Figure 1

Experimental design and TMT labelling workflow of the experiment. Human retinal and vitreous tissues were extracted from postmortem eyes of control (age: control 64.5 ± 10, n = 10) and glaucoma subjects (age: 71.5 ± 8.5, n = 10). Extracted proteins form 40 samples subjected to reduction, alkylation and subsequent digestion with Trypsin and Lys-C. Extracted peptides were quantified and labeled in a 10 plex TMT reaction. Four TMT experiments were carried out to accommodate all the biological replicates. Briefly, 2 sets of 5 control and 5 glaucoma replicates of retina tissue were used in TMT 1 and 2 experiments, and the same design was used for vitreous samples in TMT3 and TMT4 experiments. Labelled samples within each TMT experiment were pooled together, then were fractionated by basic, reversed-phase isocratic step elution using reverse phase spin columns, and analyzed by LC-ESI-MS/MS on ThermoFisher Orbitrap Fusion mass spectrometer (SPS-MS3 method). Functional pathway and protein network data analysis was performed using Ingenuity and Reactome pathway analysis.