High-Throughput Capture and Sensitive In Situ Visualization of Protein N-Phosphorylation on Membranes Based on...

N-phosphorylation is a ubiquitous post-translational modification that plays a pivotal role in regulating protein activity, function, interactions, and subcellular localization. However, its functional roles in biological systems remain largely unexplored, due to the absence of N-phospho-specific antibodies and the inherent instability of the phosphoramidate (P-N) bond. To address this challenge, we introduce the reverse-phase N-phosphoprotein array (RPNPA) as a novel and efficient solution. The RPNPA leverages a nitrocellulose membrane functionalized with a polyamidoamine (PAMAM) dendrimer, immobilized with bis(zinc(II)-dipicolylamine) (Zn(II)-DPA) to selectively capture N-phosphoproteins from complex biological samples in a high-throughput manner. The captured N-phosphoproteins were then detected using conventional antibodies, akin to traditional reverse-phase protein arrays, eliminating the need for specialized N-phospho-specific antibodies. The RPNPA offers a simple, convenient, and cost-effective approach with exceptional specificity (1:100), high sensitivity (at the nanogram level), and robust quantitative capability (R² > 0.95) for analyzing N-phosphoproteins. This innovative platform provides a powerful tool for biological and biomedical research, enabling efficient, high-throughput screening, validation, and quantification of N-phosphorylation changes at the protein level.