In-gel Tryptic Digestion of Rat Brain Proteome

In-gel digestion was determined according to the method of Losuwannarak et al. [41 (link)]. To reduce the disulfide bonds of total protein isolated from rat brains, dithiothreitol (10 mM) in ammonium bicarbonate (10 mM) was added. Reformation of disulfide bonds in proteins was blocked by alkylation with iodoacetamide (30 mM) in ammonium bicarbonate (10 mM). Protein samples were digested with sequencing-grade porcine trypsin (ratio of 1:20; Promega, Mannheim, Germany) and incubated overnight at 37 °C. Tryptic peptides were dried using a speed vacuum concentrator (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in 0.1% formic acid for nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS).