Nanoelute column

AN (25 μL, 5 μM) exposed to ONOOH as described above (section 2.3) was mixed with 75 μL 8 M urea (in 100 mM Tris, pH 8.0) and 1 μL LysC (20 ng μL⁻¹; Promega) then incubated at 21 °C for 4 h. Subsequently 150 μL 100 mM Tris (pH 8.0), 500 μL H₂O and 2 μL GluC (20 ng μL⁻¹; Promega) were added, and the samples incubated overnight at 21 °C. Following solid phase extraction with Stage-tips, samples were analyzed on a Bruker Impact II ESI-QTOF (Bruker Daltonics) mass spectrometer in the positive ion mode with a Captivespray ion source connected on-line to a Dionex Ultimate 3000RSnano chromatography systems (Thermo Fisher Scientific). Peptides were separated on a Bruker Nanoelute column (15 cm × 75 μm ID) with a solvent gradient over 65 min, using acetonitrile with 0.1% formic acid at a flow rate of 300 nL min⁻¹. The MS scan range was 150–1750 m/z with a cycle time of 2 s using MS and MS/MS sampling rates of 2 Hz. Database searches were performed with MaxQuant v1.6.1.0 using the following parameters: enzyme: GluC and LysC, with three missed cleavages; variable modification: Tyr and Trp nitration (+44.985 Da) and dinitration (89.97 Da); mass tolerance: 0.07 and 0.005 Da (first and main searches, respectively); MS/MS mass tolerance: 40 ppm (first and main searches).