Publication
The importance of glycans in biological processes are matched by their structural complexity. Coating the surface of most living cells, glycans play key roles in many biological processes, and the role they play is closely related to their structures. Structural determination of glycans remains however very challenging, due to the isomeric complexity inherent to this class of molecules. Many of the monosaccharides which make up the building blocks of glycans are isomeric, and can link together in various positions, resulting in a vast number of constitutional isomers and anomers. Furthermore, glycan synthesis is not template driven, resulting in glycans being naturally heterogenous, with a wide range of different structures occurring from cell to cell. This thesis presents a new approach, aimed at determining glycan primary structure, by combing collision-induced dissociation (CID) with cryogenic messenger-tagging infrared spectroscopy and ultra-high resolution ion mobility (IMS), performed on home-built state-of-the-art instruments. The first part of this thesis gives an overview of the instrumentation used to carry out the research presented. A detailed account of the addition of a new, ultra-high resolution ion mobility stage to the existing apparatus is provided, along with its characterization. We then investigate the generality of initial findings, showing that glycan C fragments generated by CID from disaccharides retain the anomericity of the glycosidic bond, and demonstrate that this rule extends to larger C fragments than those observed in the initial study and also applies to large, and branched parent molecules. These finding are significant as they imply that C fragments always appear to retain the anomericity of the glycosidic bond from which it was generated, a property that will greatly benefit glycan sequencing. Next, we present a methodology developed, using Y fragments generated from mobility-separated glycans, to identify which mobility-separated species correspond to the α and β reducing-end anomers. This allows us to distinguish the reducing anomers from other structures when studying a mixture of isomeric glycans by IMS. The data obtained from studying C and Y fragments of glycans can be used in a complementary way to build a spectroscopic database, which assigns exact glycan structures to specific infrared spectra. The creation of such a database would allow for rapid and exact identification of glycans, greatly advancing the field of glycomics. Finally, the cyclic oligosaccharide β-cyclodextrin was investigated by spectroscopy and IMS. The structures of its main dissociation products were computed by electronic structure calculations and compared to their experimental vibrational spectra. The fragments observed corresponded in mass to either B-type or Z-type fragments, and the calculated lowest energy structures which match the experimental data seem to indicate that the fragments observed are 2-ketone B fragments. Fur
Nicola Marzari, Lorenzo Bastonero