The pellets were washed and resuspended in urea digestion buffer (6 M urea, 2 M thiourea, in 100 mM Tris, pH 8). The proteins were reduced, alkylated, then digested with 0.2 µg of Lys-C (3 h, room temperature) followed by 0.2 µg of trypsin (overnight, room temperature, ...