Peptide separation was achieved using the 3100 OFFGEL Fractionator (Agilent Technologies) with a 24-well setup. Immobilized pH gradient (IPG) gel strips (24 cm; GE Healthcare), with a 3 to 10 linear pH gradient, were rehydrated for 15 min. Before fractionation, TMT10plex groups were dried down and resuspended in OFFGEL stock solution. Next, 150 μl of resuspended TMT10plex peptide sample was loaded into each compartment of the OFFGEL system. Peptide samples were focused until 50 kVh had accumulated (~48 hours). Liquid fractions were collected into separate Eppendorf tubes (primary recovery). To extract larger peptides retained within the IPG strips, 150 μl of ddH20/ACN/formic acid (49:50:1) was added to each compartment and incubated for a further 30 min with occasional pipette mixing. The supernatant (secondary recovery) was retrieved and added to the primary recovery. Each fraction collection was purified by a SOLA horseradish peroxidase solid-phase extraction cartridge (10 mg/1 ml; Thermo Fisher Scientific) before evaporation by centrifugation under a vacuum.

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