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.

项目摘要和摘要 由聚糖和蛋白质的特定组合产生的糖座或整体特征的收集 生物分子是一种在细胞表面上用于交换信息和直接生物过程的货币。 特定的聚糖蛋白组合创建的独特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，他们仍然对 复杂。免疫肿瘤学，病毒病理学以及超越强调的新兴证据 盲点再也无法获得。 该建议生成了一套新技术，以捕获 使用质谱（MS）和化学糖蛋白质组学中的创新的糖座。这些愿意在一起 启用对人糖座的系统尺度询问。 AIM 1引入了一个新的细胞表面平台 保留聚糖蛋白质的糖蛋白质组学，而不是依靠传统的以蛋白质为中心的方法 那忽略了聚糖组件。通过综合新的化学问题，这些问题易于富集 通过活细胞标记，我们提供化学物质的膦酸盐手柄到细胞表面糖蛋白 用于“捕获和释放”完整糖缀合物的糖蛋白质组学平台，可提供访问 组合糖代码特征。 AIM 2涉及我们的化学糖蛋白质组学方法随着进步 在用于识别和量化糖座构成的MS方法中（即糖肽，糖蛋白和 通过使用实时分析来启用自适应仪器控制，我们提高了灵敏度 和糖肽表征的吞吐量。为了完成自下而上的糖肽方法，我们也 在完整的糖蛋白上建立一种新型的自上而下方法，以进行指纹组合糖胶的修饰 使用离子离子气相化学从典型挑战物种中提取糖型信息。 在AIM 3中，我们利用这些工具来定义12种不同细胞类型的人类糖座地图集 具有专门的细胞功能的异质性糖座表达。此外，我们将此地图集扩展到 研究上皮 - 间质转变期间的动态糖座重编程以发现细胞类型 可以针对诊断和治疗策略的迁移表型的特定签名。 总的来说，该建议代表了化学糖蛋白质组学和MS方法的重大进展， 为细胞表面生物学生成了长期以来的资源，并为我提供了坚固的基础 独立的研究职业专注于人类健康和疾病中的糖座调节。关键 完成这些目标将是化学合成的培训，以制造新型的化学糖蛋白质组合试剂 在K99阶段，这将使我具备执行化学愿景所需的技能 糖蛋白质组学研究糖果。在这里提出的工作将是我过渡到的基础 独立，并将我成为系统糖生物学承诺领域的领导者。