**2.9.1 Tryptic digestion**

The protein spots were excised from the gel with a syringe and transferred to small eppendorf tubes (0.5 mL). Proteins from fluorescently stained gels were visualized and excised on a blue light transluminator (DR-180 B from Clara Chemical Research, Denver, CO, USA) wearing darkened amber glasses. After destaining and dehydration, about 25 µL trypsin (20 mg/mL in 25 mM ammonium bicarbonate, Promega, Madison, WI, USA) was added to each gel piece. To minimize autocatalytic activity, the samples were kept on ice for 30 min, prior to incubation in 37º C over night. The supernatant was transferred to a separate tube and the peptides were further extracted from the gel piece by incubation in 50% ACN/5% trifluoroacetic acid (TFA, Sigma-Aldrich) for 5 h at room temperature. The supernatant from the two steps was then pooled and dried in SpeedVac until complete dryness. If not dissolved in 5 µL 0.1% TFA for further MS preparation, the proteins were stored at -70ºC.
