Proteólisis dual mediada por proteasas acoplada a ultrafiltración para el perfilado a nivel de sistema de dianas...

Characterizing drug-binding proteins and the subsequent changes in protein complexes/interactions is pivotal to fully understanding drug mechanisms. Here, we developed a novel modification-free approach, Dual Protease-Mediated Proteolysis Coupled with Ultrafiltration (DPMU), which integrates limited proteolysis, ultrafiltration fractionation, and quantitative mass spectrometry to systematically map drug-induced structural alterations. DPMU employs thermolysin (broad specificity; prefers hydrophobic residues) and trypsin (specific for lysine and arginine residues) in a two-step workflow: thermolysin digests native proteins to generate small conformationally sensitive peptides (isolated via ultrafiltration), while trypsin subsequently digests retained intact/partially digested proteins to capture binding domains. We validated DPMU using the maltose-binding protein (MBP), demonstrating its ability to detect ligand-induced stabilization and proteolytic susceptibility shifts. Applied to cell lysates, DPMU successfully identified known targets of rapamycin (FKBP family) and geldanamycin (HSP90 family), revealing concentration-dependent binding pattern changes in protein complexes. DPMU elucidated the binding of paclitaxel to tubulin and microtubule-associated proteins, revealing its regulatory effects on the conformation of the microtubule-related complexes. In NB4 cells, DPMU identified TXNL1 as an arsenic agent target and highlighted cysteine residue enrichment, consistent with arsenic's affinity for thiol groups. Additionally, DPMU revealed the specific conformational changes of the 26S proteasome complex in response to different drugs (such as rapamycin and paclitaxel, etc.). DPMU enables system-level target discovery, conformational analysis, and mechanistic dissection, making it a versatile tool for drug development.