Characterization of Macromolecular Protein Assemblies by Collision-Induced and Surface-Induced Dissociation: Expanding the Role of Mass Spectrometry in Structural Biology
AuthorJones, Christopher Michael
AdvisorWysocki, Vicki H.
Committee ChairWysocki, Vicki H.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThis 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 collision on the fragmentation products of protein assemblies that vary by mass, number of subunits, and protein structural features. The dissociation energetics and mechanisms of protein complexes are considered by examining the influence of ion internal energy and sub-oligomeric protein structure on the dissociation process. Conditions are first established for the preservation of “native” protein quaternary structure and applied to previously characterized systems for proof-ofconcept. These conditions are subsequently extended to determine the molecular weight and subunit stoichiometry of several small heat shock proteins. Native mass spectrometry is then 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. The dissociation of protein complexes is explored by both activation methods beginning with specific and non-specific dimers with masses less than 40 kDa. These studies are extended to larger assemblies with as many as 14 subunits weighing over 800 kDa, and are applied to both homo- and hetero-oligomeric protein complexes. 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 is attributed to the rapid deposition of large amounts of internal energy deposited upon collision at a more massive target such as a surface. The ion activation time-frame and energy transfer efficiency are proposed to induce dissociation on a time-scale that precedes subunit unfolding providing access to dissociation pathways that are inaccessible by traditional means of activation. The systems studied here represent the largest ions fragmented via surface collisions within a mass spectrometer, and the fragmentation products observed by SID demonstrate its promise for expanding the role of mass spectrometry in the field of structural biology.