Capturing the Holistic Glycocode through Systems Glycobiology

项目总结及摘要

PROJECT SUMMARY & ABSTRACT The glycocode, or collection of holistic features arising from specific combinations of glycan and protein biomolecules, is a currency used at the cell surface to exchange information and direct biological processes. The unique molecular surfaces created by specific glycan-protein combinations are a critical axis of information, yet they remain poorly understood due to numerous analytical challenges associated with studying their complexity. Emerging evidence from immuno-oncology, virus pathobiology, and beyond underscore that this blind spot can no longer be afforded. This proposal generates a suite of new technologies to capture the combinatorial patterns of the glycocode using innovations in mass spectrometry (MS) and chemical glycoproteomics. Together these will enable systems-scale interrogation of the human glycocode. Aim 1 introduces a new platform for cell surface glycoproteomics that retains glycan-protein patterns rather than relying on traditional protein-centric methods that ignore the glycan component. By synthesizing novel chemical probes that append an easily enrichable phosphonate handle to cell surface glycoproteins through live cell labeling, we provide a chemical glycoproteomics platform for “catch-and-release” enrichment of intact glycoconjugates that provides access to combinatorial glycocode features. Aim 2 accompanies our chemical glycoproteomics approach with advances in the MS methods used to identify and quantify glycocode constituents (i.e., glycopeptides, glycoproteins, and glycans). Through the use of real-time analyses to enable adaptive instrument control, we improve the sensitivity and throughput of glycopeptide characterization. To complement bottom-up glycopeptide methods, we also establish a novel top-down approach to fingerprint combinatorial glycocode modifications on intact glycoproteins using ion-ion gas-phase chemistry to extract glycoform information from typically challenging denatured species. In Aim 3, we leverage these tools to define a human glycocode atlas across 12 different cell types to connect heterogeneous glycocode expression with specialized cellular functions. Furthermore, we extend this atlas to investigate dynamic glycocode reprogramming during epithelial-mesenchymal transition to discover cell-type specific signatures of migratory phenotypes that can be targeted in diagnostic and therapeutic strategies. In all, this proposal represents a significant advance in chemical glycoproteomics and MS methods, generates a long-needed resource for cell surface biology, and provides a robust foundation for me to build an independent research career focused on glycocode regulation in human health and disease. Key to accomplishing these aims will be training in chemical synthesis to make novel chemical glycoproteomic reagents during the K99 phase, which will equip me with the skills necessary to execute my vision for chemical glycoproteomics to investigate glycocode. Work proposed here will be fundamental in my transition to independence and will establish me as a leader in the promising field of systems glycobiology.