Protocol for detecting protein S-glutathionylation in Arabidopsis thaliana under oxidative stress using a non-transgenic chemical toolkit

Summary Protein S-glutathionylation is a redox-sensitive post-translational modification that regulates proteostasis and signaling during oxidative stress. However, its in vivo detection is challenging due to labile disulfide bonds and ex vivo oxidation artifacts. Here, we present a protocol for detecting protein glutathionylation in Arabidopsis thaliana using a non-transgenic toolkit. We describe steps for using propargyl glycine (PG) metabolic labeling and click chemistry. We detail workflows for global quantification, target enrichment for western blotting or liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in situ spatial visualization. For complete details on the development, use and execution of this protocol, please refer to Zhao et al.¹