Characterization of macromolecular protein assemblies by collision-induced and surface-induced dissociation: Expanding the role of mass spectrometry in structural biology

Abstract

This dissertation presents an investigation into the structure of macromolecular protein assemblies by mass spectrometry. The experiments described within are designed to systematically assess the analytical utility of surface-induced dissociation (SID) tandem mass spectrometry in the characterization of multi-subunit protein complexes. This is accomplished by studying the effects of ion-surface collisions on the fragmentation products of protein assemblies that vary by mass, number of subunits, and protein structural features.

Conditions are first established for the preservation of "native" protein quaternary structure and applied to previously characterized systems for proof-of-concept. Native mass spectrometry is subsequently combined with limited proteolysis experiments to characterize the subunit interface of a unique small heat shock protein, HSP18.5 from Arabidopsis thaliana, identifying regions of the protein essential for preservation of the native dimer.

The dissociation of non-covalent protein assemblies is then explored on a quadrupole time-of-flight (Q-TOF) mass spectrometer, modified for the study of ion-surface collisions. This instrument allows ions to be dissociated through collisions with a surface or more conventional collisions with gas atoms. Activation of a protein complex with "n" subunits through multiple collisions with inert gas atoms results in asymmetric dissociation into a highly charged monomer and complementary (n-1)-mer regardless of protein size or subunit architecture. This process is known to occur through an unfolding of the ejected subunit, and limits the amount of structural insight that can be gleaned from such studies. Collision at a surface however, results in more charge and mass symmetric fragmentation, and in some instances reflects the substructure of the protein assembly under investigation.

The differences observed between the CID and SID of protein complexes are attributed to the rapid deposition of large amounts of internal energy upon collision at a massive surface target, and reflect a dissociation process that precedes subunit unfolding. This provides access to dissociation pathways inaccessible by traditional means of activation. The fragmentation products observed by SID demonstrate promise for expanding the role of mass spectrometry in the field of structural biology.