Biotin@DpaZnMolecules Enabled Efficient Enrichmentof N‑Phosphopeptides under Neutral Conditions

Protein N-phosphorylation has been garnering increasing attention owing to its unique biological functions. The large-scale identification of protein N-phosphorylation serves as the foundation for exploring a novel function. Despite the advancements in enrichment methods under neutral conditions, persistently low enrichment efficiency has long hindered the progress of this field. In this work, Biotin@DpaZn molecules were first synthesized for liquid–liquid enrichment, which enabled the efficient enrichment of N-phosphopeptides under neutral conditions. This enrichment strategy combined the benefits of low steric hindrance, high selectivity, and high affinity inherent in pull-down techniques along with the alkali resistance of agarose microspheres, which could significantly enhance the enrichment efficiency. Compared to SiO₂@DpaZn, the number of identified N-phosphorylation sites in E. coli increased from 27 to 58. Moreover, we successfully achieved large-scale N-phosphorylation identification in Corynebacterium glutamicum under different growth conditions, greatly advancing functional studies. Overall, we developed a liquid–liquid enrichment method for protein N-phosphorylation. Our work has expanded the identification coverage of N-phosphorylation, especially in prokaryotes, facilitating the exploration of potential functions.