Combining cryognic ion spectroscopy with ion mobility for the study of glycan fragmentation

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 Î± and Î² 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 Î²-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.

Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

Natalia Nagornova Boyarkine

The three-dimensional (3D) structures of proteins and peptides in vivo largely determine their biological functions. In vitro these native structures and their heterogeneity reflect a subtle balance between noncovalent intramolecular interactions and those with the surrounding solvent molecules. Decoupling the intra- and intermolecular interactions and revealing the intrinsic structures of biomolecules in the gas phase is crucial for understanding protein-peptide (-protein, -membrane) binding processes and protein folding, and can assist in silico drug design. In addition to this, the spectroscopic studies of (bio)molecules in the gas phase provide crucial information that serves as a benchmark for validation of theoretical approaches, used for structural computations. In this thesis report we demonstrate the use of conformer-selective, Cold Ion Spectroscopy (CIS), combined with mass spectrometry and electrospray ionization technique, to reveal spectroscopic fingerprints of closed-shell (bio)molecular species in the gas phase. These fingerprints provide a stringent test for validating calculated structures of the molecules. Electronic and conformer-specific vibrational spectra of charged molecules, collisionally cooled in a linear radio frequency 22-pole ion trap have been measured using photofragment detection schemes that employs UV and/or IR laser light. A detailed set of spectroscopic and structural constraints, obtained using CIS method for an antibiotic peptide, gramicidin S (GS) allows an unambiguous determination of its 3D structure in the gas phase. The calculated gas-phase structure of GS differs substantially from its known structure in the condensed phase and, thus, provides complementary information for modeling biological activity of this antibiotic. At the next step we form hydrogen-bonded complexes of GS with a gradually increasing number of water molecules. Spectroscopy of such peptide-water clusters targets to bridge the gap between the gas phase and the in vitro structures. We have shown that both, UV and IR spectra of large GS-water clusters remain vibrationally resolved, if the species are cooled to cryogenic temperatures. The observed detailed spectroscopic fingerprints of the hydrated peptides provide a benchmark for their structural analysis. While CIS method was developed for biological species, here we demonstrate its successful application for structural determination of highly-reactive catalytic metalorganic species. The experimentally observed lack of a characteristic NH stretch vibration in the IR spectra of such a species allowed an unambiguous selection of its structures from several low-energy calculated candidates. The current approach for solving structures of biomolecules implies a comparison of a measured spectroscopic fingerprint with that, calculated for a candidate structure. Unfortunately, high accuracy structural computations become time consuming and challenging for large peptides. We revealed a specificity in electronic spectra of phosphorylated peptides, which coordinates with the docking position of a phosphogroup in peptides. This observation, if confirmed for a large variety of peptides, may allow a prediction of phosphorylation sites without a call for calculations. Regarding a relative simplicity of UV spectroscopy we see a potential of this approach as an orthogonal analytical method that assists mass spectrometry in some puzzling cases of peptide analysis. At the end of this work we tried to push the limits of CIS approach towards very large species and, for the first time, measured IR spectrum of a bare protein, cytochrome c, in the gas phase.

EPFL2011

A gas phase approach for glycan analysis: Combining ultrahigh-resolution ion mobility spectrometry with cryogenic vibrational spectroscopy

Ahmed Ben Faleh

The last decades have been punctuated by the development of revolutionary analytical technologies that have enabled the analysis of biomolecular structures and unraveled the roles they play in biological systems. However, certain classes of biomolecules remain less understood than others. In fact, despite being among the most abundant molecules on our planet, glycans remain among the least understood class of biological macromolecules due to their highly complex structure. The complexity of glycan molecules arises from their isomeric nature, which poses a significant challenge to classical analytical tools. The advent of ionization methods such as ESI and MALDI has led to an unprecedented expansion of gas phase techniques for the analysis of biomolecules. Compared to condensed phase approaches, gas phase analysis allows for isolating specific molecules and collecting highly accurate and specific structural information. In this work, we present an analytical approach for the analysis of glycan molecules based on the combination of different gas phase techniques. A central part of this thesis is the development, design, and characterization of two new instruments that have the capabilities to identify glycan isomers unambiguously. Our experimental approach is based on the combination of high-resolution ion mobility spectrometry with cryogenic IR spectroscopy. Ion mobility spectrometry allows to separate charged molecules in the gas phase according to their overall shape, serving as an isomer-selective prefilter to the IR spectroscopic interrogation process. While IMS provides a way to separate glycan isomers, vibrational spectroscopy is used to identify the mobility-separated molecules. The IR absorptions characteristic of glycan molecules lead to distinct spectroscopic fingerprints, which are perfectly suited for the identification of glycan isomers. In the first part of this work, we report the modification of an existing instrument that combined drift tube IMS with IR spectroscopy. We replaced the drift tube section by IMS device employing a relatively novel technique called SLIM, which provides the highest mobility resolution reported to date while ensuring a minimum loss of ions. This prototype instrument was used in a series of proof-of-concept experiments where we successfully demonstrate that our approach provides highly accurate results, comparable to sophisticated double-resonance spectroscopic schemes. We also describe an analytical protocol that serves to identify the isomeric content of disaccharide and tetrasacharide mixtures unambiguously, using a database approach. Furthermore, we report a series of experiments in which we separate and identify the anomers of disaccharides. In the second part of the thesis, we describe a second-generation instrument based on the same approach. The design of this instrument was aimed at maximizing its sensitivity and throughput while adding more analytical functionalities. It includes a CID section, as well as a cryogenic multi-trap allowing for the multiplexed spectroscopic analysis of glycan isomers. We hereby report the characterization results highlighting the instrument capabilities in terms of ion transmission and IMS resolution. We also report the first rapid, multiplexed, IR spectral acquisition of two tetrasacharide isomers, which emphasizes the unique capabilities of this home-built instrument.

EPFL2021

A New Strategy Coupling Ion-Mobility-Selective CID and Cryogenic IR Spectroscopy to Identify Glycan Anomers

Priyanka Bansal, Ahmed Ben Faleh, Eduardo Carrascosa Casado, Robert Paul Pellegrinelli, Thomas Rizzo, Stephan Warnke, Lei Yue

Determining the primary structure of glycans remains challenging due to their isomeric complexity. While high-resolution ion mobility spectrometry (IMS) has recently allowed distinguishing between many glycan isomers, the arrival-time distributions (ATDs) frequently exhibit multiple peaks, which can arise from positional isomers, reducing-end anomers, or different conformations. Here, we present the combination of ultrahigh-resolution ion mobility, collision-induced dissociation (CID), and cryogenic infrared (IR) spectroscopy as a systematic method to identify reducing-end anomers of glycans. Previous studies have suggested that high-resolution ion mobility of sodiated glycans is able to separate the two reducing-end anomers. In this case, Y-fragments generated from mobility-separated precursor species should also contain a single anomer at their reducing end. We confirm that this is the case by comparing the IR spectra of selected Y-fragments to those of anomerically pure mono- and disaccharides, allowing the assignment of the mobility-separated precursor and its IR spectrum to a single reducing-end anomer. The anomerically pure precursor glycans can henceforth be rapidly identified on the basis of their IR spectrum alone, allowing them to be distinguished from other isomeric forms.