Dual-Affinity HZIF-1Mo Microcolumn Platform for Quantitative Profiling of N-Phosphorylation in Breast Cancer Subtypes.

N-Phosphorylation (pHis/pArg/pLys) is a functionally significant yet technically challenging post-translational modification (PTM) due to its low abundance and acid lability. Here, we develop a hybrid zeolitic imidazolate framework nanomaterial (HZIF-1Mo) with dual-metal (Zn²⁺/Mo⁶⁺) synergy and broad pH stability. This material enables rapid enrichment of N-phosphopeptides under weakly acidic conditions (pH 2.7) via a centrifugation-assisted microcolumn strategy, achieving high selectivity and sensitivity for N-phosphopeptides. Applied to breast cancer subtypes, we identified 316 high-confidence N-phosphosites across MCF-7 (luminal A) and MDA-MB-231 (triple-negative). Notably, 38 markedly upregulated N-phosphopeptides in MDA-MB-231 implicated three functional modules: cholesterol metabolism, transporter regulation, and cytoskeletal reorganization. This work establishes a robust platform for N-phosphoproteomics and paves the way for discovering TNBC-specific diagnostic biomarkers and potential therapeutic targets.