Trypsin digestion
Proteins in the samples were trypsin-digested with the aid of Waters Corporation (Milford, MA, USA) 0.2% surfactant RapiGest™ SF. Samples were boiled for 10 min, and proteins were reduced by adding 0.5 M 1,4-dithiothreitol to a final concentration of 5 mM, vortexed and incubated for 30 min in 60°C water bath. In the next step, the proteins were alkylated with a 15 mM final concentration of iodoacetamide and incubated for 30 min in the dark at room temperature. Sequence grade trypsin Promega Biotech AB (Madison, WI, USA) Trypsin Gold (Cat No V5280) was added to the samples in a 1:100 enzyme-to-protein ratio. Samples were incubated at 37°C overnight. Samples were acidified to a final concentration of 0.5% trifluoroacetic acid and incubated for 45 min at 37°C, after which cleaved RapiGest™ SF was removed by a 10-min centrifugation at 16 000 