Phosphoproteomic analysis of X-ray-irradiated planarians provides novel insights into the DNA damage response

Phosphorylation plays a crucial role in the cellular response to radiation and cancer therapies, yet phosphoproteomics studies in planarians remain underexplored despite the organism's remarkable regenerative capacities. This study utilized advanced ion mobility mass spectrometry for 4D-label-free quantitative proteomics to identify phosphorylation sites associated with irradiation in planarians. A total of 33,284 phosphorylation sites from 15,505 phosphorylated peptides and 4710 unique phosphoproteins were identified. In the sub-lethal dose irradiation group, 1695 phosphoproteins with 3483 phosphorylation sites exhibited significant changes, while exposure to lethal doses of radiation led to significant changes in 2308 phosphoproteins with 6112 phosphorylation sites, including many kinases, transcription factors, and cytoskeletal proteins. Functional enrichment analysis revealed that the altered phosphoproteins were primarily involved in transcription, RNA biosynthesis, mRNA processing regulation, and spliceosomal complex assembly. Functional validation of five differentially phosphorylated proteins revealed that their depletion impaired stem cell regeneration after irradiation by disrupting DNA repair, suggesting that these proteins are critical to planarian biology and their radiation response. By identifying the phosphorylation state and specific sites of planarian proteins, our study lays the foundation for further research on protein phosphorylation in the radiation-induced DNA damage response. In addition, our findings provide preliminary insights into the role of calnexin, a protein involved in interacting with newly synthesized N-linked glycoproteins, in planarians.