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 糖密码，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。 复杂性来自免疫肿瘤学、病毒病理生物学等方面的新证据强调， 盲点再也无法承受。 该提案产生了一套新技术来捕获生物的组合模式 利用质谱（MS）和化学糖蛋白质组学的创新技术，这些共同将 使人类糖码的系统级询问成为可能。Aim 1介绍了一种新的细胞表面平台 糖蛋白质组学，保留聚糖-蛋白质模式，而不是依赖于传统的蛋白质中心的方法 忽略了聚糖成分。通过合成新的化学探针， 通过活细胞标记，我们提供了一种化学膦酸盐处理细胞表面糖蛋白， 用于完整糖缀合物的“捕获和释放”富集的糖蛋白质组学平台， 组合糖码特征。目标2伴随着我们的化学糖蛋白质组学方法的进展 在用于鉴定和定量糖编码成分的MS方法中（即，糖肽、糖蛋白和 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 和糖肽表征的通量。为了补充自下而上的糖肽方法，我们还 建立一种新自上而下的方法来对完整糖蛋白上的组合糖码修饰进行指纹分析 使用离子-离子气相化学从典型的具有挑战性的变性物质中提取糖型信息。 在目标3中，我们利用这些工具定义了一个跨越12种不同细胞类型的人类糖编码图谱， 具有特殊细胞功能的异质糖码表达。此外，我们将该图谱扩展到 研究上皮-间充质转化过程中动态糖码重编程以发现细胞类型 可以在诊断和治疗策略中靶向的迁移表型的特定特征。 总之，这一提议代表了化学糖蛋白组学和MS方法的重大进展， 产生了细胞表面生物学长期需要的资源，并为我建立一个强大的基础， 独立研究生涯专注于人类健康和疾病中的糖码调节。关键 实现这些目标将是化学合成的培训，以制造新的化学糖蛋白试剂 在K99阶段，这将使我具备必要的技能，以执行我的化学愿景 糖蛋白组学研究糖密码子。这里提出的工作将是我过渡到 独立性，并将建立我作为一个领导者在有前途的领域系统糖生物学。