Manually excised Coomassie stained gel spots were washed and digested according previously reported studies (20 (link), 21 (link)). The mixture of tryptic peptides (0.8 μL) derived from each protein was spotted onto a MALDI target (384 MTP Anchorchip; 800 μm Anchorchip; Bruker Daltonics, Bremen, Germany). MALDI-MS(/MS). As previously mentioned, spectra were obtained using an UltraflexTerm time-of-flight (TOF) mass spectrometer equipped with a LIFT-MS/MS device (Bruker Daltonics) at 21 kV reflector and 17 kV detector voltages, respectively (20 (link), 21 (link)). PMFs were calibrated against a standard (peptide calibration standard II, Bruker Daltonics). Flex Analysis software was used to assess the PMFs (Bruker Daltonics v.2.4). Interpretation of MS data was done using BioTools v3.2 (Bruker Daltonics). The peptide masses were searched against the Mascot search algorithm (v2.0.04, updated on 09/05/2021; Matrix Science Ltd., UK). Identified proteins were accepted as correct if they the Mascot score > 56. Because some proteins were in low abundance and did not give sufficiently powerful mass fingerprints, not all spots of interest could be recognized; some spots were mixtures of multiple proteins (30 (link), 31 (link)).
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