Native Ion Mobility–Mass Spectrometry Reports Kinetically Trapped Conformational Ensembles, Not Single Structures

Native ion mobility/mass spectrometry (IM/MS) is widely used to characterize protein structures through their collision cross sections. This Perspective examines what structural information about proteins is contained in native IM/MS spectra, and how that information can be extracted and interpreted. Commonly, the centroid cross section is interpreted as reporting on a single, static protein structure. Evidence considered in this Perspective shows that such a centroid-only interpretation can fail to reproduce conformational behavior established by solution-phase techniques, as exemplified by the canonical protein ribonuclease A. Results from emerging tandem-IMS approaches instead show that native IM/MS is fundamentally an ensemble measurement, with broad spectral features containing distinct, kinetically stable solvent-free protein conformations. When interpreted at the level of the conformational ensemble, IM/MS recovers the established solution-phase conformational behavior. We therefore conclude that native IM/MS reports a solvent-free projection of the conformational ensemble that governs protein behavior in solution and that tandem IMS methods provide experimental access to the underlying solvent-free conformational populations.