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

Protein post-translational modifications such as phosphorylation and S-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 S-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 Arabidopsis thaliana seedlings revealed 12,552 phosphorylation sites and 6,108 S-nitrosylation sites, representing the largest single-study dataset of S-nitrosylation sites to date. This approach enhances our understanding of post-translational modification dynamics in plant signaling, stress responses, and metabolism.

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