Ch3cn

Protein gel lanes were excised and cut into five pieces. Gel pieces were washed in pure H₂O followed by washes in NH₄HCO₃ (25 mM, Sigma-Aldrich) and CH₃CN (50%, Thermo Fisher Scientific). Further, gel pieces were dehydrated in CH₃CN (100%) before incubation (56°C, 45 min) in NH₄HCO₃ (25 mM) and DTT (10 mM, Sigma-Aldrich). Subsequently, samples were incubated (RT, 45 min) in NH₄HCO₃ (25 mM) containing iodoacetamide (55 mM, Sigma-Aldrich), followed by washing (RT, 5 min) in NH₄HCO₃ (25 mM) and CH₃CN (50%) and dehydration in CH₃CN (100%). Then, each piece was incubated (ice, 30 min) in 15 μl of trypsin (12.5 ng/ml, Thermo Fisher Scientific) and NH₄HCO₃ (50 mM). The excess of trypsin was removed and gel pieces were covered with NH₄HCO₃ (50 mM) and incubated over night at 37°C. The tryptic peptides were extracted the following day by incubating twice in HCOOH (5%, Thermo Fisher Scientific) and CH₃CN (50%) followed by incubation in CH₃CN (100%). Peptides from gel pieces that originated from the same sample were merged and completely dried in a speed vac. The dried peptides were resuspended in 40 μl of HCOOH (0.1%) prior to MS analysis and equal volumes (10 μl) were injected in the mass spectrometer for shotgun and targeted MS analysis.