Protein Extraction and Digestion Protocol

The sample preparation was performed as previously reported [16 (link),33 (link)]. At first, samples were thawed, briefly centrifuged and dried in a vacuum-centrifuge (Concentrator plus, Eppendorf AG, Hamburg, Germany). Afterwards, samples are refilled with formic acid (FA) (VWR International GmbH, Darmstadt, Germany) and incubated in FA for 20 min at room temperature. After incubation, samples were sonicated in an ice-cooled sonication bath (USC300TH, VWR International GmbH, Darmstadt, Germany) for five minutes and vacuum-dried. After that, samples were refilled with ammonium bicarbonate (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), reduced with dithiothreitol for 30 min at 56 °C (AppliChem GmbH, Darmstadt, Germany), and acetylated with iodoacetamide (Merck KGaA, Darmstadt, Germany) at room temperature in the dark. Trypsin (SERVA Electrophoresis GmBH, Heidelberg, Germany), diluted in ammonium bicarbonate to 0.1 µg/µL per 1,000,000 µm² tissue, was then added to the samples according to the collected tissue area and digestion was performed at 37 °C overnight. The digestion was stopped after ~16 h by adding trifluoroacetic acid (Merck KGaA, Darmstadt, Germany). Next, samples were vacuum-dried, and peptides were stored in 20 µL of 0.1% TFA at −80 °C.