Protein Purification and Oxidation

The culture supernatants containing the secreted proteins were centrifugated (1 h, 30,000 × g, 10°C) and sterilized by filtration (0.22 µm; Express Plus; Merck Millipore). The supernatants were equilibrated to pH 7 with 0.5 mM NaOH, and the proteins were purified using two ion exchange chromatographies (Fig. S1): first, an anion exchange on DEAE Sephadex A-25 (Cytiva) and a cation exchange on CM Sephadex A-25 (Cytiva) for proteins not retained on the anion exchanger. Both resins were initially equilibrated with 20 mM sodium phosphate buffer (pH 7). Elution of the secreted proteins was performed with 1 M NaCl. Fractions from both anion and cation exchangers were pooled, desalted on Sephadex G-25 (PD-10 Desalting Columns, Cytiva) in 50 mM sodium acetate (pH 5.2), and concentrated using Vivaspin polyethersulfone membrane (3 kDa cutoff, Sartorius). Protein concentrations were determined using the Bradford method (Bio-Rad), and the secretomes were used immediately. Secreted proteins (50 µg) were incubated 30 min with 1 mM hydroxylamine, desalted on Sephadex G-25 (PD SpinTrap, Cytiva), then incubated with 100 mM of H₂¹⁸O₂ (Sigma-Aldrich) for 1 h in the dark, then desalted. Initial concentration of commercial H₂¹⁸O₂ was estimated to be ∼450 mM using FOX method (51 ). Protein concentrations were determined, and the samples were stored at −20°C.