Standard peptide calibration standard 2

As previously described, the Coomassie Blue-stained gel spots were washed and digested [12 (link)]. In the end, 0.8 μL from a mixture of tryptic peptides derived from each protein was spotted onto a MALDI target (384 MTP Anchorchip) (800 μm Anchorchip; Bruker Daltonics, Bremen, Germany). The spectra were collected with an UltraflexTerm time-of-flight (TOF) mass spectrometer outfitted with a LIFT-MS/MS device (Bruker Daltonics, Bremen, Germany) at reflector and detector voltages of 21 kV and 17 kV, respectively, as described previously [12 (link)]. The PMFs were calibrated against a standard peptide calibration standard II (Bruker Daltonics, Bremen, Germany). The PMFs were assessed using Flex Analysis software (version 2.4, Bruker Daltonics, Bremen, Germany). The MS data were interpreted using BioTools v3.2 (Bruker Daltonics, Bremen, Germany). The peptide masses were searched against the Mascot search algorithm (v2.0.04, updated on 9 May 2021; Matrix Science Ltd, London, UK). The identified proteins were screened for Mascot scores higher than 56 and p < 0.05.