Design and Characterization of Novel Tandem Ion Mobility Mass Spectrometry Instrumentation

Abstract

Native ion mobility (IM) mass spectrometry (MS) is a gas-phase structural biology technique that couples low-pressure, electrophoretic separation of ions with mass detection to provide low-resolution information on the size, shape, and charge of biomolecules such as peptides and protein assemblies. Soft ionization techniques that generate ions from aqueous solutions at biologically relevant pH values minimize the disruption of solution-phase noncovalent interactions in protein ions during gas-phase analysis. In tandem IM, multiple dimensions of IM are separated by ion selection regions that are used to isolate subpopulations of the initial ion distribution. Ion trapping can also be used to store ions between dimensions of IM, which spatially focuses and realigns ion distributions. Tandem IM-MS analysis increases the amount of structural information that can be gained from a sample. Chapter 1 provides an overview of native IM-MS and tandem IM-MS studies of proteins to contextualize the current work. Recently, our lab used the emerging Structures for Lossless Ion Manipulations (SLIM) architecture to construct a tandem IM instrument; this architecture enables operationally unique methods to select and store ions. In Chapter 2, those ion selection and trapping processes are characterized using tandem IM experiments and ion trajectory simulations. It is demonstrated that the DC potentials applied to select ions influence the resulting subpopulations that are isolated. Briefly trapping ions after selection spatially focuses and realigns ion subpopulations, removing contributions from diffusion during the previous IM dimension. Ion trajectory simulations described in Chapter 3 demonstrate that the DC and RF potentials used during ion storage in a trap influence both the position of ions within the trap and also the effective temperature of those ions. Exposure to increasingly high fields can cause higher-energy collisions between the background gas and highly mobile ions, increasing the effective temperature of those ions. The lessons learned from Chapters 2 and 3 were incorporated into the design of a new tandem IM-MS instrument constructed from SLIM devices and described in Chapter 4. This new instrument uses longer IM paths in both IM-MS and tandem IM operational modes and uses SLIM devices to perform all ion selection, trapping, and field-dependent mobility analyses. Analysis of proteins and protein complexes using this new instrument show good agreement with previously characterized IM-MS instrumentation. Additionally, tandem IM analysis of protein ions shows the isolation of subpopulations that contain distinct protein structures. The results of this dissertation will be used in the future to leverage ion trapping and selection to probe the structural stability of isolated protein structures in the gas phase.