The TMT samples were fractionated using off-line high pH reverse phase chromatography: samples were loaded onto a 4.6 x 250 mm Xbridge™ BEH130 C18 column with 3.5 µm particles (Waters). Using a Dionex bioRS system, the samples were separated using a 25-minute multistep gradient of solvents A (10 mM formate at pH 9) and B (10 mM ammonium formate pH 9 in 80% acetonitrile), at a flow rate of 1 ml/min. Peptides were separated into 48 fractions, which were consolidated into 24 fractions. The fractions were subsequently dried and the peptides re-dissolved in 5% formic acid and analysed by LC-MS.
