HydrophilicInteraction Liquid Chromatography-BasedEnrichment Method for Deciphering the N‑Phosphorylated ProteomeLandscape

Protein N-phosphorylation, especially in mammals, was gradually studied and attracted widespread attention. However, the lack of effective enrichment methods resulted from the intrinsic lability of the P–N bond under acidic conditions, which seriously hindered biological function elucidation. In this work, hydrophilic two-dimensional nanocomposites with enhanced hydrophilicity and coordination interaction with phosphate were designed as the HILIC stationary phase, and then the HILIC-based strategy was developed for enrichment of N-phosphopeptides under neutral conditions. Compared to the previous enrichment methods, the number of N-phosphorylation sites identified in Escherichia coli was increased from less than 30 to 298, which demonstrated that the strategy could improve identification coverage. Furthermore, we implemented N-phosphorylation analysis of subcellular and more than 200 N-phosphorylation sites were identified from the HeLa cell membrane. Finally, the dynamic change of N-phosphorylation was studied in lung cancer cells with high brain metastasis, and possible sites involved in regulating invasion were discovered, which could provide more potential targets for tumor diagnosis or treatment. Taken together, this work not only provided a new method for the tool box of N-phosphorylation enrichment but also enabled profiling of various samples of the N-phosphoproteome landscape.