Uniting Mass Spectrometry and Glycoscience to Investigate Cancer Biology

项目概要/摘要。 蛋白质糖基化是一种普遍的、化学复杂的、生物多样性的翻译后修饰过程。 修饰（PTM）涉及广泛的细胞功能。糖基化在调节中起重要作用 糖基化的变化是细胞增殖和代谢过程的普遍特征， 恶性转化和肿瘤进展。转移，或癌症扩散到非相邻部位 身体，是许多侵袭性癌症的一个特别隐蔽的特征。有证据表明， 糖基化的变化可能反映肿瘤进展和转移潜力的适应性，使得全球 糖基化的表征对于理解癌症侵袭/转移的分子基础至关重要。 尽管糖基化在癌症研究中至关重要，但目前用于表征糖基化的技术还不成熟。 这个PTM还不发达质谱法（MS）是用于分析PTM的金标准，但是化学分析方法是不可靠的。 相对于其它修饰，糖基化的复杂性显著减缓了MS技术的进展。 该建议引入了活化离子电子转移解离（AI-ETD）作为一种新的串联质谱方法 用于完整糖肽的全面表征。红外光活化和 AI-ETD中电子驱动的自由基断裂产生肽序列和聚糖组成信息 在单一串联MS事件中。这消除了在制造过程中所必需的每个前体的多次串联MS扫描。 目前的方法，提高灵敏度和有效地增加一倍的吞吐量。在此应用中，AI-ETD将 在最新一代Orbitrap MS系统（Fusion Lumos）上实施，以利用其强大的数据- 将开发用于糖蛋白组学实验的高通量AI-ETD方法。 这些方法将用于三种等基因人类癌细胞系的糖蛋白质组学表征， 代表黑色素瘤的非转移、中度转移和高度转移形式。通过使表征 数百个糖基和数千个聚糖，这项工作将是最全面的糖蛋白质组学 比较癌细胞系，允许研究癌症侵袭的糖基化特征， 在糖蛋白质组中前所未有的广度和深度。该数据集将促进对 在分子水平上研究癌症转移，并揭示了糖基化在癌症中的作用的新见解。 完成这个项目后，我将寻求在一个卓越的癌症糖生物学博士后培训， 实验室，补充我的研究生研究的技术开发重点。的组合 MS仪器开发和癌症糖科学方面的专业知识将使我能够进行多产的 职业生涯作为一个独立的科学家在生物学和技术的接口和癌症研究的前沿。

Project Summary/Abstract. Protein glycosylation is a prevalent, chemically complex, and biologically diverse post-translational modification (PTM) involved in a wide array of cellular functions. Glycosylation plays essential roles in regulation of cellular proliferation and metabolic processes, and changes in glycosylation are universal features of malignant transformation and tumor progression. Metastasis, or the spread of cancer to non-adjacent parts of the body, is a particularly insidious characteristic of many aggressive cancers. Evidence suggests that specific changes in glycosylation may reflect fitness of tumor progression and metastatic potential, making global characterization of glycosylation crucial to understanding the molecular basis of cancer aggression/metastasis. Despite the critical importance of glycosylation in cancer research, current technology for characterizing this PTM is underdeveloped. Mass spectrometry (MS) is the gold standard for analysis of PTMs, but the chemical complexity of glycosylation has significantly slowed progress of MS technology relative to other modifications. This proposal introduces activated-ion electron transfer dissociation (AI-ETD) as a new tandem MS approach for comprehensive characterization of intact glycopeptides. The combination of infrared photo-activation and electron-driven radical fragmentation in AI-ETD generates peptide sequence and glycan composition information in a single tandem MS event. This eliminates multiple tandem MS scans per precursor that are necessary in current approaches, improving sensitivity and effectively doubling throughput. In this application, AI-ETD will be implemented on the newest generation of Orbitrap MS systems (Fusion Lumos) to capitalize on its robust data- acquisition platform, and high-throughput AI-ETD methods for glycoproteomic experiments will be developed. These methods will be utilized in glycoproteomic characterization of three isogenic human cancer cell lines that represent non-, intermediate-, and highly-metastatic forms of melanoma. By enabling characterization of hundreds of glycosites and thousands of glycans, this work will be the most comprehensive glycoproteomic comparison of cancer cell lines yet, allowing investigation of glycosylation signatures of cancer aggression with unprecedented breadth and depth across the glycoproteome. This data set will advance the understanding of cancer metastasis at a molecular level and reveal new insights into the role of glycosylation in cancer. Upon completion of this project, I will seek postdoctoral training in a preeminent cancer glycobiology laboratory, complementing the technology development focus of my graduate research. The combination of expertise in MS instrumentation development and the glycoscience of cancer will uniquely equip me for a prolific career as an independent scientist at the interface of biology and technology and the forefront of cancer research.