External TOF calibration was performed with red phosphorous which was spotted next to the sections. TIMS was external calibrated in electrospray ionization (ESI) mode using Tune Mix (Agilent, Santa Clara, USA). Single MS/MS spectra were acquired directly from tissue for some selected precursor ions at 50 µm laser pitch. For that, the mass range was set from m/z 50-1300 Da using a TIMS ramp from 1/K0 0.5 to 1.7 V•s/cm 2 with 200 ms ramp time. 600 laser shots were acquired per pixel in negative ion mode. Quadrupole isolation windows and collision energies were adapted for each precursor individually. If required, spectra from several pixel were accumulated, and collision energies were stepped from lower to higher voltages.