Clean up thermo pierce c18 spin columns

The resultant proteins were precipitated by adding five volumes of cold acetone and incubated overnight at −80 °C. They were then equilibrated at room temperature for 10 min, centrifuged at 16,000× g for 15 min at 4 °C, and the supernatant was discarded. The resulting pellet was washed three times with cold 80% acetone. Subsequently, the protein pellet was allowed to dry in a rotary concentrator. The samples were resuspended in 30 μL of 8 M urea and 25 mM of ammonium bicarbonate. Then, they were reduced with DTT to a final concentration of 20 mM in 25 mM of ammonium bicarbonate and incubated for 1 h at room temperature. Next, they were alkylated by adding Iodoacetamide to a final concentration of 20 mM in 25 mM of ammonium bicarbonate and incubated for 1 h in the dark at room temperature. Subsequently, the samples were diluted 8 times with 25 mM of ammonium bicarbonate. Digestion was performed with trypsin sequencing grade (#V5071, Promega, Madison, WI, USA) in a 1:50 protease/protein ratio (mass/mass) and incubated for 16 h at 37 °C, the digestion reaction was stopped by adding 10% formic acid. The samples were then subjected to Clean Up Thermo Pierce C18 Spin Columns (cat cod 89870, Thermo Scientific, Waltham, MA, USA), according to the supplier’s instructions. Subsequently, the cleaned peptides were dried in a rotary concentrator at 1000 rpm overnight at 10 °C.

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)].