Analyzing glycans cleaved from a biotherapeutic protein using a combination of ion mobility spectrometry and cryogenic ion spectroscopy

The characterization of biomolecules plays a central role in biomedical research. The development of state-of-the-art technologies is needed to make such analyses faster, more sensitive, and more accurate. While mass spectrometry continues to play a central role in biomolecular analysis, the use of multidimensional approaches that combine mass spectrometry, ion mobility spectrometry, and ion spectroscopy have been rapidly developing. This thesis investigates the application of such an approach for the routine analysis of glycans. The first part of this thesis describes combining ultrahigh-resolution ion mobility with cryogenic IR spectroscopy and mass spectrometry for analysis of N-linked glycans. This approach is demonstrated by characterizing four N-glycans cleaved from the therapeutic fusion protein Etanercept that differ in abundance from 1% to 22% of the total N-glycan content. Our results demonstrate that the sensitivity, speed, and resolution of vibrational spectra are sufficient for large-scale N-glycan characterization and identification. In addition to the successful demonstration of our approach for identifying known N-glycans, we developed a strategy to synthesize and identify commercially unavailable positional isomers of N-glycans. This work demonstrates the potential of the technique to become a broadly accessible analytical tool for glycan analysis. In the following part, the ability of our approach to quantify the glycan mixtures is investigated. Spray instability, different ionization efficiency of analytes, and their tagging efficiency complicate the process of quantification. Proposed solutions to these issues are discussed. We also present a study demonstrating that cryogenic infrared spectra of alkali metal complexes of carbohydrates of different complexity, including disaccharides and N-glycans can serve as fingerprints. We showed that the identity of the metal cation has no effect on the IR spectral complexity. In the last part, cryogenic infrared spectroscopy with isotopic substitution in positive and negative ion modes was used for structural characterization of the reverse peptides GRGDS and SDGRG. The recorded cryogenic spectra represent a useful benchmark for theoretical structural predictions due to their sharp and distinct features. This work represents the first step towards the structural determination of these peptides, with ongoing efforts focused on quantum chemical calculations.