A Detergent-Free Grinding Sample Preparation Method Dramatically Enhances PELSA for Mapping Integral Membrane Proteins–Ligand Interaction

The conventional use of detergent-containing buffers for integral membrane proteins (IMPs) extraction always disrupts native protein conformations, potentially compromising the performance of ligand target identification. Consequently, there is a lack of reliable, unbiased, detergent-free proteomic methods that enable the sensitive identification of ligand binding IMPs. In this study, we developed DFG-PELSA (Detergent-Free Grinding sample preparation method coupled with Peptide-centric Local Stability Assay), an innovative workflow that integrates mechanical grinding, extensive trypsinization, and DIA mass spectrometry for the proteome-wide identification of ligand binding proteins and binding regions with enhanced ability to identify IMP targets. Our method successfully recovered over 1000 transmembrane proteins from HeLa cells while maintaining their native conformations. This method successfully mapped the interactions and binding regions of AMP-PNP with IMPs such as SERCA2 and ABCB6. DFG-PELSA also revealed the on-target and off-target mechanisms of EGFR tyrosine kinase inhibitors (EGFR-TKIs), providing valuable insights for drug discovery and optimization. Additionally, DFG-PELSA identified stability shifts induced by the binding of cardiotonic steroids to the transmembrane region of ATP1A1, demonstrating its effectiveness in studying challenging IMP targets. These applications highlight DFG-PELSA’s unique capability in studying ligand-IMP interactions, offering a useful platform for structure-based drug discovery and target validation.