Offline High pH Reversed-Phase Fractionation
TMT-labelled peptides were fractionated using an Ultimate 3000 rapid separation (RS) nano UHPLC system (Thermo), generating 12 combined fractions. The system is equipped with a Kinetex Evo C18 column (Phenomenex) that has 2.1 mm in internal diameter (ID) and is 25 cm in length, filled with C18 bound silica particles with diameter of 1.7 μm. Mobile phase was HPLC grade H2O, acetonitrile, and ammonium formate (pH 10). The concentration of ammonium formate was maintained at 20 mM while concentration of acetonitrile gradually increased throughout the fractionation elution programme. Starting from 2.7% (v/v), acetonitrile concentration increased to 21% in the first 10 min, to 36% after 24 min 15 sec of elution, then to 51% after 33 min of elution. Acetonitrile concentration was subsequently increased and maintained at 90% for 10 min to wash the column. The flow rate was 400 ml/min and the elution was performed at 45°C. UV absorbance was monitored at 280 nm and fractions were collected into 96 well microplates using the integrated fraction collector. Fractions were recombined orthogonally in a checkerboard fashion, combining alternate wells from each column of the plate into a single fraction, and commencing combination of adjacent fractions in alternating rows. Wells were excluded prior to the start or after the cessation of elution of peptide-rich fractions, as identified from the UV trace. This resulted into two sets of 12 combined fractions, which were dried in a vacuum centrifuge and resuspended in 10 ml solvent (4% acetonitrile and 5% formic acid) prior to LC-MS3.