Advancing Glycan Analysis: Cryogenic Ion Spectroscopy for Database-Driven Identification

Glycans play a pivotal role in both physiological processes; however, the field of glycobiology remains relatively understudied within biochemistry. The intricate complexity of glycans, coupled with their abundance of isomeric forms, presents formidable challenges in their analysis, thereby impeding a comprehensive analysis of their functional significance. The first step towards understanding the precise function of each glycan is the identification of its primary structure. To tackle this, substantial research efforts have been directed towards the development of methods for glycan structural elucidation. Gas phase techniques offer a significant advantage for the detailed characterization of glycans, primarily due to their ability to isolate distinct isomeric forms. However, traditional analytical approaches still fall short as they do not provide complete structural information. This necessitates the employment of new tools to unravel the intricacies of glycans.This thesis demonstrates the use of ion mobility, mass spectrometry, and cryogenic IR spectroscopy for the comprehensive analysis and unambiguous identification of glycans. The initial focus of this research is the enhancement of the sensitivity and speed of these techniques. Subsequently, we introduce a spectroscopic database approach that enables the discrimination of isomeric glycans that have been separated through ion mobility. Additionally, we present the application of spectral decomposition to resolve glycans with overlapping ion mobility profiles.Next, we illustrate the utilization of Hadamard transform to advance the throughput of our technique. This approach allows for simultaneous acquisition of the IR spectra of diverse glycans separated by ion mobility within a single laser scan. After establishing a robust identification procedure for glycans against our database, we showcase the combination of ion mobility spectrometry, collision-induced dissociation, and cryogenic IR spectroscopy for identifying isomeric human milk oligosaccharides not represented in our database. This method involves fragmenting the glycans, identifying the resulting fragments, and subsequently reconstructing the structure of the precursor molecule. This innovative approach expands our glycan database without the necessity for additional standards.The final part of this research focuses on the online integration of liquid chromatography with cryogenic IR spectroscopy. We demonstrate that the advances in acquisition speed facilitate real-time identification of peaks as they elute during liquid chromatographic analysis.