Gel-based approach

40 μg of cytoplasmic proteins were separated by one dimensional SDS polyacrylamide gel electrophorese (1D SDS PAGE) according to Laemmli [42] with the following modifications: the loading buffer consisted of 3.75% (v/v) glycerol, 1.25% (v/v) ß-mercaptoethanol, 0.6% (w/v) SDS, 0.0014% (w/v) bromophenol blue, 16.5 mM Tris-HCl (pH 6.8). The separation gel contained 12% (w/v) acrylamide gel (with 0.32% bisacrylamide), 0.375 M Tris-HCL (pH 8.8), 0.255% (w/v) SDS, 0.062% (w/v) APS, and 0.062% (v/v) TEMED and the stacking gel 5% (w/v) acrylamide (with 0.13% (w/v) bisacrylamide), 0.125 M Tris-HCl (pH 6.8) 0.25% (w/v) SDS, 0.075% (w/v) APS, and 0.075% (v/v) TEMED.

Proteins were fixed with 40% (v/v) ethanol and 10% (v/v) acetic acid for one hour and subsequently stained with colloidal coomassie [43] for one hour. In-gel digestion using trypsin and extraction of the peptides were carried out as described by Lerch et al. [43] with an additional extraction step using acetonitrile.

For digestion with Lys-C, a buffer containing 25 mM TRIS/HCl and 1 mM EDTA (pH 8.5) was used. The applied enzyme concentration was 1/40 of the total protein concentration. Digestion of AspN was performed in 10 mM Tris-HCl (pH 8.0) with a final AspN concentration of 1/50 of the total protein concentration.