Nanogel Electrophoresis for Micro and Nanoscale Analyses of Glycosylation - Glycosylation, and in particular sialylation, is fundamental to molecular structure, function, stability, and signaling. Although glycosylation is a critical biomarker and drug target to improve human health, it is a challenging post-translational modification to study. This is because of the difficulty in distinguishing structurally similar monomeric residues which are connected to form linkage and positional isomers. Moreover, these structures are labile, easily degraded, and reference standards are costly and of limited availability. Research in the Holland lab addresses these challenges to glycan research through miniaturized native enzymatic reactions that are integrated in real-time with separation-based assays. Biocompatible nanogels are used to advance nanoscale reactions by enabling new separation modalities which are critical to evaluating and harnessing enzyme activity. These self-assembled nanogels are thermally reversible separation materials that maintain the biological function of complex biomolecules and provide a means to create and embed multifunctional assays in capillary electrophoresis. Nanogel separations create nanoliter reaction zones to interrogate biomolecules in seconds. The 5-year program outlined in this proposal centers on 3 fundamental goals. Goal 1 generates fast, low-volume assays that quantify enzyme activity in complex systems with optical detection and mass spectrometry under physiological conditions. Monovalent and multivalent interactions that model viral infections are characterized in-capillary at the picogram level. Goal 2 introduces new serial nanoscale processing of glycoproteins in microscale channels by integrating high efficiency nanogel electrophoresis with enzymes and lectins. Heterogeneous glycoprotein ligands are systematically sorted to report the structural distribution of glycans prior to mass spectrometry. Goal 3 creates a glycan foundry; thereby, providing a tool to customize standard synthesis through an automated microscale enzyme-based remodeling. An automated capillary electrophoresis method is adapted to trimming and rebuilding custom glycan substrates and ligands within minutes. The overall vision of the research program is to develop enabling biotechnology tools to advance fundamental studies of glycosylation. With new microchannel separations the nanoliter reaction zones can be used to elucidate biomolecular interactions that lead to infection and disease, interrogate glycan structures with unprecedented accuracy, and provide a new platform to researchers to customize the synthesis of glycoforms in-house to advance research in human health related to glycobiology.
