On-gel fluorescent visualization and the site identification of S-nitrosylated proteins

Abstract

Mounting evidence indicates that S-nitrosylation of critical cysteine residues in a protein represents a common feature of protein function regulation and cell signaling. However, the progress in studying the exact role of S-nitrosylation has been hampered by a lack of rapid and accurate methods for the detection of these S-nitrosylated proteins and the exact modification sites. In this article, we report a fluorescence-based method in which the S-nitrosylated cysteines are converted into 7-amino-4-methylcoumarin-3-acetic acid (AMCA) fluorophore-labeled cysteines—termed the AMCA switch method. The labeled proteins are then analyzed by nonreducing SDS–PAGE, and the S-nitrosylated proteins can be readily detected as brilliant blue bands after the activation of ultraviolet light. Furthermore, the sites of modification can be determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after in-gel tryptic digestion of the fluorescent band, and the recognizable AMCA tag in the MS spectra ensures the accurate site identification of the nitrosocysteines. Therefore, our method offers some apparent advantages by direct visualization of on-gel image of S-nitrosylated proteins, shorter experiment time by skipping the anti-biotin immunoblotting step, and elimination of the potential interference of endogenous biotinylated proteins. Based on this method, we detected the S-nitrosylation and the modified site in bovine serum albumin and gankyrin after in vitro S-nitrosylation. Overall, our results indicate that the AMCA switch method is a fast and accurate method to identify the S-nitrosylated protein and the modification sites.