Single-Ion Imaging Native Mass Spectrometry: Unraveling the Structural Features and Dissociation Energetics of Macromolecular Assemblies.

We explore mass-resolved imaging of fragments generated from single macromolecular assembly (MMA) ions on a custom-built Orbitrap/time-of-flight (TOF) mass spectrometer with integrated UV photodissociation (UVPD) and a position- and time-sensitive Timepix3 imaging detector assembly. We postulated that the 2D detector images provide information about the 3D geometry of the MMAs in the gas phase as the TOF analyzer has the ability to retain the relative positions of the product ions following the fragmentation process and until they reach the imaging detector, when the fragmentation occurs at the level of single-precursor MMA ion. We demonstrate that the Orbitrap/TOF mass spectrometer enables fragmentation at the single-precursor MMA ion level using dimeric and tetrameric noncovalently bound assemblies. Timepix3-derived relative position data from single-precursor fragmentation events of two distinct tetrameric MMAs reveal different higher-order structural signatures that enable their differentiation. Furthermore, mapping these single-precursor fragmentation events to possible dissociation pathways provides insight into the underlying dissociation mechanisms. Overall, this study demonstrates the potential of single-ion mass-resolved imaging to understand UVPD dissociation mechanisms, fragmentation pathways of MMA ions, and their higher-order structure.