Lipidomic Analysis by High-Resolution Mass Spectrometry

Both high-resolution (R=100 000 at m/z 400) and low-energy CID tandem mass spectrometry experiments were conducted on a Thermo Scientific (San Jose, CA) LTQ Orbitrap Velos mass spectrometer (MS) with Xcalibur operating system. Lipid extracts in chloroform/methanol (2/1) were infused (1.5 μl/min) to the ESI source, where the skimmer was set at ground potential, the electrospray needle was set at 4.0 kV and temperature of the heated capillary was 300 °C. The automatic gain control of the ion trap was set to 5 × 10⁴, with a maximum injection time of 50 ms. Helium was used as the buffer and collision gas at a pressure of 1 × 10⁻³ mbar (0.75 mTorr). The MSⁿ experiments were carried out with an optimized relative collision energy ranging from 25 to 45% and with an activation q value at 0.25, and the activation time at 10 ms to leave a minimal residual abundance of precursor ion (around 20%). The mass selection window for the precursor ions was set at 1 Da wide to admit the monoisotopic ion to the ion-trap for collision-induced dissociation (CID) for unit resolution detection in the ion-trap or high resolution accurate mass detection in the Orbitrap mass analyzer. Mass spectra were accumulated in the profile mode, typically for 3–10 min for MSⁿ spectra (n = 2, 3, 4).