Advancing 'omics discovery via trapped ion mobility spectrometry

To truly appreciate how cells function, we must fully understand the delicate interplay between large macromolecular protein machines and small molecules that modulate their function, biosynthesis or even capturing their short-lived interactions. Among these small molecules are complex branched sugars attached to specific sites on a protein, lipids that specifically interact with proteins that are found on the surface of cells and metabolites that regulate cell homeostasis and signalling pathways. The analysis of these biomolecules is crucial for understanding how chemistry regulates dynamic biological processes as even small changes in a chemical bond can have significant effects in cellular function. However, owing to their inherent structural complexity, this remains a principal challenge in analytical bioscience and consequently we know comparatively little of their biological functions. Another technical challenge is the ability to characterise transient protein interactions, which can be captured using fast reacting crosslinking chemicals that physically attach interacting partners permanently and facilitates their characterisation. Mass spectrometry (MS) is the workhorse for the analysis of these compounds and technical developments in instrumentation are fundamental for progressing our ability to study structure-function relationships and elucidate their roles in cells, tissues and whole organisms.

Sydney Brenner famously stated that "Progress in science depends on new techniques, new discoveries and new ideas, probably in that order" and our proposal is founded on this principle. This new mass spectrometer is perfectly suited for the analysis of these biomolecules and offers several advantages over the current state-of-the-art platforms that cannot accurately identify all structures present in a complex sample. One of the key benefits of the instrument is how isomeric structures, which are molecules with the same atomic composition but assembled in different orientations, can be accurately characterised by the separation of their ion species inside the instrument. This mass spectrometer has a proven track record and has been used extensively for studying proteins; here we aim to exploit this technology for the study of oligosaccharides, lipids, metabolites and chemically crosslinked protein fragments.

The new instrument will be integrated into our laboratory in the Department of Chemistry in Oxford that is dedicated towards the structural analysis of protein complexes - the new instrument is potentially transformative and will have an immediate impact for our research and that of our collaborators. It will also provide a much-needed resource to BBSRC-funded researchers to undertake experiments in specialised fields which are not widely supported (or even absent) in the UK. As biological discoveries hinges on access to state-of-the-art equipment, we are confident our proposal will support BBSRC's remits and priorities, including transformative technologies and frontier bioscience. This multi-user and interdisciplinary instrument will ring a step-change in the analysis of complex small molecules and in our ability to determine their physiologically relevant functions in health and disease processes.

Determining the structure of small, non-linear biomolecules, namely glycans, lipids and metabolites remains a considerable challenge, especially as their biosynthesis is not template driven, like DNA or proteins, and therefore cannot be predicted. This necessitates technologies capable of distinguishing their complex, and often isomeric, structures by first principles. Additionally, many functions are achieved by short-lived interactions among (glyco)protein complexes that are difficult to resolve with conventional structural biology methods. Chemical crosslinking is a powerful method capable of trapping these interactions, yet their characterization by MS also remains difficult due to enormous sample heterogeneity.

The timsToF addresses these issues and represents a potentially transformative technology across several OMICs fields. This is founded on its unique capability, namely trapped ion mobility spectrometry (tims), that helps over overcome sample complexity to enable next-generation analysis of glycans, glycopeptides, lipids, metabolites and chemically crosslinked (glyco)peptides. These ions have distinctive gas-phase properties that enable their separation, including among structural isomers, that not only adds in their characterisation but also their quantification. This additional dimension sets the timsToF above conventional 2D LC-MS/MS instruments, all while maintaining high scan speeds. It achieves this by parallel accumulation serial fragmentation (PASEF), a propriety technology that maintains sensitivity (no ion loss) but also mobility-aligns precursors with their fragmentation data (a key step in structural assignments), further aiding analysis. We have demonstrated firsthand how the timsToF outperforms the most advanced LC-MS and other ion mobility systems for glycans; these proof-of-principle data are significant as characterization represents the first step in linking structure to function among some of the most complex biomolecules in nature.