The mouse lenses were lysed by vortexing for 2 minutes with glass beads (0.5, 1, and 3 mm in diameter) in 8 M urea and 50 mM triethylammonium bicarbonate (TEABC). Protein lysates were centrifuged at 16,000g at 4°C to exclude cell debris (pelleting at the bottom), and protein concentration was estimated using a bicinchoninic acid (BCA) assay. A total of 100 μg each sample was reduced with 10 mM dithiothreitol at room temperature for 1 hour and alkylated with 30 mM iodoacetamide for 20 minutes in the dark. The protein samples were digested overnight at 37°C using sequencing-grade trypsin (1:50) followed by desalting and labeling with 10-plex TMT reagents according to the manufacturer's instructions (Thermo Fisher Scientific, Rockford, IL, USA), and the first six channels (126, 127N, 127C, 128N, 128C, and 129N) were used for labeling. The labeling reaction was performed for 1 hour at room temperature, followed by quenching with 100 mM Tris-HCl (pH 8.0). The digested and labeled peptides from all six time points were pooled and desalted with C18 Sep-Pak (Waters Corporation, Milford, MA, USA).
The peptides were fractionated by basic pH reversed-phase liquid chromatography (bRPLC) into 96 fractions, followed by concatenation into 24 fractions by combining every 24th fraction. Agilent (Santa Clara, CA, USA) 1260 offline LC system was used for bRPLC fractionation, which includes a binary pump, variable wavelength detector, an autosampler, and an automatic fraction collector. Lyophilized samples were reconstituted in solvent A (10 mM triethylammonium bicarbonate, pH 8.5) and loaded onto XBridge C18, 5-μm 250 × 4.6-mm column (Waters Corporation). Peptides were resolved using a gradient of 3% to 50% solvent B (10 mM triethylammonium bicarbonate in 90% acetonitrile, pH 8.5) at a flow rate of 1 mL/min over 50 minutes, collecting 96 fractions. Subsequently, the fractions were concatenated into 24 fractions followed by vacuum drying using SpeedVac (Thermo Fisher Scientific, San Jose, CA, USA). The dried peptides were suspended in 15 μL of 0.1% formic acid, and all 15 μL was injected.