PROJECT SUMMARY An extensive array of glycosylation covers the extracellular milieu of higher level organisms. These glycan structures are attached to half of the human proteome and are critical for their function in nearly all biological processes. Monitoring alterations in the structures of glycosylation on glycolipids and glycoproteins can identify new ways of inhibiting infections and pinpoint new targets for detecting and treating autoimmune diseases and cancer. Despite their biological importance, our ability to monitor the vast array of glycan structures and their exact role in disease states remains limited. This is primarily due to the challenges associated with analyzing complex carbohydrate structures, which present an unparalleled level of structural diversity. Tools for large-scale glycomic studies do not provide the level of structural detail necessary to effectively resolve subtle differences in glycan structures. On the other hand, techniques capable of detailed structural elucidation of glycans are slow, experimentally intensive, and often require multiple approaches. This proposal seeks to develop an analytical platform that will enable rapid glycan characterization with a level of structural detail that is unavailable with existing techniques. This proposal aims to incorporate ion mobility (IMS) and gas-phase hydrogen/deuterium exchange (HDX) for glycan structural analysis into a robust mass spectrometry platform. We will use this platform with novel internal standards to analyze a library of monosaccharides, oligosaccharides, glycan chains from glycoproteins, and glycolipids to assemble a database of the ion mobility (Aim 1) and hydrogen/deuterium exchange (Aim 2) properties of biologically relevant carbohydrate ions. Molecular modeling will be used to help interpret and rationalize the different behaviors of each structure and expand our structural reference database in silico (Aim 3). The combination of ion mobility and hydrogen/deuterium exchange will be implemented into a LC-MS platform for Glycan Structural Elucidation on a Chromatographic Timescale “GlySECT” (Aim 4). In parallel, we will develop the software for extracting the data and searching against the databases established in this project to aid in structural elucidation of the glycan chains. This technology has the potential to enable rapid, site-specific, glycan structural determination and provide a much-needed tool for glycobiology, biomarker discovery, and therapeutic protein characterization.