Ion Mobility-Mass Spectrometry Reveals the Influence of Subunit Packing and Charge on the Dissociation of Multiprotein Complexes

摘要

The composition, stoichiometry, and organization of proteinrncomplexes can be determined by collision-inducedrndissociation (CID) coupled to tandem mass spectrometryrn(MS/MS). The increased use of this approach in structuralrnbiology prompts a better understanding of the dissociationrnmechanism(s). Here we report a detailed investigation ofrnthe CID of two dodecameric, heat-stable and toroidallyrnshaped complexes: heat shock protein 16.9 (HSP16.9)rnand stable protein 1 (SP-1). While HSP16.9 dissociatesrnby sequential loss of unfolded monomers, SP-1 ejects notrnonly monomers, but also its building blocks (dimers), andrnmultiples thereof (tetramers and hexamers). Unexpectedly,rnthe dissociation of SP-1 is strongly charge-dependent:rnloss of the building blocks increases with higherrncharge states of this complex. By combining MS/MS withrnion mobility (IM-MS/MS), we have monitored the unfoldingrnand dissociation events for these complexes in the gasrnphase. For HSP16.9 unfolding occurs at lower energiesrnthan the ejection of subunits, whereas for SP-1 unfoldingrnand dissociation take place simultaneously. Wernconsider these results in the light of the structuralrnorganization of HSP16.9 and SP-1 and hypothesize thatrnSP-1 is unable to unfold extensively due to its particularrnquaternary structure and unusually high charge density.rnThis investigation increases our understanding of thernfactors governing the CID of protein complexes andrnmoves us closer to the goal of obtaining structuralrninformation on subunit interactions and packing fromrngas-phase experiments.