Sample preparation for mass spectrometry

Proteins were precipitated with trichloroacetic acid and incubated 10 min at 4°C. The protein pellet was washed twice with cold acetone and resuspended with 4 M urea. Then the samples were treated with 5 mM of tris(2-carboxyethyl)phosphine (TCEP) for 30 min at 37°C in order to reduce disulfide bonds. After incubation, iodoacetamide (1.8 mg/ml final) was added to the samples to irreversibly prevent the formation of disulfide bonds and incubated for 30 min at 25°C in the dark. The samples were subsequently diluted with 0.1 M ammonium bicarbonate to have a final concentration of urea of 1.6 M. For digestion, the proteins were incubated overnight at 37°C in presence of 1 μg of trypsin. After acidification with trifluoroacetic acid (TFA, 1% final), the peptides were loaded on a C-18 column (The Nest Group, SS18V) pre-equilibrated with buffer A (0.1% TFA). The column was washed 3 times with buffer C (5% acetonitrile / 95%water (v/v) and 0.1% TFA) and peptides were eluted with buffer B (50% acetonitrile / 50%water (v/v) and 0.1% TFA). The peptides were finally dried under vacuum and kept at—80°C. Before LC-MS/MS mass analysis, samples were resuspended in 0.1% formic acid by sonication.