Sequencing grade modified trypsin trypsin gold

For each of the nuclear (N) and cytoplasmic (C) samples, 60 μg of total protein was separately resolved by 10% SDS-PAGE under reducing conditions. Proteins were visualized by silver staining with a ProteoSilver Plus kit (Sigma-Aldrich, Poole, U.K.).^{39 (link)} At least 30 horizontal bands were excised from each gel lane and processed on a 96-well Progest plate (Digilab, Huntingdon, U.K.). Gel bands were processed with the Progest Investigator (Digilab) using established protocols for reduction and alkylation.^{40 (link)} Finally, gel plugs were rehydrated in 20 μg/mL sequencing-grade modified-trypsin (trypsin-gold) (Promega, Southampton, U.K.) that was prepared by adding (1:50 v/v) trypsin/25 mM ammonium bicarbonate and incubated overnight at 37 °C. Fifty microliters of 0.1% formic acid was added to stop the tryptic digestion, the extracted tryptic peptides were collected in siliconized Eppendorf tubes (Sigma-Aldrich, Poole, U.K.) and vacuum-dried to ~20 μL, the volume was adjusted to ~30 μL with 0.1% formic acid, and the sample was analyzed by Orbitrap LC–MS/MS.