Whole cell lysate protein digestion
Cells were washed with PBS once, typrsinised, neutralised with complete DMEM, pelleted, and lysed (6M Guanidine [Thermo]/50 mM HEPES [Sigma] pH 8.5). Lysates were sonicated for 2.5 min at constant 4°C cooling with Bioruptor Pico (Diagenode), and cell debris was removed by centrifugation at 21,000 xg for 10 min at 4°C. To reduce protein, dithiothreitol (DTT, Sigma) was added and samples were incubated for 20 min at room temperature. Cysteines were alkylated with 14 mM iodoacetamide (IAA, Sigma), incubated 20 min at room temperature in the dark, and excess IAA was quenched with DTT for 15 min. Guanidine concentration was lowered to 1.5M with 200 mM HEPES (pH 8.5), then protein samples were digested with LysC protease (Wako) at a 1:100 protease-to-protein ratio for 3 h at room temperature. Guanidine concentration was further lowered to 0.5M and Trypsin (Thermo) was then added at a 1:100 protease-to-protein ratio followed by overnight incubation at 37 °C with constant shaking. Trypsin was quenched with 5% formic acid. Samples were then centrifuged at 21,000 xg for 10 min at 4°C to remove undigested protein. Peptides were subjected to octadecyl carbon chain (C18) solid-phase extraction (SPE, Sep-Pak, Waters) and dried with a speed-vac (Thermo).