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.