Affinity peptide ligands: new tools for chasing non-canonical N-phosphoproteome

The enrichment of protein N-phosphorylation encounters substantial challenges due to the inherent instability of the N-P bond, severely impeding the manifestation of its biological functions. Traditional enrichment methods often rely on antibodies, organic solvents and metal ion interactions, which are limited by lack of universality, potential degradation of sample integrity, or reduced selectivity for N-phosphorylation. To overcome these challenges, we innovatively capitalized phage display technology to identify affinity peptides that specifically bind to the N-PO₃ group. By functionalizing magnetic nanoparticles with the affinity peptide, we developed a novel, organic solvent- and metal-free enrichment strategy that enhanced both the selectivity and efficiency for all three types of N-phosphopeptide capture under neutral conditions, ensuring superior preservation of sample integrity and allowing more accurate proteomic analysis. This strategy has demonstrated robust enrichment capabilities for both prokaryotic and eukaryotic samples. In HeLa cells, 1995 novel N-phosphorylation sites were identified, representing a substantial increase of 2- to 5-fold in detection depth over previous approaches and significantly expanding the scale of the N-phosphoproteome database. Additionally, it was discovered that N-phosphorylation modification was highly concentrated in the nucleus. By integrating the nuclear isolation technique, 1296 N-phosphorylation sites were identified for the first time, offering new leads for uncovering the functions of N-phosphorylation in nuclear proteins. Finally, in conjunction with the quantitative proteomics method, the dynamic changes in N-phosphorylation modification during the progression of Alzheimer's disease were investigated, providing fresh perspectives on the research of AD pathogenesis. Overall, this work not only presents a new approach for efficient enrichment of N-phosphopeptides but also advances the functional study of N-phosphorylated proteins in physiological and pathological processes.