植物学报 ›› 2019, Vol. 54 ›› Issue (4): 497-502.doi: 10.11983/CBB19108

• 技术方法 • 上一篇    下一篇

植物蛋白质S-亚硝基化修饰的检测与分析

陈立超,詹妮,李彦莎,冯健,左建儒*()   

  1. 中国科学院遗传与发育生物学研究所, 北京 100101
  • 收稿日期:2019-06-13 接受日期:2019-06-20 出版日期:2019-07-01 发布日期:2020-01-08
  • 通讯作者: 左建儒 E-mail:jrzuo@genetics.ac.cn
  • 基金资助:
    国家自然科学基金(31830017);国家自然科学基金(31521001);中国科学院战略先导B项目(XDB27030207)

Detection and Analysis of Protein S-nitrosylation in Plants

Chen Lichao,Zhan Ni,Li Yansha,Feng Jian,Zuo Jianru*()   

  1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
  • Received:2019-06-13 Accepted:2019-06-20 Online:2019-07-01 Published:2020-01-08
  • Contact: Zuo Jianru E-mail:jrzuo@genetics.ac.cn

摘要:

S-亚硝基化是一种重要的蛋白质翻译后修饰方式, 是指一氧化氮(NO)基团共价连接至靶蛋白特定半胱氨酸残基的自由巯基, 从而形成S-亚硝基硫醇(SNO)的过程。S-亚硝基化修饰广泛存在于各有机体中, 通过改变蛋白质生化活性、稳定性、亚细胞定位以及蛋白质-蛋白质相互作用等机制而调控不同的生物学过程或信号通路。在蛋白质S-亚硝基化检测分析方法中, 最为广泛使用的是生物素转化法(biotin switch assay), 其基本原理是首先封闭未被修饰的自由巯基, 进而将被修饰的SNO基团特异地还原为自由巯基并使用生物素将其特异标记。被生物素标记的半胱氨酸残基(即被修饰位点)可进一步通过蛋白质免疫印迹和/或质谱等方法进行检测分析。该文详细描述了植物蛋白质样品的体内和体外生物素转化法的实验流程, 并对实验过程中的注意事项进行了讨论。

关键词: 一氧化氮, S-亚硝基化, 生物素转化法, 植物

Abstract:

S-nitrosylation is an important protein posttranslational modification, involved in covalently linking a nitric oxide (NO) molecule to the thiol group of a cysteine residue to generate S-nitrosothiols. S-nitrosylation regulates multiple biological processes by modulating protein activity, stability, subcellular localization and protein-protein interactions. The biotin-switch assay is one of the most-often used methods to detect and analyze protein S-nitrosylation. In principle, the free thiols in a target protein are first blocked, followed by reducing the S-nitrosothiols of the target protein to free thiols by ascorbate, which are subsequently labelled by biotin to form biotinylated proteins. The biotin-labelled sample was assayed by immunoblotting and mass spectrometry. Here, we present detailed experimental procedures for the in vitro and in vivo biotin-switch methods and give advice on key troubleshooting solutions.

Key words: nitric oxide, S-nitrosylation, biotin-switch assay, plants

图1

牛血清白蛋白(BSA)体外S-亚硝基化修饰的分析检测 使用biotin-maleimide标记BSA样品, 经SDS-PAGE胶分离后进行免疫印迹分析。分别采用anti-biotin和anti-BSA抗体作为第一抗体(primary antibodies; 1:20 000稀释), anti-mouse IgG为第二抗体(secondary antibodies)。样品信号用SuperSignal Western Femto Maximun Sensitivity Substrate Kit检测。曝光时间分别为20秒(上)和30秒(下)。GSH: 谷胱甘肽; GSNO: S-亚硝基谷胱甘肽; Asc: 抗坏血酸盐"

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