Capturing Low-Abundance Glycopeptides for Decoding the Glycoproteome

摘要 糖基化是最常见的蛋白质修饰之一，对细胞生存至关重要。糖蛋白 包含关于细胞发育和疾病状态的大量有价值的信息。全球 蛋白质糖基化分析有助于更好地了解糖蛋白的功能和分子 疾病的机制，并导致将糖蛋白确定为生物标志物。然而，它是 由于多糖的异质性，对糖蛋白的综合分析具有极大的挑战性 以及许多糖蛋白丰度较低。该项目的目标是开发一种创新和 丰富多种糖链结构的糖肽，特别是低丰度糖肽的有效方法， 并将该方法应用于蛋白质N-糖基化和O-糖基化的全局性和定点质谱法分析 光谱(MS)。在强劲的初步数据指导下，这一目标将通过追求四个具体目标来实现 目标。1)通过使用不同类型的 树状大分子。基于每个糖链都含有多个羟基的共同特点，提出了一种新的方法 受益于一个多糖和多个硼酸(BA)分子之间的协同作用 连接到一个树枝状大分子将被开发来捕获低丰度的糖肽。不同类型的 将合成和测试树枝状大分子，特别是从包含1→3分支基序的单体中 将增加BA在树枝状大分子表面的密度，并加强与多糖的相互作用。2) 通过最小化空间效应和形成三元络合物来增强协同作用。 将研究不同种类的BAS，特别是小尺寸的乙烯基硼酸。这会降低立体感 阻碍和加强一种多糖与bas之间的整体相互作用。此外，该组织的形成 将对三元络合物进行研究，以进一步增强相互作用。3)O-的全球和特定地点的分析 含有糖链结构信息的糖蛋白。通过可逆的共价相互作用，富含糖肽 含有完整的多聚糖，可以利用多聚糖的结构信息对O-糖蛋白进行定点分析。 这对于O-糖基化尤其重要，因为缺乏一种酶来普遍裂解O-糖链和 生成通用标签。4)组织和血清中糖蛋白的综合分析 卵巢癌。将所提出的方法与多重蛋白质组学相结合，检测临床样本中的糖蛋白 将进行系统的、定量的分析。这些结果将提供对分子的洞察。 疾病的机制，并导致发现早期检测的生物标记物。最终，最好的 与右旋BA偶联的树枝状大分子将使我们能够有效地捕获低丰度的糖肽。 由于该方法操作简单，不受样品限制，在生物医学领域将有广泛的应用前景。 生物和生物医学研究领域，并将极大地推动糖科学。

SUMMARY Glycosylation is one of the most common protein modifications and is essential for cell survival. Glycoproteins contain a wealth of valuable information regarding the development and disease statuses of cells. Global analysis of protein glycosylation aids in a better understanding of glycoprotein functions and the molecular mechanisms of disease, and leads to the identification of glycoproteins as biomarkers. However, it is extraordinarily challenging to comprehensively analyze glycoproteins because of the heterogeneity of glycans and the low abundance of many glycoproteins. The objective of this project is to develop an innovative and effective method to enrich glycopeptides with diverse glycan structures, especially those with low abundance, and apply this method to globally and site-specifically analyze protein N- and O-glycosylation by mass spectrometry (MS). Guided by strong preliminary data, this objective will be fulfilled by pursuing four specific aims. 1) Effective enrichment of glycopeptides through the synergistic interactions using different types of dendrimers. Based on the common feature that every glycan contains multiple hydroxyl groups, a novel method benefiting from the synergistic interactions between a glycan and multiple boronic acid (BA) molecules conjugated to one dendrimer will be developed to capture low-abundance glycopeptides. Different types of dendrimers will be synthesized and tested, especially from monomers containing the 1→3 branching motif that will increase the density of BA at the dendrimer surface and enhance the interactions with a glycan. 2) Enhancement of the synergistic interactions by minimizing the steric effect and forming the ternary complex. Different kinds of BAs will be studied, especially vinylboronic acids with a small size. This will decrease the steric hindrance and strengthen the overall interaction between one glycan and BAs. Moreover, the formation of the ternary complex will be studied to further enhance the interactions. 3) Global and site-specific analysis of O- glycoproteins with glycan structure information. Through reversible covalent interactions, enriched glycopeptides contain intact glycans, allowing for site-specific analysis of O-glycoproteins with glycan structure information. This is especially important for O-glycosylation due to the lack of an enzyme to universally cleave O-glycans and generate a common tag. 4) Comprehensive analysis of glycoproteins in tissues and sera from patients with ovarian cancer. Combining the proposed method with multiplexed proteomics, glycoproteins in clinical samples will be systematically and quantitatively analyzed. The results will provide insights into the molecular mechanisms of the disease and lead to the discovery of biomarkers for early detection. Eventually, the best dendrimer conjugated with the right BA will enable us to effectively capture low-abundance glycopeptides. Because of the ease of operation and no sample restrictions, the method will have extensive applications in the biological and biomedical research fields and will significantly advance glycoscience.