MALDI Imaging for Lipid and Metabolite Annotation

Additional MALDI imaging experiments were performed to aid in lipid and metabolite annotation [34 (link),47 (link)] using a SolariX XR 7T-ESI/MALDI-Fourier-transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam II 2 kHz laser. All imaging experiments were performed at a lateral resolution of 100 µm, using a total of 100 laser shots per pixel with a small laser spot size. Data were collected in positive ionization mode across the m/z range of 300–1000 using 2 million data points, while negative MS spectra were collected over the m/z range of 100–850 using 1 million data points. Ion transmission voltage parameters were set as follows—funnel RF amplitude 120.0 Vpp, RF amplitude TOF 350.0 Vpp, TOF 0.6 ms, and RF frequency transfer optic 4 MHz. All the methods were externally calibrated through the electrospray ion source and NaTFA clusters. Internal calibration was applied using different lock masses—the [PC (16:0/18:1) + H]⁺ (m/z 760.58508), the [PC (16:0/18:1) + Na]⁺ (m/z 782.56702), and [PC (16:0/18:1) + K]⁺ (m/z 798.54096) were used for the MALDI-positive FT-ICR MSI analysis, and the 9-AA cluster ion (m/z 193.0771) for the MALDI-negative FT-ICR MSI experiments.