Capturing the Holistic Glycocode through Systems Glycobiology

项目总结与摘要 糖码，或由糖和蛋白质的特定组合产生的整体特征的集合 生物分子，是一种在细胞表面用于交换信息和指导生物过程的货币。 由特定的糖蛋白组合产生的独特的分子表面是信息的关键轴， 然而，由于与研究其相关的许多分析挑战，人们对它们仍然知之甚少 复杂性。来自免疫肿瘤学、病毒病理生物学和其他领域的新证据强调了这一点 我们再也不能承受盲点了。 这一提议产生了一套新技术来捕获 利用质谱学(MS)和化学糖蛋白组学的创新技术开发的糖码。把这些意志放在一起 实现对人类糖码的系统规模的讯问。目标1介绍一种新的细胞表面平台 保留糖-蛋白质模式而不是依赖于传统的以蛋白质为中心的方法的糖蛋白质组学 忽略了多聚糖的成分。通过合成新的化学探针，将易富集的 通过活细胞标记处理细胞表面糖蛋白，我们提供了一种化学 糖蛋白组学平台，用于“捕捉并释放”浓缩完整的糖偶联物，从而提供获得 组合糖码特征。AIM 2伴随着我们的化学糖蛋白组学方法取得进展 在用于鉴定和定量糖码成分(即糖肽、糖蛋白和 多聚糖)。通过使用实时分析来实现自适应仪器控制，我们提高了灵敏度 和糖肽表征的吞吐量。为了补充自下而上的糖肽方法，我们还 建立一种新的自上而下的完整糖蛋白指纹组合糖码修饰方法 使用离子-离子气相化学从典型的具有挑战性的变性物种中提取糖形信息。 在目标3中，我们利用这些工具定义了12种不同细胞类型的人类糖码图谱以进行连接 具有特殊细胞功能的异质性糖码表达。此外，我们将这本地图集扩展到 研究上皮-间充质转化过程中的动态糖码重编程以发现细胞类型 可作为诊断和治疗策略目标的迁徙表型的特定特征。 总而言之，这一提议代表了化学糖蛋白组学和MS方法的重大进步， 为细胞表面生物学提供了长期需要的资源，并为我构建 独立研究生涯专注于糖码调节对人类健康和疾病的影响。关键是 实现这些目标将是在化学合成方面的培训，以制造新的化学糖蛋白质组试剂 在K99阶段，这将使我具备实现我的化学愿景所需的技能 研究糖码的糖蛋白组学。这里提出的工作将是我过渡到 独立，并将使我成为系统糖生物学这一前景光明的领域的领导者。

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.