In-Gel Trypsin Digestion and LC-MS/MS Analysis

Following SDS–PAGE, entire gel lanes were sliced into 30 pieces per lane and subjected to in-gel digestion with trypsin as described7 (link). Peptide samples were analysed by liquid chromatography-tandem MS using a nanoACQUITY UltraPerformance liquid chromatography system (Waters) coupled online to an LTQ Velos (Thermo Fisher Scientific). Peptides were concentrated and desalted on a Symmetry C₁₈ preparative column (20 mm × 180 μm inner diameter, 5-μm particle size; Waters) and separated on a bridged ethyl hybrid C₁₈ analytical column (250 mm × 75 μm inner diameter, 1.7-μm particle size; Waters) using a 45-min linear gradient from 1 to 25% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nl min⁻¹. Peptides were selected for fragmentation automatically by data-dependent analysis.