**2.5 2D-gel electrophoresis**

IEF (first dimension) was carried out on non-linear wide-range immobilized pH gradients (pH 4-7; 7 cm long IPG strips; GE Healthcare, Uppsala, Sweden) and achieved using the EttanTM IPGphorTM system (GE Healthcare, Uppsala, Sweden). Analytical-run IPG-strips were rehydrated with 125μg� of total proteins in 125μl of rehydratation buffer (urea 8 M, CHAPS 2%, 0,5% (v/v) IPG Buffer, bromophenol blue 0,002%) for 12 h at 20°C. The strips were then focused according to the following electrical conditions at 20°C: 500 V for 30 min, from 1000 V for 30 min, 5000 V until a total of 15000 Vt was reached. After focusing IPG strips were equilibrated for 15 min in 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.05 M Tris-HCl, pH 6.8, 2% (wt/vol) DTT, and subsequently for 15 min in the same urea/SDS/Tris buffer solution but substituting the 2% (wt/vol) DTT with 2.5% (wt/vol) iodoacetamide. The second dimension was carried out on 12% polyacrylamide gels at 25 mA/gel constant current until the dye front reached the bottom of the gel. MS gel was stained with colloidal comassie.
