NP-DiaMS: A High-Throughput Equilibrium Dialysis-LC-MS Platform for Protein-Directed Screening of Natural Products

Target-directed discovery is widely used in modern drug development, and protein-directed screening of natural products is an effective route for identifying bioactive small molecules. However, crude natural product extracts present substantial analytical challenges due to compositional complexity and matrix interference. Common affinity-based mass spectrometry approaches, such as affinity ultrafiltration-mass spectrometry, rely on centrifugation and washing steps that may reduce throughput and weaken reversible interactions, leading to incomplete interaction profiling. Here we developed a Natural Product-Dialysis and Mass Spectrometry (NP-DiaMS) workflow for protein-directed screening of small molecules in complex natural product systems. The method uses diffusion-controlled equilibrium partitioning combined with small-molecule-selective separation to reduce matrix effects while preserving weak binding events. Using pyruvate kinase M2 (PKM2) as an initial model target, the workflow was established with standard alkaloid mixtures and applied to crude herbal extracts, leading to the identification of three PKM2-associated small molecules with different binding strengths. The binding interactions were validated by surface plasmon resonance measurements, and the cellular relevance of the identified ligands was further examined by cell-based assays. Comparative experiments indicated that the equilibrium dialysis workflow retained additional interaction-related features while maintaining overall screening consistency, corresponding to an approximately 12-16-fold increase in sample processing throughput compared with tube-based ultrafiltration workflows. These results indicate that NP-DiaMS is well suited for active component discovery from complex extracts, with practical advantages in throughput, weak-interaction retention, and matrix tolerance.