Protein Extraction, Reduction, and Tryptic Digestion

Approximately 1 mg of total soluble protein extract was reduced with 5 mM dithiothreitol for 2 h at 37°C, cooled and then proteins were alkylated with 14 mM iodoacetamide for 30 min at room temperature in the dark. Unreacted iodoacetamide was quenched by increasing dithiothreitol concentration to 10 mM with a further incubation for 15 min at room temperature in the dark. Proteins were diluted to 1.5 M urea with 50 mM triethylammonium bicarbonate (TEAB) buffer (Sigma-Aldrich) and incubated at 50:1 ratio with sequencing-grade modified trypsin (Promega, Madison, WI, USA) overnight at 37°C with gentle agitation. Protein digestion was stopped by acidification of the mixture to pH 2.0 with trifluoroacetic acid. Acidification serves to precipitate lipids that would interfere with downstream purification and importantly to prepare the samples for desalting which requires peptide mixture to be acidic (Hsu et al., 2009 (link)). Peptides were then purified using Sep-Pak Vac tC18 100 mg cartridge (Waters, Milford, MA, USA), as described previously (Groen et al., 2013 (link)), and completely dried in a Speed Vac concentrator (Thermo Scientific, Bremen, Germany).