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).