Development of Charge Manipulation Nanoelectrospray Ion Mobility-Mass Spectrometry Techniques for Multiprotein Complex Analysis.

Abstract

Macromoleclar protein complexes comprise a critical class of biomolecules unique in both their importance in biology and their relative impenetrability to detailed structural probes. Nanoelectrospray coupled to ion mobility-mass spectrometry (nESI-IM-MS) is an emerging tool for determining size and structure of protein complexes. However, its usefulness in such endeavors is largely dependent on the ability to accurately measure and correlate both intact assemblies and their protein building blocks in the gas phase to biologically-relevant structures in solution. Previous data have indicated that protein complex charge state has a demonstrated, yet currently unclear, influence on the dissociation pathways available to complexes upon collisional activation. Here, experiments designed to evaluate many different methods for ion charge state manipulation are described in the context of their potential applications in structural biology. In addition, the charge state-dependent mechanisms by which the building blocks of protein complexes are revealed via collisional activation in the gas phase are studied in detail, uncovering new intermediates and predicative correlations. Following a comprehensive introductory chapter, Chapter 2 describes a detailed set of experiments aimed at evaluating the relative merits of different charge manipulation protocols for protein complex structure analysis. Gas-phase methods, such as ion-neutral chemistry performed in the source region of the instrument, are found to provide a superior ability to reduce protein charge without leading to unwanted protein unfolding. In Chapter 3, the dissociation pathways of two protein complexes that take near-identical product ion formation pathways when high charge states are considered, are studied in detail revealing the role of previously-unknown compact states in their dissociation mechanisms upon charge state reduction. In Chapter 4, ion-ion chemistry is used to rapidly screen a relatively large number of charge-reduced protein complexes for charge states at which collision induced dissociation and unfolding energy thresholds converge, producing the first relationship capable of predicting the amount of charge reduction necessary to shift the dissociation mechanism of collisionally activated protein complexes generally toward the ejection of compact, native-like product ions. In Chapter 5, a final summary of this work is presented, along with a projected outlook of future endeavors in this area.