MALDI-MSI and MALDI-FTICR Imaging Protocol

MALDI-MSI of the TMAs in this study was acquired with a rapifleX MALDI TOF instrument (Bruker Daltonik) in reflectron positive ion mode in the mass range of m/z 700–3000 Da. The pixel size was set to 50 μm, and 300 spectra were averaged per pixel with a laser frequency of 5 KHz. We also acquired high mass resolution MALDI–Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging data of a representative TMA (case #3, see Table 1) using a custom-designed 21T FTICR instrument (48 (link)) build around Velos Pro linear ion trap (Thermo Fisher Scientific) with MALDI imaging source (Spectroglyph) (49 (link)), which included MS/MS data-dependent acquisition (DDA). Detailed acquisition protocols are described in Supplemental Methods, Section 6.