Varun Sharma: Enhancing mass spectrometry for structurally resolved spatial and shotgun lipidomics: Improving sensitivity, specificity, and molecular coverage

Abstract

Direct-infusion mass spectrometry provides an efficient approach to molecular analysis by eliminating chromatographic separation and enabling rapid, high-throughput data acquisition. However, its application to complex biological samples is often limited by insufficient sensitivity, restricted molecular coverage, and challenges in achieving confident structural annotation, particularly for low-abundant and isomeric lipid species. This thesis aims to address these limitations by advancing direct-infusion mass spectrometry in both bulk and mass spectrometry imaging (MSI) workflows to accurately detect and identify isomers and challenging lipid classes, such as steroids and oxidised neutral species. 

A major component of this work explores the use of silver cationisation to enhance sensitivity and structural specificity. Silver cationisation substantially improves the detection of steroid isomers by inducing gas-phase conformational changes, which increases collisional cross-sections in ion mobility mass spectrometry and promotes charge-retention fragmentation in tandem mass spectrometry. In particular, for steroids, charge-retention fragmentation occurs along the steroid backbone and α-cleavage at the carbon adjacent to the hydroxyl group, enabling reliable localisation of functional groups and differentiation of structural isomers. When coupled with liquid-liquid extraction, silver cationisation extends the molecular coverage of shotgun lipidomics to steroids and oxidised neutral lipids. Moreover, integration with pneumatically assisted nanospray desorption electrospray ionisation mass spectrometry imaging, silver adduct fragmentation enables structurally resolved spatial mapping of biologically relevant steroids. 

This thesis also addresses broader challenges associated with tandem mass spectrometry imaging (MS2I), including acquisition speed, scale, and interpretability. Specifically, a parallel acquisition workflow was established and described that includes simultaneous acquisition of high-resolution MSI and large-scale (>100) MS2I datasets, and increased confidence in annotation. For the latter, a spatial-correlation-based approach, spatial similarity networking (SSN), was developed to deconvolute chimeric MS2 spectra by grouping product ions based on their shared spatial distributions. The combination of MS2I and SSN provides a previously unexplored dimension for structural interpretation in MSI.

Collectively, the developments and workflows presented in this thesis expand the analytical capabilities of direct-infusion mass spectrometry, enabling more sensitive detection, greater structural specificity, and broader molecular insights in both spatial and shotgun lipidomics.