MALDI Imaging of Frozen Tissue

Fresh frozen tissue collection, matrix spraying, data acquisition and MALDI imaging were performed as described previously [17 (link)]. The matrix solution used was 20 mg/mL 5-dihydroxybenzoic acid (#149357-20G Sigma-Aldrich Inc, Missouri, USA) in 100% acetone and 0.1% trifluoroacetic acid (#302031 Sigma). The tissue was scanned with both MS and TIMS settings at a resolution of 20 μm. The MS settings were: scan range 20–2500 m/z in positive MS scan mode. The TIMS settings were: 1/K0 0–8 − 1.89 V×s/cm2, ramp time of 200 ms, acquisition time of 20 ms, duty cycle = −10%, and ramp rate of 4.85 Hz. Acquired raw data were initially processed with SCiLS lab 2021a (Bruker Scientific LLC, Billerica, MA, USA) and the file was exported to Metaboscape 2021b (Bruker, USA) for annotations and further downstream analysis.