Shotgun glycomics: linking glycan structure and function

描述(由申请人提供)：我们提交这项资助申请是为了回应美国国立卫生研究院宣布，恢复法资金可用于竞争性修订申请，作为母公司Eureka Grant奖的补充，标题为“鸟枪糖类药物：连接多糖结构和功能”。Shotgan Glycomics是一种纳米级的方法，它允许我们1)荧光标记从细胞或组织糖共轭中释放的所有游离多糖；2)纯化多糖；3)创建代表细胞或组织糖类的标记糖链文库(TGL)。由于多聚糖可能作为效应器分子结合的表面，标记的多聚糖被打印成多聚糖微阵列，并被生物相关的多聚糖结合蛋白(GBP)或生物体所识别。对低聚糖进行测序是极其困难的，只有高度专业化的研究人员才能用高度复杂的设备进行测序。与核酸和蛋白质不同，多聚糖不能被放大。因此，知道序列的价值有限。鸟枪Glycomics使我们能够获得可供审问的微阵列上的多糖结构的档案库，以获得有关功能的知识，以便我们将测序和合成努力集中在相关的多糖上。当我们开发TGL并开始用GBP询问它们时，我们认识到从已知特异性的GBP与未知的糖链结构的结合中可以获得大量的结构信息。例如，植物凝集素的精致专一性几十年来就已为人所知，并已有文献记载。因此，用特定的凝集素在微阵列上分析数百个分离的多糖可以产生每个糖的元数据，并用不同特异性的额外凝集素重复测定，迅速增加了元数据量。我们推断，如果我们包括其他参数，我们应该能够用简单的微阵列分析来确定多糖结构。因此，我们正在请求支持，以开发一种新的方法来研究多糖结构，我们将其命名为“用于多糖结构的元数据分析”。这种方法只需要分子生物学和蛋白质组实验室常用的设备，我们将开发计算工具和算法来将糖-蛋白质结合的元数据转换为糖结构。 与公共卫生相关：与核酸和蛋白质不同，多糖不能被放大。因此，了解多糖序列的价值是有限的。鸟枪Glycomics为功能分析提供了糖链结构的档案库。然而，相同的多聚糖阵列可用于通过使用微阵列多聚糖、多聚糖特定蛋白质和用于元数据解释的计算工具来获得关于所有未知多聚糖的结构数据。

DESCRIPTION (provided by applicant): We are submitting this grant application in response to the NIH announcement of the Availability of Recovery Act Funds for Competitive Revision Applications as a supplement to the parent EUREKA Grant Award entitled, "Shotgun Glycomics: Linking Glycan Structure and Function." Shotgun Glycomics is a nanoscale approach that allows us to 1) fluorescently tag all free glycans released from cell or tissue glycoconjugates; 2) purify the glycans; and 3) create tagged glycan libraries (TGLs) that represent the glycome of the cell or tissue. Since glycans probably function as surfaces for effector molecules to bind, the tagged glycans are printed as glycan microarrays and interrogated for recognition by biologically relevant glycan-binding proteins (GBPs) or organisms. Sequencing oligosaccharides is extremely difficult and only done by highly specialized researchers with highly sophisticated equipment. Unlike nucleic acids and proteins, glycans cannot be amplified. Thus knowing sequences is of limited value. Shotgun Glycomics allows us to obtain an archival library of glycan structures on a microarray accessible to interrogation to gain knowledge about function so that we focus our sequencing and synthetic efforts on relevant glycans. As we developed TGLs and began interrogating them with GBPs, we recognized that massive amounts of structural information were available from the binding of GBPs with known specificity to unknown glycan structures. The exquisite specificity of plant lectins, for example, has been known and documented for decades. Thus, analyzing hundreds of isolated glycans on a microarray with a specific lectin could produce metadata for each glycan, and repeating the assay with additional lectins with different specificities rapidly increased the amount of metadata. We reasoned that if we included other parameters, we should be able to define glycan structures with simple microarray analyses. Thus, we are requesting support to develop a novel approach to glycan structure that we have termed "Metadata Analysis for Glycan Structure". This method only requires equipment common to molecular biology and proteomic labs, and we will develop computational tools and algorithms to convert metadata on glycan-protein binding to glycan structure. PUBLIC HEALTH RELEVANCE: Unlike nucleic acids and proteins, glycans cannot be amplified. Thus, knowing glycan sequences is of limited value. Shotgun Glycomics provides archival libraries of glycan structures for functional analysis. However, the same glycan array can be used to obtain structural data on all of the unknown glycan using "Metadata Analysis for Glycan Structure," a novel approach to glycan structure using microarrayed glycans, glycan specific proteins, and computational tools for metadata interpretation.