Capturing the Holistic Glycocode through Systems Glycobiology

通过系统糖生物学捕获整体糖码

• 批准号: 10505658
• 负责人: Nicholas M Riley
• 金额: $ 10万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10505658
• 关键词: Address; Aldehydes; Architecture; Atlases; Binding; Biological; Biological Process; Biology; Cell physiology; Cell surface; Cells; Chemicals; Chemistry; Collection; Complement; Complex Mixtures; Custom; Development; Diagnostic; Disease; Epitopes; Fingerprint; Foundations; Gases; Glycobiology; Glycoconjugates; Glycopeptides; Glycoproteins; Goals; Gold; Health; Heterogeneity; Human; Human Biology; Immunooncology; Investigation; Ions; Knowledge; Label; Link; Mass Spectrum Analysis; Membrane Glycoproteins; Methods; Modification; Molecular; Outcome; Pathology; Pattern; Phase; Phenotype; Polysaccharides; Proteins; Proteome; Reagent; Regulation; Research; Resources; Retinal blind spot; Role; Scaffolding Protein; Solid; Surface; System; Technology; Therapeutic; Time; Training; Translating; Virus; Vision; Work; base; biological systems; career; cell type; chemical synthesis; combinatorial; encryption; epithelial to mesenchymal transition; extracellular; glycoproteomics; glycosylation; glycosyltransferase; high throughput analysis; improved; innovation; instrument; new technology; novel; novel strategies; phosphonate; prevent; programs; sedentary; skills; tool

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.

项目概要和摘要 糖码，或由聚糖和蛋白质的特定组合产生的整体特征的集合 生物分子是细胞表面用来交换信息和指导生物过程的货币。 由特定聚糖-蛋白质组合产生的独特分子表面是信息的关键轴， 然而，由于与研究它们相关的许多分析挑战，它们仍然知之甚少。 来自免疫肿瘤学、病毒病理学等的新证据强调了这一点。 再也不能承受盲点了。 该提案产生了一套新技术来捕获组合模式 结合使用质谱 (MS) 和化学糖蛋白组学的创新技术进行糖代码分析。 目标 1 引入了一个新的细胞表面平台，能够对人类糖码进行系统规模的询问。 糖蛋白质组学保留聚糖蛋白质模式，而不是依赖传统的以蛋白质为中心的方法 通过合成附加易于富集的新型化学探针，忽略聚糖成分。 通过活细胞标记磷酸盐处理细胞表面糖蛋白，我们提供了一种化学物质 用于“捕获并释放”富集完整糖复合物的糖蛋白组学平台，可提供 Aim 2 伴随着我们的化学糖蛋白组学方法的进步。 用于鉴定和定量糖码成分（即糖肽、糖蛋白和 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。 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方法来指纹识别完整糖蛋白的组合糖码修饰 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型信息。 在目标 3 中，我们利用这些工具定义跨 12 种不同细胞类型的人类糖码图谱以连接 此外，我们将该图谱扩展到具有特殊细胞功能的异质糖码表达。 研究上皮-间质转化过程中的动态糖码重编程以发现细胞类型 迁移表型的特定特征可以作为诊断和治疗策略的目标。 总而言之，该提案代表了化学糖蛋白组学和 MS 方法的重大进步， 产生了细胞表面生物学长期需要的资源，并为我建立一个坚实的基础 独立研究生涯专注于人类健康和疾病关键的糖码调节。 实现这些目标将进行化学合成方面的培训，以制造新型化学糖蛋白组试剂 在 K99 阶段，这将使我具备实现化学愿景所需的技能 这里提出的研究糖蛋白组学的工作将是我向糖代码过渡的基础。 独立性将使我成为系统糖生物学这一有前途的领域的领导者。