Widespread protein N-phosphorylation in organism revealed by SiO₂@DpaZn beads based mild-acidic enrichment method

Protein phosphorylation is one of the most commonly studied and ubiquitous post-translational modifications (PTMs), and defining site-specific phosphorylation is essential to understand basic and disease biology. However, the chemical properties and biological activities hamper the detection of non-canonical N-phosphorylation from biological samples, and the study of N-phosphorylation over the last half century has lagged behind canonical O-phosphorylation. Here, a mild-acidic method based-on SiO₂@DpaZn beads was developed for protein N-phosphorylation sites identification. The method was verified as an effective complement for neutral enrichment science the stability of N-phosphorylation varied with the protein context. We firstly verified the feasibility of the mild-acidic enrichment strategy by standard peptides. Totally, 301 and 1476 N-phosphorylation sites were identified from E. coli and HeLa, respectively, verifying the robust of the method. The results greatly enriched N-phosphorylation site database. Furthermore, the method provided the peptide sequence motif of the N-phosphorylation sites and the biological functions of the identified proteins. The work represented an important step forward in studying the role of N-phosphorylation and other labile phosphorylation.