A proteomic approach for the analysis of S-nitrosylated proteins using a fluorescence labeling technique

摘要

S -nitrosylation, a post-translational modification of the thiol group of cysteine residues by nitric oxide (NO), has emerged as a new mode of signal transduction and regulation of protein function. It has recently been shown that S -nitrosylation may result in various protein dysfunctions. However, an improved S -nitrosylation analysis method is needed to achieve high sensitivity and quantitative accuracy. We hypothesized that an analysis method using fluorescence dye could detect S -nitrosylated proteins at a higher sensitivity than that of the conventional method. In this study, we developed a procedure for analyzing S -nitrosylated proteins using CyDye (the CyDye switch method). This CyDye switch method for detecting S -nitrosylated proteins was developed based on the biotin-switch method for analyzing S -nitrosylated proteins. We analyzed NO donor-induced S -nitrosylated proteins in a model protein and at the cellular level. We demonstrated that this CyDye switch method could detect specific S -nitrosylated proteins using SDS-PAGE and mass spectrometry. Our results indicate that the optimized CyDye switch method is suitable for the detection of the post-translational S -nitrosylation of proteins.