Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry

We have combined ion mobility spectrometry–mass spectrometry with tandem mass spectrometry to characterise large, non‐covalently bound macromolecular complexes in terms of mass, shape (cross‐sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision‐induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring‐like three‐dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on‐pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross‐sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X‐ray structure of the coat protein building blocks. Comparing the cross‐sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3‐fold and the 5‐fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti‐viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non‐covalently bound macromolecular complexes and their assembly pathways. Copyright © 2010 John Wiley & Sons, Ltd.