2.5. Trypsin digestion

The isolated PLM were subjected to enzyme digestion following a previously described protocol, with minor deviations.11 In brief, all PLM preparations were diluted in microsomal storage buffer to a working solution of 7 µg PLM/µl. In Eppendorf LoBind tubes, 10 µl of diluted PLM was added to 10 µl of H₂O and 5 µl of IS‐peptides (200 fmol/µl 1A2, 200 fmol/µl 2D25, 600 fmol/µl 2E1, and 200 fmol/µl 3A29 dissolved in 0.1 M AmBic with 20% acetonitrile (ACN) added according to the manufacturer's instructions). For denaturation, 5 µl of a 12% sodium deoxycholate (SDC) solution was added to the mix and incubated at 80°C for 10 min with vigorous rotational shaking. The denatured mix was cooled to 60°C, and 5 µl of 35 mM TCEP was added, followed by incubation for 20 min at 60°C. The reduced proteins were cooled to room temperature and alkylated by incubation with 5 µl of 80 mM MMTS for 20 min. The solution was diluted with 70 µl of 25 mM AmBic, and 10 µl of trypsin mix (0.033 µg/µl trypsin prepared in 25 mM AmBic with 2 mM CaCl₂, 1:30 w/w trypsin:protein) was added. The digestion proceeded for 4 h at 37°C, after which the reaction was stopped, and SDC was precipitated by the addition of formic acid (FA) to a final concentration of 0.3% FA. The solution was vortexed vigorously followed by centrifugation at 15,000g for 2 min, after which the supernatant was moved to a glass vial and analyzed by LC–MS/MS.