Investigation of Heterogeneous Proteins and Protein Complexes with Native Ion Mobility-Mass Spectrometry and Theory
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Date
2022-10-04
Authors
Rolland, Amber
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Publisher
University of Oregon
Abstract
Native ion mobility-mass spectrometry (IM-MS) offers many advantages for the study of biomolecules and their complexes. High mass accuracy and sensitivity enable unambiguous determination of complex stoichiometries with respect to subunit composition as well as bound ligands. Ion mobility spectrometry adds an additional dimension of separation and can provide some structural information. Native IM-MS experiments are also fast with minimal sample requirements. Because of these reasons, native IM-MS has become an important tool in structural biology, able to investigate challenging samples that may not be amenable to study by other techniques.
However, there are still some major challenges for using native IM-MS in the study of biomolecules. Heterogeneity—arising from the presence of multiple conformations, subunit compositions, ligands and small molecules, for example—results in complicated native mass spectra that can be difficult or even impossible to deconvolute and interpret. Characterizing the heterogeneity of these samples is desirable, as reports of lipids, small drugs, and metals being important for physiological structure and function continue to accumulate. Additionally, interpretation of structural information from IM data has remained largely qualitative, and more fundamental questions about this technique persist, including detailed understanding of the nature of gas-phase protein structure and behavior and how it might differ from solution-phase. Investigation into this aspect is required to make structural interpretation from native IM-MS data quantitative.
In the first half of this dissertation, strategies to overcome the challenges of heterogeneity are explored, and computational methods are developed to solve the quantitation problem. With these methods, key features of gas-phase protein ion compaction are revealed, allowing more informed interpretation of structural details from this technique. The second half of this dissertation illustrates the wealth of information that can be accessed for challenging, heterogeneous biomolecules in native IM-MS experiments upon application of these computational methods. With results from both experiment and computation, oligomeric states of the membrane pore-forming protein toxin Cytolysin A are identified, and the composition and topology of multimeric β-crystallin protein complexes, which are implicated in cataract formation, are characterized.
This dissertation includes previously published and unpublished co-authored material.
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Keywords
gas-phase ion compaction, heterogeneity, ion mobility, molecular dynamics simulations, native mass spectrometry, protein complex