Here we present a new native ion mobility top-down mass spectrometry (nIM-TDMS) approach which uses tandem IM to selectively isolate single native protein conformers in-instrument for conformation-specific top-down fragmentation using structure-sensitive electron-based fragmentation. In doing so, this method enables confident structural interpretation of the resulting fragmentation patterns by correlating them directly to specific protein conformational states. The approach was implemented on a cyclic ion mobility (cIM)-enabled quadrupole ToF instrument with postmobility electron capture dissociation (ECD) capabilities, shown in Figure . ECD is an electron-based dissociation method wherein a stream of electrons is released from a filament, resulting in their interaction with the analyte molecule and, in the case of proteins, subsequent backbone cleavage. For readers interested in the fundamental principles and applications of this technique, we recommend consulting a number of excellent reviews. ECD was selected as our chosen dissociation method due to its proven track record in mapping fragmentation patterns to structure whilst retaining protein:ligand interactions. The geometry of this instrument allows nIM-TDMS of proteins (blue line) and their complexes (orange line) through the workflows detailed in Figure . The tandem IM capabilities of the cIM instrument combined with top-down capabilities further enables the characterization of ligands associated with single conformer protein complexes (Figure red line), enabling for the first time to our knowledge MS n capabilities on a quadrupole ToF instrument.