**2.2. 2DGE-based western blot detection of nitroproteins**

2DGE: The precast IPG strips (pH 3–10 NL; 180 × 3 × 0.5 mm) and 18-cm IPGstrip holder was used for 2DGE first dimension—isoelectic focusing (IEF) on an IPGphor instrument (GE Heathcare) to separate protein sample. After IEF, the IPG strip was processed for 2DGE second dimension—sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), namely, the equilibrated proteins in the IPG strips were separated by molecular weight during electrophoresis on in 12% PAGE resolving gel (250 × 215 × 1.0 mm), and visualized with silver-staining [9, 36] or Coomassie brilliant blue G staining [35].

2DGE-based Western blotting: The 2DGE-separated proteins were transferred to a PVDF membrane, which were blocked by BSA, incubated with an anti-human nitrotyrosine antibody and secondary antibody, then visualized with 1-Step™ nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Thermo Product No. 3404).

Image analysis of a 2D gel and of 2D-Western blotting: The scanned images of the 2D gels and the Western blot membranes were input to a PDQuest analysis system (Bio-Rad, version 7.1, Hercules, CA) to synthesize image. Gaussian spots were applied to all subsequent spotmatching and analyses. Each spot volume was normalized to the total optical density (OD) to minimize experimental factors on a spot volume [30].

### **2.3. MS/MS characterization of tryptic nitropeptides from nitroproteins**

LC-ESI-MS/MS: The nitrotyrosine-positive gel-spots were excised, digested, purified, eluted, air-dried, redissolved. Then, the peptide mixture was subjected to LC-ESI-quadrupole-time of flight (LC-ESI-qTOF) or LTQ-OrbiTrap Velos MS/MS analyses. The detailed operation process and parameter settings have been described [35].

MALDI-MS/MS: The immunopositive 2D gel spots were excised, digested, purified, eluted, airdried, redissolved, and were spots onto MALDI-plate, which were subjected to MALDI-MS/ MS analysis [37]. The detailed procedure has been described [9, 11, 36].
