The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Top‐down mass spectrometry (TD‐MS) provides unique information on compositions, structures, and functions of proteins or protein complexes and has been recognized as a powerful approach to complement conventional tools in protein analysis. Employing microfluidic chips in TD‐MS workflows offers unique advantages including flexible integration and automation of multiple sample treatment functions, lowered sample consumption, compatibility with high‐sensitive ionization and higher throughput. Here we reviewed the reported microchips developed for TD‐MS in aspects of design of ionization, separation and mixing modules, and fabrication approaches; we also attempted to summarize the design considerations that may inspire development of microchips with better performance and wider applications.