On-beads and in-gel protein digestion

Beads with extracted proteins were washed 3× by 50 mM ammonium bicarbonate buffer and subjected to digestion by trypsin (2 h, 37 °C; sequencing grade, Promega). Tryptic peptides were extracted into LC-MS vials by 2.5% formic acid (FA) in 50% ACN with addition of polyethylene glycol (20,000; final concentration 0.001%) and concentrated in a SpeedVac concentrator (Thermo Fisher Scientific). 1D gel lines for sample and negative control were divided into 10 gel areas separating gel areas with high and low abundant proteins. Individual gel areas were excised manually and after destaining and washing procedures each band was incubated with 125 ng trypsin (sequencing grade; Promega). The digestion was performed for 2 h at 40 °C on a Thermomixer (750 rpm; Eppendorf). Tryptic peptides were extracted as mentioned above.