A Potassium Phosphoramidate-Based Strategy for Compiling and Profiling the Noncanonical N -Phosphoproteome of Nasopharyngeal Carcinoma

Protein N-phosphorylation, especially in eukaryotes, plays a critical role in cell signal transduction and tumorigenesis. However, the N-phosphoproteome has not been extensively profiled due to its low abundance and chemical lability. Herein, we developed a potassium phosphoramidate (PPA)-based strategy for the generation of high-quality N-phosphorylation spectral libraries to profile the N-phosphoproteome. This approach relies on the chemical phosphorylation of basic amino acid residues (lysine, arginine, and histidine) by PPA, followed by peptide-level fractionation, phosphopeptide enrichment, spectral library generation, and data-independent acquisition mass spectrometry (DIA-MS) analysis. To develop this method, the feasibility and reproducibility were first validated using N-phosphorylated bovine serum albumin (N-pho-BSA). Phosphoproteome analysis of HEK293T lysates further demonstrated that the PPA-based library data-independent acquisition (PAlibDIA) achieved superior coverage and quantification reproducibility compared with conventional data-dependent acquisition (DDA) and direct DIA. This PAlibDIA approach was then employed to characterize N-phosphosites in human nasopharyngeal carcinoma (NPC), resulting in 493 pHis, 714 pLys, and 557 pArg sites; 85% are novel sites that were not previously reported. Further data analysis revealed that differentially regulated N-phosphosites were associated with RNA splicing, chromatin remodeling, nucleosome assembly, and multiple signaling pathways. Together, our PAlibDIA has great potential for comprehensive and in-depth analysis of the N-phosphoproteome, offering new opportunities to uncover regulatory mechanisms and identify potential therapeutic targets.