Combining ion mobility spectrometry and cryogenic ion spectroscopy with enzymatic digestion/synthesis for the study of N-linked glycans

Glycans play an essential role in numerous physiological and pathological processes of living organisms. Despite their significant biological relevance, glycobiology remains one of the least explored fields of biochemistry. The intrinsic isomeric complexity of glycans poses a great analytical challenge that limits the study of their functions. For this reason, increasing efforts have been directed toward developing new methods that provide a fast, sensitive and accurate analysis of glycans. In this work, we demonstrate a new multidimensional approach for glycan identification based on their unique infrared fingerprints. Our experiments rely on state-of-the-art technology that combines cryogenic messenger-tagging infrared (IR) spectroscopy and ultra-high resolution ion mobility spectrometry (IMS). Adding a spectroscopic dimension to the current databases allows differentiating even subtle structural details between glycan isomers.The first part of this thesis describes a spectroscopic database approach, in particular, the mechanism for assigning IR spectra to structures that are not contained in our database. The combined implementation of selective enzymatic digestion and collision-induced dissociation (CID) with cryogenic IR spectroscopy provides an unambiguous identification of the primary structure of parent glycan molecules. Each time we add a new species to the database, we can identify it from a mixture based on its spectral fingerprint and obtain a new core structure that can serve to identify larger glycans.In the next part, we demonstrate the combination of ultra-high resolution ion mobility with IR spectroscopy for the identification of isomeric glycans. We used a selective chemoenzymatic approach to synthesize a pure glycan standard that could not be obtained commercially and then used its IR fingerprint spectra to assign mobility-separated positional isomers. Using these results, we then investigated the impact of the host cell line on the glycan profile of a monoclonal antibody (mAb) at the isomer level. We demonstrated that our technique can monitor glycosylation patterns in mAbs and can be used to complement, or even replace, existing methods for establishing the similarity of glycan profiles between biological drugs and their biosimilars. In the last part, we apply our IMS-IR approach to study sialylated N-linked glycans. One part of this work is focused on searching for new glycan biomarkers in blood serum samples obtained from female donors with different stages of breast cancer. Exploiting the differences in glycosylation between healthy and sick individuals, we identified a potential glycan biomarker candidate. Moreover, we showed that adding a CID dimension to IMS-IR provides a way for isomer identification without the need for glycan standards. In the second part of this work, we described the relative quantification of sialylated glycans released from follicle-stimulating hormone (FSH) using ion mobility spectrometry. This method allows a fast and reliable analysis of the content of glycans in their native form directly from a complex mixture.