Protein Precipitation, Reduction, and Trypsin Digestion

The remaining proteins were precipitated overnight at −80 °C with 5 volumes of cold acetone. After this step, the tubes were acclimated at room temperature for 10 min and centrifuged at 16,000× g for 5 min at 4 °C. The supernatant was discarded, and the pellet was washed 3 times with cold 80% acetone. The samples were subsequently left to dry in a rotatory concentrator. Next, the samples were resuspended in 30 uL of 8 M urea and 25 mM ammonium bicarbonate and then reduced with 20 mM DTT in 25 mM ammonium bicarbonate for 1 h at room temperature. Proteins were then alkylated with iodoacetamide at a final concentration of 20 mM in 25 mM ammonium bicarbonate for 1 h at room temperature and in darkness. After this, the samples were diluted 8 times with 25 mM ammonium bicarbonate to dilute the alkylating agent. Digestion was carried out with sequencing-grade trypsin (#V5071, Promega, Madison, WI, USA) in a 1:50 protease/protein (mass/mass) proportion at 37 °C for 16 h. After this time, 10% formic acid was added to stop the reaction (by a pH change). The samples were then washed in “Clean Up” Thermo Pierce C18 Spin Columns (#89870, ThermoFisher, Waltham, MA, USA) following the provider’s instructions. Finally, the peptides were dried in a rotatory concentrator overnight at 1000 rpm and 10 °C [22 (link)].