Proteomic approach for identification of protein S-nitrosation in mouse gastric mucosa treated with S-nitrosoglutathione

Nitric oxide, endogenously generated and exogenously supplied to the stomach, plays an important role in gastric physiological and pathological processes, including epithelial secretion, barrier function, immune response, and carcinogenesis. One of the mechanisms by which NO and related nitroso-compounds transmit their signals is S-nitrosation-mediated protein modification. To screen and identify gastric mucosal proteins that are uniquely sensitive to S-nitrosation, we reacted mouse gastric mucosal lysates with S-nitrosoglutathione, a physiologically relevant nitrosating agent, then performed proteomic analysis including the biotin-switch assay. The results showed that more than sixty protein spots on 2-DE were detected, and thirty-two of these were identified by MALDI-TOF MS and PMF. Eight of these identified proteins were unique S-nitrosated proteins not previously reported. Using Western blot technique, we further confirmed S-nitrosation especially in four proteins such as carbonic anhydrase-2, nucleoside diphosphate kinase B, Raf kinase inhibitor protein, and galectin-2, all known to be closely related to gastric mucosal protection and integrity, cell migration, and tumor metastasis. In addition, ex vivo study closer to in vivo situation also demonstrated these four proteins significantly S-nitrosated with S-nitrosoglutathione in mouse gastric mucosa. These findings will provide useful information regarding the linkage of protein S-nitrosation to gastric physiology and pathophysiology.