Ion Mobility - Mass Spectrometry Methods for Studying Membrane Proteins

Abstract

embrane proteins are important therapeutic targets which play vital roles in cellular function. As protein structure is closely linked to its function, the characterization of membrane proteins is vital to elucidating their involvement in disease and potential druggability. Despite this, membrane proteins are underrepresented in structural databases due to challenges associated with their hydrophobicity and difficulty in obtaining high purity samples. There is an imminent need for methods to study the dynamic structure, folding, and ligand interactions of membrane proteins. Native mass spectrometry (MS) has emerged as a method capable of handling the polydispersity of membrane proteins samples to yield details of their structure and function. Particularly, ion mobility MS (IM-MS) has shown promise in assessing the organization of membrane protein complexes and, through collision induced unfolding (CIU) experiments, the relative stability of gas phase membrane proteins. However, challenges remain in developing robust, high-throughput methods for the study of membrane proteins through IM-MS. In Chapter 2, we develop a workflow for studying ligand binding of the integral translocator protein (TSPO) with the aim of revealing the identity of an endogenous ligand. As part of this workflow, a system for automated de-noising of CIU fingerprints is presented, allowing for the quantitative stability characterization of eight different lipids and ligands. For the first time, quantitative CIU measurements of stability and classification are used to determine that the ligands endogenously bound to TSPO are a mixture of phospholipid isoforms bearing the PG head group. In Chapter 3, we present a workflow for elucidating key differences among disease associated protein variants through IM-MS. Seven mutant forms of peripheral myelin protein (PMP22) are shown to possess differences in dimer formation and gas phase stability that correlate with previously published data about cellular trafficking and disease state. Further, from our analysis, we construct a potential mechanism of how dysregulation of PMP22 leads to disease in which mutations cause destabilization of PMP22 monomers leading to the formation of dimeric complexes that are poorly trafficked in cells. In Chapter 4, we present methods to study three mutant forms of the voltage sensing domain of the KCNQ1 potassium channel. We find that CIU of the KCNQ1 mutants classify well according to their observed level of trafficking in cells, indicating a role for specific structural triggers in KCNQ1 trafficking. The function-based classification represents a more broadly applicable method for studying structure differences in proteins with many known mutations. In Chapter 5, we explore the fundamental relationship between membrane protein solubilization techniques and IM-MS. The integral transmembrane proteins, PMP22 and TSPO, and a monotopic membrane protein, cytochrome P450 3A4, are prepared in multiple solubilization techniques, including micelles, bicelles and, nanodiscs. Noticeable differences in oligomerization are found for TSPO samples liberated from detergent micelles and nanodiscs, and for shifts in the observed charges states are noted. Upon CIU analysis of the complexes, we find that differences in unfolding trajectories and gas phase stability exist for both PMP22 and CYP3A4 when liberated from the varying solubilization agents, which we interpret to relate to the local protein environment. Our experiments do not show that one solubilization method is universally superior at preserving the gas phase structure of membrane proteins as each protein system had different patterns of compaction in terms of CCS, number of CIU transitions, and stability across the solubilization methods.