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The structural complexity of glycans makes their characterization challenging, not only because of the presence of various isomeric forms of the precursor molecule but also because the fragments can themselves be isomeric. We have recently developed an IMS-CID-IMS approach using structures for lossless ion manipulations (SLIM) combined with cryogenic infrared (IR) spectroscopy for glycan analysis. It allows mobility separation and collision-induced dissociation of a precursor glycan followed by mobility separation and IR spectroscopy of the fragments. While this approach holds great promise for glycan analysis, we often encounter fragments for which we have no standards to identify their spectroscopic fingerprint. In this work, we perform proof-of principle experiments employing a multistage SLIM-based IMS-CID technique to generate second-generation fragments, followed by their mobility separation and spectroscopic interrogation. This approach provides detailed structural information about the first generation fragments, including their anomeric form, which in turn can be used to identify the precursor glycan.

Glycan analysis has evolved considerably during the last decade. The advent of high-resolution ion-mobility spectrometry has enabled the separation of isomers with only the slightest of structural differences. However, the ability to separate such species raises the problem of identifying all the mobility-resolved peaks that are observed, especially when analytical standards are not available. In this work, we report an approach based on the combination of IMSn with cryogenic vibrational spectroscopy to identify N-glycan reducing-end anomers. By identifying the reducing-end alpha and beta anomers of diacetyl-chitobiose, which is a disaccharide that forms part of the common core of all N-glycans, we are able to assign mobility peaks to reducing anomers of a selection of N-glycans of different sizes, starting from trisaccharides such as Man-1 up to glycans containing nine monosaccharide units, such as G2. By building an infrared fingerprint database of the identified N-glycans, our approach allows unambiguous identification of mobility peaks corresponding to reducing-end anomers and distinguishes them from positional isomers that might be present in a complex mixture.

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.

The high isomericcomplexity of glycans makes them particularlydifficult to analyze. While ultra-high-resolution ion mobility spectrometry(IMS) can offer rapid baseline separation of many glycan isomers,their unambiguous identification remains a challenging task. One approachto solving this problem is to identify mobility-separated isomersby measuring their highly resolved cryogenic vibrational spectra.To be able to apply this approach to complex mixtures at high throughput,we have recently developed a Hadamard transform multiplexed spectroscopictechnique that allows measuring vibrational spectra of all speciesseparated in both IMS and mass spectrometry dimensions in a singlelaser scan. In the current work, we further develop the multiplexingtechnique using ion traps incorporated directly into the IMS devicebased on structures for lossless ion manipulations (SLIM). We alsoshow that multiplexed spectroscopy using perfect sequence matricescan outperform standard multiplexing using Simplex matrices. Lastly,we show that we can increase the measurement speed and throughputfurther by running multiple multiplexing schemes using several SLIMion traps in combination with simultaneous spectroscopic measurementsin the segmented cryogenic ion trap.

A method for analyzing a plurality of molecules with cryogenic vibrational spectroscopy including the steps of providing a packet of molecules in a ionized form, injecting the packet into an ion mobility section, spatially separating the ions of the packet into subpackets according to their collisional cross section (CCS), recompressing the subpackets, by removing an empty space between them, loading the ions into a cryogenic ion trap by keeping subpackets with different collisional cross section in a respective separate compartment, cooling the ions in collisions with a buffer gas, tagging the ions by attaching a messenger molecule, sending a pulse to the trap to excite vibrations of the cold, trapped, and messenger-tagged ions, and separately ejecting ion subpacket from the trap into an extraction region of a time-of-flight mass spectrometer and measuring the number of remaining messenger-tagged ions and untagged ions for each subpacket.

A method, system, and computer-readable medium for identifying and creating a database of isomers and isobars of molecules including the steps of performing isomer or isobar separation on molecules to obtain separate isomeric or isobaric molecules, measuring mass-to-charge ratios (m/z) to obtain IR fingerprints of the separate isomeric or isobaric molecules, and storing first data on the mass-to-charge ratios (m/z) and/or the IR fingerprints of the separate isomeric or isobaric molecules to a database.