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

Toshihiro Yoshimura, K. Kurogi, Ming-Cheh Liu, M. Suiko, Y. Sakakibara
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引用次数: 1

Abstract

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.
利用荧光标记技术分析s -亚硝基化蛋白质的蛋白质组学方法
s -亚硝基化是一氧化氮(NO)对半胱氨酸残基巯基的翻译后修饰,已成为一种新的信号转导和蛋白质功能调节模式。最近有研究表明,s -亚硝基化可导致多种蛋白质功能障碍。然而,需要一种改进的s -亚硝基化分析方法来实现高灵敏度和定量准确性。我们假设使用荧光染料的分析方法能够以比传统方法更高的灵敏度检测s -亚硝基化蛋白。在这项研究中,我们开发了一种使用CyDye (CyDye开关法)分析s -亚硝基化蛋白的方法。这种检测s -亚硝基化蛋白的CyDye开关方法是在分析s -亚硝基化蛋白的生物素开关方法的基础上发展起来的。我们在模型蛋白和细胞水平上分析了NO供体诱导的s -亚硝基化蛋白。我们证明这种CyDye开关方法可以通过SDS-PAGE和质谱检测特定的s -亚硝基化蛋白。结果表明,优化后的CyDye开关法适用于蛋白质翻译后s -亚硝基化的检测。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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