一种用于同时分析蛋白质磷酸化和亚硝基化的新型蛋白质组学工作流程。

A novel proteomics workflow for simultaneous analysis of protein phosphorylation and -nitrosylation.

作者信息

Zhang Wenyang, Wang Yanjiao, Li Wenyan, Wu Shaowen, Chen Yuanyuan, Ye Mingyang, Huang Wenjie, Fernie Alisdair R, Yan Shijuan

机构信息

State Key Laboratory of Swine and Poultry Breeding Industry, Guangdong Key Laboratory for Crop Germplasm Resources Preservation and Utilization, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640 China.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangdong Laboratory for Lingnan Modern Agriculture, College of Life Sciences, South China Agricultural University, Guangzhou, 510642 China.

出版信息

aBIOTECH. 2025 Jul 15;6(3):452-465. doi: 10.1007/s42994-025-00227-2. eCollection 2025 Sep.

DOI:10.1007/s42994-025-00227-2

PMID:40994443

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12454217/

Abstract

UNLABELLED

Protein post-translational modifications such as phosphorylation and -nitrosylation regulate protein functions and cellular programs in eukaryotes. Moreover, extensive evidence suggests crosstalk between these modifications. However, we lack a comprehensive method for the simultaneous detection and analysis of multiple post-translational modifications. Here, we present an optimized workflow that identifies phosphorylation and -nitrosylation sites using a novel phosphate affinity tag switch technique. Validation with model proteins and complex biological samples confirmed the high sensitivity, coverage, and reproducibility of this method. Applying this method to seedlings revealed 12,552 phosphorylation sites and 6,108 -nitrosylation sites, representing the largest single-study dataset of -nitrosylation sites to date. This approach enhances our understanding of post-translational modification dynamics in plant signaling, stress responses, and metabolism.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s42994-025-00227-2.

摘要

未标记

蛋白质的翻译后修饰，如磷酸化和亚硝基化，可调节真核生物中的蛋白质功能和细胞程序。此外，大量证据表明这些修饰之间存在相互作用。然而，我们缺乏一种同时检测和分析多种翻译后修饰的综合方法。在此，我们提出了一种优化的工作流程，该流程使用一种新型的磷酸亲和标签切换技术来识别磷酸化和亚硝基化位点。用模型蛋白和复杂生物样品进行的验证证实了该方法的高灵敏度、覆盖率和可重复性。将该方法应用于幼苗，发现了12552个磷酸化位点和6108个亚硝基化位点，这是迄今为止关于亚硝基化位点的最大单研究数据集。这种方法增强了我们对植物信号传导、应激反应和代谢中翻译后修饰动态的理解。