Uniting Mass Spectrometry and Glycoscience to Investigate Cancer Biology

项目摘要/摘要。 蛋白质糖基化是一种普遍存在的、复杂的化学和生物多样性的翻译后 修饰(PTM)涉及广泛的细胞功能。糖基化在调节中起着至关重要的作用 细胞增殖和代谢过程的改变以及糖基化的变化是 恶变和肿瘤进展。转移，或癌症向非邻近部位的扩散 身体，是许多侵袭性癌症的一个特别隐蔽的特征。证据表明，特定的 糖基化的变化可能反映了肿瘤进展的适宜性和转移潜力，使其成为全球性的 糖基化的特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 这种PTM还不发达。质谱(MS)是分析PTMS的金标准，但化学成分 糖基化的复杂性大大减缓了MS技术相对于其他修饰的进展。 该方案引入了活化离子电子转移解离(AI-ETD)作为一种新的串联MS方法 用于完整糖肽的全面表征。红外光活化和红外光敏技术的结合 AI-ETD中电子驱动的自由基碎裂产生肽序列和糖链组成信息 在单个串联MS事件中。这消除了每个前体的多次串联MS扫描，这是 目前的方法，提高了敏感度，有效地使吞吐量翻了一番。在此应用中，AI-ETD将被 在最新一代的Orbitrap MS系统(Fusion Lumos)上实施，以利用其强大的数据- 将开发用于糖蛋白质组实验的高通量AI-ETD方法和获取平台。 这些方法将用于三个等基因人类癌细胞株的糖蛋白组学鉴定 代表非转移性、中转移性和高度转移性黑色素瘤。通过启用以下特性 数百个糖位和数千个糖链，这项工作将是最全面的糖蛋白组学 癌症细胞系的比较，允许研究癌症侵袭性的糖基化特征 糖蛋白质组中前所未有的广度和深度。这一数据集将促进对 在分子水平上对癌症转移的研究，揭示了糖基化在癌症中的作用。 在这个项目完成后，我将在卓越的癌症糖生物学方面进行博士后培训。 实验室，补充我研究生研究的技术开发重点。这两种技术的结合 在MS仪器开发和癌症的糖学方面的专业知识将独特地装备我，使我能够 作为一名独立的科学家，在生物和技术的交界处和癌症研究的前沿。

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.