Enabling Ion Mobility Mass Spectrometry for Glycomics

In the post-genomic era it has become increasingly apparent that biology is far more complicated to what was imagined just a few years ago. In parallel to the discovery of non-coding genetic material and DNA modifications that have revolutionised our view of genomics, the ubiquity of gene-product modifications is necessitating us to reassess the complexity of these proteins. The attachment of sugars to proteins (glycosylation) is the most prevalent of all protein modifications. These so-called glycans influence protein function, ability to binds other biomolecules as well as protein transport within and outside the cell. Furthermore, changes in glycan structure are associated with a wide range of diseases, and these sugar molecules are interesting targets as biomarkers. Despite their great importance however, the identification and assignment of glycans remains a great challenge, due to the complexity of their structures.

Glycans are typically composed of 10-15 monosaccharide residues, arranged as intricate branched structures. As their assembly in the cell is not a template driven, glycans are incredibly diverse assemblies of sugar building blocks, rendering their structural elucidation very difficult. Characterization of a given glycan generally requires the combination of multiple analytical approaches built around the cornerstones of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Current strategies for these experiments are essentially unchanged in >15 years, with the significant improvements in MS instrumentation over that time having had little impact on our ability to characterize glycan structures. Furthermore, the data obtained from these experiments is often complicated and requires considerable time and expertise for its interpretation.

We propose to capitalise on the advent of a new type of MS approach, ion mobility MS (IM-MS) to develop a new tool for modern glycoscience. IM is a unique technology that can separate different glycan structures by their size and shape in an inert gas. We present here proof-of-principle data to demonstrate the potential utility of IM for glycomics, and formulate a new tool for its application. This will be used to impart separation of structures, and through measuring their collision cross-sections (CCS), improve confidence in assignment of unknown glycans. Our proposal is founded on our exploratory studies into understanding how glycan ions migrate in the gas phase. We have shown that the separation, and therefore assignment, of glycans by IM is critically dependent on the presence of specific metal ion adducts. This enables us to delineate a strategy whereby we will measure CCS values of glycan ions and adducts from synthetically and common biologically derived structures.

Importantly, current commercially available IM-MS instrumentation is not capable of absolute CCS measurements and therefore requires calibration prior to analysis. Measurements will be performed on unique instrumentation in Oxford that is able to determine CCS values without the need for calibration. These absolute values will be made available to the community through our database GlycoMob providing both a suitable calibrant dataset and a framework for integrating IM into the LC-MS glycomics pipeline. Finally we will couple IM-MS to HPLC and gas-phase fragmentation for unsurpassed levels of analysis, imparting four characteristic pieces of information for each sample set to 'fingerprint' specific glycans. We anticipate that this tool could revolutionise our approach to glycomics discovery, and the diverse fields of research it underpins.

We propose to develop a novel multi-dimensional analytical tool for glycoanalysis based on ion mobility mass spectrometry (IM-MS). This technology will not only improve current limits in detection but will also significantly add confidence in structural assignments by imparting four specific improvements to current MS approaches. Firstly, owing to unique drift properties of glycan ions compared to non-carbohydrate material; it is possible to probe glycan-specific data alone. Secondly, the ion mobility drift or arrival time of individual glycan structures can be converted to absolute glycan-specific collision cross section (CCS) values that are independent of instrument or laboratory variability. The power of this approach is through cross-referencing a glycan ion precursor mass and fragmentation spectrum to its corresponding CSS value which will help overcome the difficulties arising from the presence of structural isomers. Thirdly, the power of IM-MS will be extended through HPLC coupling to provide quantitative glycan information and high-throughput capabilities. Lastly, to support data interpretation of IM and MS/MS data we will populate a new CCS database, GlycoMob, as well as supplement the existing glycan fragmentation database Unicarb-DB with measurements of glycan standards.

Glycan analysis using IM-MS is a relatively new approach and applications for CCS measurements have principally employed home-built instruments. As a consequence the wide-spread use of IM-MS for glycomics analysis on commercially available instruments has been hindered. Furthermore the added benefit offered from coupled HPLC has yet to be explored. To establish IM-MS as a valuable and robust glycomics method, we will systematically establish a calibration framework for commercial instruments, measure CCSs of diverse synthetic and purified glycan standards and develop a novel LC-IM-MS tool capable of delivering glycan structural information unmatched by current methodologies.

The proposed work is highly relevant to the current call for the development of novel tools and resources and is directly applicable to five BBSRC strategic priorities: 'technology development for the biosciences', 'data-driven biology', 'systems approach to biological research', 'ageing research: lifelong health and wellbeing', and 'increased international collaboration'. We are committed to ensuring that our publicly funded research will allow wide and substantial consequences, in particular with respect to translating fundamental research into impacts on the health and economic prosperity of UK society.

Basic Science:
Glycoscience is a highly interdisciplinary field with implications for research ranging from human health to material science and the development of bioenergy sources. However its study is currently hampered by the limitations of the currently available analytical tools. We propose here to develop a novel approach that has the potential to transform glycan identification, thereby having a profound impact across the glycoscience community. >>100 UK research groups are interested in the study of glycans and oligosaccharides, and at present >60 ion mobility mass spectrometry (IM-MS) instruments are housed in academic and industrial laboratories in this country. However, the lack of an established method and calibration strategy for glycans limits their use. We will take advantage of our unique drift tube IMS instrument (that allows the determination of absolute IM values) to measure a wide range of calibrants and populate a freely available reference database. This will therefore enable unprecedented levels of information for glycoscience research, but also enable new uses for the significant existing IM-MS infrastructure base in the UK

Industry:
Our work is of direct interest to biotherapeutic companies. As of 2010, 77 of the 147 approved biopharmaceutical drugs were glycoproteins, including monoclonal antibodies, growth factors, hormones and fusion proteins, with global annual sales of these drugs at roughly US$50 billion. Furthermore, more stringent guidelines from the European Medicines Agency and US Food and Drug Administration have required detailed reporting of the glycan structures, while the market for rapid, quantitative and accurate methods has experienced a recent and rapid expansion especially with the emergence of biosimilar and chimeric glycoproteins. The impact for industry of novel methods for glycan analysis and the availability of a curated database of known biopharmaceutical glycans to cross-reference experimental values will be highly valuable
Scientific instrument vendors rely on novel applications to stretch their development capabilities. The UK is world-leading in design of MS equipment, but the analysis of glycans has to a certain extent been overlooked due its complexity. However, we think that our novel approach will simplify glycan data interpretation to make it not only attractive to companies that own existing instances of IM-MS instrumentation (>40 in the UK), but also to stimulate the market for such equipment, and future developments thereof. These are sentiments echoed by our collaborator Waters Corporation, a leading MS manufacturer, who are supporting this proposal.

Training:
This proposal will offer excellent learning and hands-on training opportunities for both the PDRA and PhD student through our industrial and academic collaborations. The PhD student will acquire highly sought-after glycan analytical skills from the researcher Co-I, PIs and from Waters which will position her for continued success after the completion of the project. This project will also offer great possibilities for the researcher Co-I, particularly in managing a research programme and translating primary data into a useable resource for the wider community, while interaction with industry and international collaborators will provide an important career building opportunity.