using the Orbitrap analyzer operated under the target mass resolution of 100,000 (full width at half-maximum defined at m/z 400) with automatic gain control set to 5.0 × 105 as target value. Lipids were identified by in-house-developed LipidXplorer software, relying on their accurately determined m/z (error <3.5 ppm), which met specific lipid class sum composition constraints.