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.