Shotgun glycomics: linking glycan structure and function

描述（由申请人提供）：我们提交此拨款申请是为了响应NIH关于竞争性修订申请恢复法案基金可用性的公告，作为标题为“Shotgun Glycomics：链接聚糖结构和功能”的父EUREKA拨款奖的补充。散弹枪糖组学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结合物释放的所有游离聚糖；2)纯化多糖；3)创建标记的聚糖文库（TGLs），代表细胞或组织的糖。由于聚糖可能作为效应分子结合的表面，标记的聚糖被打印成聚糖微阵列，并被生物学上相关的聚糖结合蛋白（GBPs）或生物体识别。测序寡糖是极其困难的，只有高度专业化的研究人员与高度复杂的设备完成。与核酸和蛋白质不同，聚糖不能被扩增。因此，知道序列的价值是有限的。Shotgun Glycomics允许我们在微阵列上获得聚糖结构的档案库，以获取有关功能的知识，以便我们将重点放在相关聚糖的测序和合成工作上。当我们开发TGLs并开始用GBPs询问它们时，我们认识到，从已知特异性的GBPs与未知聚糖结构的结合中可以获得大量的结构信息。例如，植物凝集素的独特特性已经为人们所知并记录了几十年。因此，用特定的凝集素在微阵列上分析数百个分离的聚糖可以产生每个聚糖的元数据，并且用不同特异性的其他凝集素重复分析可以迅速增加元数据的数量。我们认为，如果我们包括其他参数，我们应该能够通过简单的微阵列分析来定义聚糖结构。因此，我们请求支持开发一种新的聚糖结构方法，我们称之为“聚糖结构元数据分析”。该方法只需要分子生物学和蛋白质组学实验室常用的设备，我们将开发计算工具和算法，将有关聚糖-蛋白质结合的元数据转换为聚糖结构。

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.