Developing Ion Mobility-Mass Spectrometry for Measurements of Solution-Relevant Protein Structures.

Abstract

Ion mobility-mass spectrometry (IM-MS) has emerged as a robust tool in structural proteomics. However, native solution protein structure must be retained in the gas phase for accurate models to be determined by IM-MS.

To meet this challenge, we screen a broad set of anions and cations separately for their ability to stabilize protein structure in the absence of bulk solvent in Chapter 2 and 3, respectively, and find different mechanisms of stabilization. Cations tend to tightly bind protein complexes and act to reduce Coulombic unfolding. By contrast, anion-protein complexes exhibit a ‘dissociative cooling’ type mechanism. These differences prompt us to study the combined effects of tuned salt-pairs in Chapter 4, where we further reveal cation stabilization mediated by tethering the regions of protein structure. In Chapter 5, we use cation charge carriers to demonstrate that reduced charge mobility is a key parameter in altering the energetic thresholds associated with the gas-phase compaction for ring-like multiprotein complexes.

In addition to the challenges delineated above, many proteins exist in a range of conformational states in solution that subtly depend upon the local environment. In chapter 6, we report on the ability of a lectin tetramer, concanavalin A, to misfold in solution by exposure to denaturants, such as acid and organic solvents, or by multiple freeze-thaw cycles. We then demonstrate that this misfolded tetramer can be recovered to a more native-like state by adding specific Hofmeister-type salts in solution, and that these transitions can be followed using electrospray ionization coupled to IM-MS.

Moreover, a significant challenge in using MS to define the stoichiometry of unknown protein complexes involves the formation of non-specific protein-protein interactions during protein ionization/desolvation. In Chapter 7, we highlight the capacity of IM-MS to distinguish specific versus nonspecific quaternary structures in the case of bovine glutamate dehydrogenase and serum amyloid P component, by identifying those conformers with a clear concentration dependence.

Future endeavors will be made to develop new general strategies to stabilize proteins in the gas phase, and further explore IM-MS to distinguish monoclonal antibody disulfide variants and dimerized assemblies, critical in the process and formulation development of biotherapeutics.