Combining multi-stage ion mobility spectrometry with cryogenic infrared spectroscopy for glycan analysis

Structural analysis of biomolecules is essential to understanding biological processes. Among the various classes of biomolecules, glycans have gained significant attention recently due to their ubiquitous roles in biological systems. However, study of glycans has lagged behind that of other biomolecules due to the slow development of appropriate analytical tools. Even a small variation in structure can profoundly affect their biological functions, making detailed glycan characterization important. They have a high degree of structural complexity due to isomeric monosaccharide building blocks, various linkage sites (each with alpha or beta stereochemistry), and branching. Conventional analytical approaches often do not provide complete structural information of glycans, necessitating new tools.This thesis presents a new tool for analyzing glycans by combining ultrahigh-resolution ion mobility spectrometry (IMS), collision-induced dissociation (CID), and cryogenic infrared (IR) spectroscopy. The approach enables one to perform CID of mobility-separated species and further mobility separation of isomeric fragments prior to measuring their IR fingerprint. This would help build a spectroscopic database for identifying fragments, which can then be used to reconstruct the precise isomeric form of the parent glycan.The first part of this thesis describes the incorporation of fragmentation capabilities into a pre-existing home-built instrument combining ultrahigh-resolution IMS using structures for lossless ion manipulations (SLIM) and cryogenic IR spectroscopy. We report the design of the first SLIM-IMS-CID device containing a series of on-board traps to perform CID at millibar-range pressures and further mobility separation of the resulting fragment isomers. We then characterize the on-board CID process by comparing the fragmentation pattern of a pentapeptide to that obtained from a commercial mass spectrometer. Furthermore, we demonstrate the approach combining SLIM-based IMS-IMS with cryogenic IR spectroscopy allows us to identify isomeric fragments generated from human milk oligosaccharides, including the anomericity of the glycosidic linkage.In the following part, we present a strategy to identify positional isomers of N-glycans for which isomerically pure standards are difficult to obtain. It is based on generating structurally diagnostic fragments specific to each isomer and comparing their IR fingerprints to a database of small, commercially available standards. The database can be populated by adding the IR fingerprints of newly-identified structures which can be diagnostic of larger glycans. Using a similar approach, we identify reducing-end anomers of several N-glycans by identifying their Y-fragments via comparison of their IR fingerprints to those of small, analytical standards. Using the IR spectra of newly-identified anomers to identify those of larger glycans eliminates the need for a reference in every case. This work demonstrates the potential of IR fingerprinting for glycan analysis without heavy reliance on analytical standards.The last part focuses on development and implementation of SLIM-based (IMS)^3 combined with cryogenic IR spectroscopy. This approach enables reconstruction of parent glycan structures through identification of second-generation fragments using a spectroscopic database. This is instrumental for cases when we do not find a spectral match for the first-generation fragments.