Comprehensive Lipidomics Profiling with UHPLC-Orbitrap

For both lipidomics analyses, a Dionex Ultimate 3000 RS UHLPC system (Thermo Scientific, San Jose, CA) was employed. Ionization was performed with heated electrospray ionization probe (HESI II) and mass spectra acquired using a Q-Exactive Orbitrap (Thermo Scientific). Source parameters for lipidomics, in positive and negative polarity are provide in Table S-3. Samples were maintained at 4ºC in the autosampler. 2 μL of sample was injected onto a Waters Acquity BEH C18 column (50 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA) maintained at 30ºC. For negative ion mode, 5 μL of sample was injected onto the column and analyzed with the same mass spectral parameters (Table S-2). A gradient ramp (Table S-1) was employed consisting of mobile phase C (60:40 acetonitrile:water, volume fraction) and mobile phase D (90:8:2 isopropanol:acetonitrile:water, volume fraction), both with 10 mmol/L ammonium formate and 0.1% formic acid.
Mass spectra were acquired in full scan mode using data-dependent top 5 analysis (ddMS²-top5) in both positive and negative polarity with a mass resolution of 70,000. Full scan and ddMS²-top5 scan parameters are shown in Table S-2. Before each analysis, the instrument was externally calibrated and at least 3 blanks were analyzed. Internal mass calibrants (lock masses) were used in positive ion mode and consisted of diisooctyl phthalate (m/z 391.2842) and polysiloxanes (m/z 371.1012 and 445.1200). No stable lock mass was observed to be used in negative ion mode. To compare iterative exclusion (IE) with traditional ddMS²-top5 for lipidomics, a minimum of 4 sequential injections were analyzed by ddMS²-top5 with IE and 4 without IE, for both negative and positive polarity analysis of Red Cross and substantia nigra lipid extracts. For excluding ions previously selected for fragmentation and placed on an exclusion list, a 10 ppm exclusion tolerance was used.