Tandem mass tag (TMT) labeling and fractionation

For TMT labeling, the peptide samples of the 4 groups were separately labeled using 4 labeling reagents of the 6-plex label sets (Thermo Scientific, USA). Each sample containing 30 μg protein was resuspended in 50 μl TEAB buffer containing 60% acetonitrile (ACN) and mixed with 20 μl of the corresponding TMT reagent for 2 h at 26 °C. The labeling reactions were then quenched by adding 5% hydroxylamine for an additional 30 min incubation. Four labeled peptide samples were mixed in equal amounts and dried in a centrifugal evaporator.

The mixed TMT-labeled peptides were resuspended in 110 μl of mobile phase A (20 mM ammonium formate buffer, 3% ACN, pH 10.0) and separated using a 1.7 μm × 2.1 mm × 100 mm BEH C18 column (Waters, USA) in a Dionex Ultimate 3000 RSLC system with a 60 min gradient starting from 4% mobile phase B (ACN) to 64% B at a flow rate of 0.25 ml·min^-1. Eluted fractions were pooled into 10 peptide samples over the gradient at 1 min intervals and were dried to completion. The peptide samples were reconstituted in 0.1% formic acid for the subsequent LC-MS/MS analysis.