Tuneplus software

All MS experiments were performed on a MALDI-LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with 60 Hz 337 nm N₂ laser. Full scan mass resolution of 60,000 (at m/z 400), laser energy of 18 μJ and microscans of 4 were used for all analyses. MS/MS were performed in HCD mode with normalized collision energies of 45 and isolation window of 3 m/z (unless otherwise stated). Monoisotopic precursor selection was enabled. Different dynamic exclusion durations were tested and optimized. The multiplex MS imaging method was set up in Xcalibur software (Thermo Scientific, Bremen, Germany) and the imaging position file was defined in TunePlus software (Thermo Scientific, Bremen, Germany).

All MALDI-MSI experiments were performed on a MALDILTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a 60 Hz 337 nm N₂ laser. Full MS experiment was performed in positive-ion mode with m/z range of 450−1000, mass resolution of 30 000 (at m/z 400), using a laser energy of 20 μJ and 2 microscans/step. A spatial resolution of 75 μm was used for all acquisitions. The precursor ions of target lipids in the full MS scan were isolated and subsequently fragmented by higher-energy collisional dissociation (HCD) with the normalized collisional energy of 25. Instrumental methods were set up in Xcalibur software (Thermo Scientific, Bremen, Germany) and the imaging position file was defined with TunePlus software (Thermo Scientific, Bremen, Germany).