Tandem Mass Tag (TMT) labeling and cation exchange-based fractionation of peptides
The digested lysates were desalted using C18 Omix tips (catalog no A57003100, Agilent) and dried down in 50 μg portions. For each of the conditions, 50 μg of digested peptides were labeled with the TMT 10plex reagent (catalog no 90113, ThermoFisher). Dried pellets were resuspended in 50 μL of 1X phosphate buffered saline and to each tube, one aliquot of the respective TMT reagent, which was resuspended in 41 μL of acetonitrile, was added. The reaction was allowed to proceed at 21 °C for one hour and then quenched via the addition of 8 μL of 1 M ammonium bicarbonate. Samples were dried down and then desalted using Omix tips. The individual TMT labeled samples were pooled and fractionated using strong cation exchange chromatography on an AKTA Pure 10 (GE Healthcare) equipped with a Luna 5 μm 100 angstrom 150 x 2.1 mm strong cation exchange (SCX) column (catalog no 00F-4398-B0, Phenomenex). Buffer A was 5 mM KH2PO4 in 30% acetonitrile (catalog no 34998, Sigma Aldrich), p H 2.7. Buffer B was 350 mM KCl (catalog no PX1405 EM Science) in buffer A. A 200 μL/min gradient was run from 0% B to 50% B over 10 ml, then up to 100% B over 1 ml. A total of 12 fractions were collected during the peptide elution portion of the gradient.