Robert Paul Pellegrinelli
The importance of glycans in biological processes are matched by their structural complexity. Coating the surfaceof most living cells, glycans play key roles in many biological processes, and the role they play is closely relatedto their structures. Structural determination of glycans remains however very challenging, due to the isomericcomplexity inherent to this class of molecules. Many of the monosaccharides which make up the building blocksof glycans are isomeric, and can link together in various positions, resulting in a vast number of constitutionalisomers and anomers. Furthermore, glycan synthesis is not template driven, resulting in glycans being naturallyheterogenous, with a wide range of different structures occurring from cell to cell. This thesis presents a newapproach, aimed at determining glycan primary structure, by combing collision-induced dissociation (CID) withcryogenic messenger-tagging infrared spectroscopy and ultra-high resolution ion mobility (IMS), performed onhome-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 isprovided, along with its characterization.We then investigate the generality of initial findings, showing that glycan C fragments generated by CID fromdisaccharides retain the anomericity of the glycosidic bond, and demonstrate that this rule extends to larger Cfragments 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 theglycosidic 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, toidentify which mobility-separated species correspond to the α and β reducing-end anomers. This allows us todistinguish 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 aspectroscopic database, which assigns exact glycan structures to specific infrared spectra. The creation of sucha 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 itsmain dissociation products were computed by electronic structure calculations and compared to theirexperimental vibrational spectra. The fragments observed corresponded in mass to either B-type or Z-typefragments, and the calculated lowest energy structures which match the experimental data seem to indicatethat the fragments observed are 2-ketone B fragments. Further investigation of B fragments generated fromother systems may indicate a correlation between the structure of these fragments and the type of glycosidicbond from which they are produced. If this turns out to be the case, then B fragments can also be added to thespectroscopic database as identifiers for glycan structures.
EPFL2021
