Selective enrichment tandem β-elimination assisted strategy for N-phosphorylation analysis

N-phosphorylation modifications are crucial for prokaryotic signal transduction and verified as intermediate for several metabolic enzymes, yet the landscape of N-phosphorylation remains an obstacle due to the lack of effective identification strategies. One of the difficulties derives from the labile phosphoramidate bond (P-N) under acidic conditions, making it easily hydrolyze during the routine analysis. Meanwhile, O-phosphopeptides influence the accurate identification of N-phosphorylation sites during mass spectrometry (MS) analysis. Herein, a selective enrichment tandem β-elimination assisted identification (abbreviated as EnaBe) strategy was established to address the difficulties. Firstly, N-phosphoproteins could be captured within 10 min by SiO₂@DpaZn microspheres under neutral conditions, which was benefited from rapid mass transfer. Secondly, the β-elimination efficiency could reach over 92% within 3 h under the optimized condition, and MS signals of N-phosphopeptides were significantly enhanced by integrating the β-elimination treatment. Finally, N-phosphoproteins from E. coli lysates were analyzed by EnaBe approach and 16 N-phosphoproteins with high confidence were identified. Furthermore, functional analysis showed that the proteins played vital roles in phosphorylation process and bacterial primary metabolic processes including glucose, purine and ADP metabolism. All above results demonstrated the superiority of the EnaBe strategy.