Experimental spectra were obtained on a LTQ linear ion trap mass spectrometer, a hybrid LTQ-FT-ICR mass spectrometer (Thermo Fisher Scientific) and a 6530 QTOF mass spectrometer (Agilent). All lipid standards were obtained from Sigma/Aldrich and Avanti Polar Lipids. The infusion of lipid standards and extracted lipid samples was performed using a chip based nano-electrospray infusion (Advion Nanomate). Plasma lipids were extracted using methyl-tert-butyl ether (MTBE)24 (link). In brief, methanol (225 μL) was added to 30 μL blood plasma and shaken with an additional 750 μL of methyl-tert-butyl ether solvent. Phase separation of this extract was induced by adding 187.5 μL of water, vortexing and centrifuging the mixture at 14,000 g for 2 min. The upper organic phase was collected and dried in a vacuum centrifuge. After adding 10 μL of 100 mM ammonium acetate to 90 μL of the supernatant, lipid extracts were infused into the mass spectrometers using an Advion Nanomate chip-based infusion system (nanoESI). Ion trap mass spectra were collected in low resolution mode (1,500 resolving power) on the linear ion trap. The data collection method performed a full scan and a data dependent MS/MS scan of the most abundant ions. Different CID voltages in the range from 0V to 100V were used for evaluation of spectra. For abundance calculations standard spectra were scanned in low-resolution mode with 15V, 20V, 25V, 35V, 45V and 55V CID voltage to obtain specific MS/MS fragmentations. All spectra were recorded with the Thermo Xcalibur software. An infusion time of 30 seconds was set up in full scan mode with 0V CID with an additional 30 seconds of data dependent MS/MS scans to obtain tandem mass spectra for the largest peaks. For each sample, around 50 MS/MS scans were averaged. NIST SRM 1950 blood plasma samples were infused for around 10 minutes to allow the acquisition of a higher number of MS/MS scans.
The 6530 QTOF mass spectrometer for measurement of reference compounds was operated with the following parameters. An Agilent JetStream electrospray source was used in infusion mode at a flow rate of 0.25 ml/min for acquiring QTOF MS and MS/MS spectra. Data were collected with a 0.25 s scan rate in both profile and centroid modes, and mass calibration was maintained by constant infusion of reference ions at 121.0509 and 922.0098 m/z. MS/MS data was generated utilizing data-dependent MS/MS triggering with dynamic exclusion. Precursor ions, with a minimum 1 k signal intensity were isolated with a 4 m/z isolation width (medium setting), and a variable collision energy was applied based on precursor ion m/z (10 eV + 0.03 eV × ion m/z). Data were exported into the open exchange format mzXML. Samples were measured in negative and positive mode. For lipid profiling with liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-MS/MS) we used settings from an external reference25 (link), except we choose a scan rate of 4-8 spectra per scan event and collision energies ranging from 20-40eV.