Project Report

Glycosylation is an exceedingly common biological process in which a glycan (essentially, a large carbohydrate chain) is enzymatically attached to a protein. Glycosylated proteins, aka glycoproteins, are involved in a variety of important biological roles ranging from structural organization to cellular signaling and molecular transport. Alterations in the molecular structure of glycans are associated with a number of biological changes including obesity, inflammation, pregnancy/lactation, and cancer. However, previous attempts to study specific glycan/glycoprotein structure and function have been hampered by the sheer variety and complexity of glycosylation. This project looked for ways to utilize new technologies for structural analysis of biomolecules, such as nano-flow ultra high performance liquid chromatography (nano-UHPLC), ion mobility spectrometry (IMS), and electron-transfer dissociation (ETD), to obtain more information about glycan and glycoprotein structure. By incorporating these new technologies into established structural analysis techniques such as mass spectrometry (MS) and collision-induced dissociation (CID), a synergistic increase in structural information was achieved. Isomeric glycans with identical compositions and only slight differences in molecular structure were separated and differentiated. The newly developed methods were applied to the separation of glycans and glycopeptides from complex biological fluids such as human serum and bovine milk. As expected, the integration of these new technologies into existing structural analysis techniques provided far more information than any one technique by itself. In particular, nano-UHPLC and IMS, combined with MS and CID, provided effective and reliable separation and differentiation of structurally-similar glycans. Using this combination, complex mixtures of glycans were subjected to four nearly-orthogonal dimensions of separation: hydrophobicity (from nano-UHPLC), cross-sectional area (from IMS), mass (from MS), and fragmentation pattern (from CID). The multiple dimensions of separation each contribute to create a comprehensive view of the analyte. The incorporation of IMS, in particular, had an unexpected benefit—it identified and removed contaminant molecules that had passed through nano-UHPLC and MS. Though both of these techniques act as a filter, they are not always 100% effective. IMS acted as a third filter which separated contaminants from analytes and improved the specificity of the analysis. The technology platforms developed by this project have immediate applications to the glycomics and glycobiology community as a whole. Using this type of multidimensional analysis, complex glycan mixtures can be quickly profiled down to the structural level. In this way, it will be possible to more comprehensively explore glycosylation and its biological roles.

National Science Foundation (NSF)
Office of International and Integrative Activities (IIA)
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Carter Kimsey
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Hua Serenus
United States
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