Protein digestion was performed as already reported with few modifications [44 (link)]. Briefly, 400 µg of proteins for each sample were diluted with ammonium bicarbonate (NH4HCO3, Sigma-Aldrich, ≥99.0%, Darmstadt, Germany) buffer solution at 50 mM and with RapiGestTM SF Surfactant (Waters Corporation, Milford, MA, USA) at a final concentration of 0.1%. The proteins were reduced by adding DL-dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, MO, USA, ≥99.5%) at a final concentration of 40 mM and incubated for 45 min at 56 °C; carbamidomethylation reaction was carried out by 55 mM iodoacetamide (IAA) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min in the dark.
Trypsin (Trypsin from the porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA) was added to the sample for protein digestion at an enzyme/protein ratio of 1:100 and incubated overnight at 37 °C. The digestion was stopped by adding trifluoroacetic acid (TFA) (Honeywell, Seelze, Germany) at a final concentration of 0.5% to reach an acidic pH (<2). RapiGestTM SF surfactant removal was operated by incubating the samples for 30 min at 37 °C followed by centrifugation at 13,000× g rpm for 10 min. The supernatant which contained the peptides was finally collected and analysed.
Trypsin (Trypsin from the porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA) was added to the sample for protein digestion at an enzyme/protein ratio of 1:100 and incubated overnight at 37 °C. The digestion was stopped by adding trifluoroacetic acid (TFA) (Honeywell, Seelze, Germany) at a final concentration of 0.5% to reach an acidic pH (<2). RapiGestTM SF surfactant removal was operated by incubating the samples for 30 min at 37 °C followed by centrifugation at 13,000× g rpm for 10 min. The supernatant which contained the peptides was finally collected and analysed.
Free full text: Click here
