Enhanced Protein Digestion Protocol

The protein digestion was performed following a standard enhanced, filter-aided sample preparation protocol (FASP) [32 (link)]. Briefly, after washing, proteins were reduced and alkylated. On-filter digestion was performed with 1.2 µg Trypsin/Lys-C mix (Promega, Madison, USA) for 14 h at 37 °C. Digested peptides were recovered, dried, and redissolved in 0.1% aqueous trifluoroacetic acid. After desalting and sample cleanup by C18 ZipTips (Sigma-Aldrich), all samples were spiked with standardized indexed retention time reference peptides for facilitation of retention time alignment (iRT kit, Biognosys, Switzerland). For each liquid chromatography (LC)–mass spectrometer (MS) analysis, 3 µg of digested protein was loaded onto the LC column.