Native Top-Down Mass Spectrometry Links Structure and Gas-Phase Stability of ERα-LBD

Abstract Defining how protein assemblies form and dissociate is central to understanding biomolecular function. The ligand binding domain (LBD) of estrogen receptor α (ER) forms obligate head-to-head homodimers in solution. The high-affinity leucine zipper that comprises this dimerization interface hinders our ability to measure the kinetics and energetics of oligomer formation. Here, native top-down mass spectrometry (nTDMS) reveals a predominant dimeric ER-LBD assembly under ligand-free conditions. Controlled desolvation voltage drives energy-dependent disassembly to a monomer, enabling the assessment of dimer stability in the gas phase and dissociation pathways. Top-down fragmentation of both dimeric and monomeric species provides sequence-level characterization while preserving assembly context. These results establish nTDMS as a unified approach to link the structure and gas-phase stability of protein assemblies.