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

This study is led by Professor Shijuan Yan (Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, China). The authors introduce a novel method, phosphate affinity tag-switch (PAT-switch), for simultaneous profiling of phosphorylation and S-nitrosylation (SNO) – two pivotal yet chemically distinct post-translational modifications (PTMs) in plants.

The PAT-switch workflow integrates three core steps: blocking free thiols with iodoacetamide while preserve S-nitrosylated cysteines and disulfide bonds, selective PAT labeling of S-nitrosylated cysteines, and reduction/alkylation of disulfide bonds. Post-enzymatic digestion, both phosphopeptides and PAT-labeled peptides are co-enriched by immobilized metal ion affinity chromatography (IMAC), and subsequently analyzed by nanoLC–MS/MS. Distinct mass shifts (79.97 Da for phosphorylation and 221.08 Da for PAT-tag) permit search engines to confidently discriminate between the two PTMs.

The method exhibited high specificity and sensitivity, as validated by S-nitrosylated BSA, where 30 SNO sites were detected with minimal background interference. When applied to Arabidopsis thaliana seedlings, 2,442 S-nitrosylated peptides and 4,959 phosphopeptides were identified from 300 μg protein input, achieving coverage comparable to cutting-edge single-PTM profiling methodologies. Further expansion of the PTM landscape was achieved through high-pH reversed-phase fractionation, enabling large-scale mapping of 12,552 phosphosites and notably 6,108 SNO sites, including 3,795 previously unreported sites. The study also revealed 968 proteins bearing dual-PTMs in Arabidopsis , including 378 novel S-nitrosylated targets—such as TOR kinase, PPC isoforms, BSK/MAPK families—and 392 proteins with newly identified PTM sites. Functional enrichment highlighted these dual-modified proteins involved in carbon metabolism, photosynthesis, and stress signaling, suggesting putative PTM crosstalk in regulating plant physiology. Overall, the PAT-switch workflow provides a powerful tool for analyzing phosphorylation and S-nitrosylation, thereby facilitating a deeper understanding of PTM-mediated regulation in plants.

See the article:

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

https://link.springer.com/article/10.1007/s42994-025-00227-2