Development of Ion Mobility Mass Spectrometry Methods for High Resolution Structural Characterisation of Oligonucleotides and Proteins

Abstract

Ion mobility mass spectrometry (IM-MS) is one of the fundamental techniques for understanding the structural biology of proteins and nucleic acids. It allows the characterisation of biomolecular structure and conformational dynamics, which is essential for the investigation into disease mechanisms, pharmaceutical drug development and chemical synthesis. Moreover, collision cross section (CCS) values that can be derived from ion mobility measurements provide a robust descriptor that is platform independent and can be easily compared between different instruments. In comparison, retention time (RT) in liquid chromatography (LC) is not as standardisable across systems, for example, if different column chemistries are used. However, IM-MS is yet to become a mainstream technique in the industry. In this thesis, I focus on developing IM-MS methods for characterisation of single stranded oligonucleotides with phosphorothioate modifications, antibody-drug conjugates and protein structure in general. In the introductory chapter, I discuss the role of IM-MS in the conformational characterisation of biomolecules, from monomeric and multimeric proteins with varying degrees of order to intrinsically disordered proteins. The technique's advantages over traditional methods like X-ray crystallography and NMR in studying protein conformational dynamics are highlighted. Chapter 2 introduces a method for the separation and characterisation of isomers of a model 6-mer phosphorothioate oligonucleotide using IM-MS, focusing on the separation of isobaric diastereoisomers into distinct conformer subsets and the exploration of instrumental parameters' influence on the arrival time distribution. Stereoconfigurations were assigned through comparison with a stereopure standard which allowed to examine the metastable and differing nature of diastereoisomeric ion conformations. Chapter 3 extends the investigation to coupling ion mobility mass spectrometry experiments with molecular dynamics simulations for phosphorothioate-modified oligonucleotides and their non-modified counterparts. The groundwork laid for this integrated approach suggests a potential predictive framework for better understanding the conformational space and dynamics of these biomolecules. Insights into the preference for 5' end phosphate and phosphorothioate deprotonation and the resulting conformational diversity are discussed, highlighting the distinct structural variety between modified and unmodified sequences. In Chapter 4, the focus shifts to protein analysis, employing IM-MS alongside 213 nm photodissociation and collisional activation to dissect networks of non-covalent interactions in native-like gas phase protein structures. Facilitated by the integration of multivariate analysis to interpret complex data sets and mapping distinct photofragments onto available crystal structures allowed localising structural changes on protein folding/unfolding. Chapter 5 explores the application of Horner-Wadsworth-Emmons (HWE) olefination for the site-specific modification of proteins and glycoproteins, showcasing the versatility and efficiency of developed chemistry. This chapter also details the optimization of bioconjugation processes for various biological and pharmaceutical applications, supported by IM-MS and LC-MS analyses. CCS measurements and activated ion mobility experiments confirmed preservation of substrate structure at both functionalisation and bioconjugation stages, while intact antibody MS confirmed effective functionalisation of glycan chains targeted by the chemistry. The concluding chapter synthesises the work, reflecting on the use of IM-MS across different projects to analyse biomolecules. It also outlines future directions for further development of ion mobility methods coupled with simulation and their expanded application to biomolecules, particularly in enhancing the analysis of oligonucleotides and understanding their fragmentation mechanisms.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Perdita Barran (Supervisor)