*2.3. n-UPLC-MS/MS*

Protein concentrations of AH samples were determined by a dye-binding method based on the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA) (Table 1), and samples were further diluted in 1× phosphate-buffered saline (PBS) to a final concentration of 0.1 μg/μL. Samples were prepared as per the SMART digestion kit protocol from ThermoFisher Scientific (Waltham, MA, USA) and cleaned up using solid-phase extraction (SPE) plates from ThermoFisher. The resulting peptides collected from the filters were dried in a vacuum centrifuge and stored at −80 ◦C. Then, 50 μL of diluted AH samples was resuspended in 0.1% formic acid and analyzed by n-UPLC-MS/MS. Tryptic peptides were loaded into an LTQ-Orbitrap mass spectrometer with a nanoelectrospray ionization source (Thermo Electron, MA, USA) connected to a nanoACQUITY UPLC system (Waters, MA, USA). Peptide samples were separated on a 25 cm × 75 μm BEH130 C18 column (Waters) with a 0–95% segmented gradient of 3–40% B for 168 min, 40–95% B for 2 min, and 95% B for 10 min at a flow rate of 0.5 μL/min. Mobile phase A was 0.1% formic acid in water, while mobile phase B was 0.1% formic acid in acetonitrile. The mass spectrometer was set to the data-dependent acquisition method (isolation width: 1.5 Da). As per the data-dependent acquisition method, the first ten most intensively charged peptide ions were selected and fragmented using a collision-induced dissociation (CID) method (Figure 1).

**Table 1.** Demographic characteristics of enrolled patients with a single risk factor, those with Double risk factors, and cataract controls.

