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

The conventional use of detergent-containing buffers for integral membrane protein (IMP) extraction may disrupt native protein conformations, potentially compromising the performance of ligand target identification. As a result, there is a lack of reliable, unbiased, detergent-free proteomic methods that enable the 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 target proteins. Our method successfully recovered over 1,000 transmembrane proteins from HeLa cells while maintaining their native conformations. This method successfully mapped the interaction and binding regions of AMP-PNP with IMPs such as SERCA2 and ABCB6. DFG-PELSA also revealed the target and off-target mechanisms of tyrosine kinase inhibitors (TKIs), providing valuable insights for drug discovery and optimization. Additionally, DFG-PELSA identified stability shifts induced by cardiotonic steroids binding 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-membrane protein interactions, offering a useful platform for structure-based drug discovery and target validation.