Sizing up viruses: Application of emerging structural mass spectrometry approaches for the characterisation of proteins, nucleic acids and their interactions

Recombinant adeno-associated viruses (rAAVs) have emerged as key gene delivery vehicles and are of growing interest for future gene therapy applications. However, their structural heterogeneity and complex assembly present significant challenges for characterisation, particularly at the intact capsid level. These include resolving capsid protein (VP) stoichiometry, conformational diversity derived from disordered regions, and higher-order structure (HOS). Addressing these challenges requires analytical approaches capable of probing these large, heterogeneous assemblies at both the intact particle and residue level.
Structural mass spectrometry (MS) provides a powerful platform for interrogating such systems. In this thesis, complementary MS-based methodologies, including conventional native MS (nMS), ion mobility nMS (IM-nMS), Orbitrap-based Fourier transform charge detection mass spectrometry (FT-CDMS), and fast photochemical oxidation of proteins (FPOP), were applied to investigate the structure and dynamics of genome-encapsulating rAAV8 capsids. Together, these approaches enable access to intact particle mass, charge distribution, gas-phase stability, and residue-level solvent accessibility.
FT-CDMS is a recent addition to the structural MS toolbox and remains under active development, requiring continued validation, including evaluation of the charge measurement calibration and of the systematic errors in modal mass and modal charge determination, for which comparison with results from conventional nMS is required. Subsequently, this single-particle technique was applied to measure the modal mass and charge of heterogeneous populations. Charge distributions were used here as descriptors of capsid conformational states, providing a semi-quantitative framework for assessing structural heterogeneity in megadalton ensembles.
Under solution conditions designed to mimic stages of transduction, capsid conformational changes driven by pH and endoprotease activity were investigated by FT-CDMS, potentially involving rearrangements of the disordered N-termini of VP1 and VP2. Additionally, genome-containing rAAV8 capsids exhibited destabilisation under physiologically relevant conditions, consistent with a bimodal uncoating pathway. Together with VP stoichiometry analysis, the identification of a putative stable disassembly intermediate provided insight into rAAV assembly.
The mechanism of genome ejection was further investigated by top-down native MS, under charge-modifying and in-source collisional activation conditions using FT-CDMS. These gas-phase stability experiments, informed by complementary results from IM-nMS measurements on model protein complexes, supported a mechanism in which local VP-VP destabilisation facilitates DNA release from the capsid.
Orthogonally, oxidative footprinting, introduced here for the residue-level characterisation of intact virus-like particles, was used to investigate relevant conformational states guided by FT-CDMS findings. Interpretation of oxidation patterns provided semi-quantitative evidence of VP1 and VP2 N-terminal externalisation under solution conditions mimicking the late endosomal environment. In addition, differential hydroxyl radical oxidation, reflecting relative solvent accessibility, indicated a transient protein-nucleic acid interaction, supported by statistical analysis.
Overall, this work establishes a framework for the characterisation of the HOS of megadalton virus like particles under solution conditions that mimic key stages of transduction. This was achieved by combining laser-induced oxidative labelling with single-particle MS approaches. The FPOP workflow enabled residue-level probing of intact ensembles with mapping of amino acid-nucleotide interactions. The CDMS approach enabled the detection of conformers through independently measured ion charge, with interpretations supported by new insights into the electrospray ionisation mechanism. Together, these MS-based methods offer complementary characterisation of capsid structure, stability and assembly.