Development of Isotopically Labeled Glycoproteins for use as Internal Standards

项目摘要/摘要 准确定量结合在糖蛋白上的糖链的能力有很大的范围。 这意味着什么。过去40年的大量研究表明，这一反常现象 糖基化在几乎所有类型的人类癌症中都会发生，并展示了使用 无论是诊断还是预后方面的糖链标志物。重组人的糖基化反应 蛋白质疗法也被认为具有深远的影响，其中一个更著名的例子 是糖工程导致促红细胞生成素(EPO)血清半衰期延长。因此， 糖蛋白多糖的定量在新的发现中起着重要的作用 各种治疗药物发展的诊断/预后标记物。 目前进行定量糖组分的一个障碍是缺乏广泛可用的 标准糖蛋白和同位素标记试剂，以实现准确的定量。这一问题 由人类进行的实验室间研究强调了多糖的定量 蛋白质组组织(HuPo)和生物分子资源设施协会(ABRF)。 这两项研究都表明，在分析 各参与实验室对中低水平的葡聚糖进行了比较。无法准确地 定量低水平的多糖尤其令人担忧，因为通常是低丰度的多糖 有最大的影响，就像治疗性的人类静脉免疫球蛋白G一样 (IVIG)。 这项提议的重点是开发一系列具有良好特征的标准同位素标记 糖蛋白，使N-和O-连接的糖链的准确、可靠和可重复的分析在 相对和潜在的绝对水平。选择了一组糖蛋白来覆盖 生物制药和学术研究人员的需求。在这里，已知数量的 同位素标记的糖蛋白可以直接添加到样品中，以任何标准进行分析 包括质谱学和葡聚糖定量的程序将通过比较 天然离子与同位素标记离子的比率。

Project Summary/Abstract The ability to accurately quantitate the glycan chains attached to glycoproteins has wide-ranging implications. Numerous studies over the past 40 years have demonstrated that abnormal glycosylation occurs in virtually all types of human cancers, and demonstrates the potential of using glycan markers in either a diagnostic or a prognostic manner. The glycosylation on recombinant protein therapeutics is also known to have profound effects, with one of the better-known examples being the increased serum half-life of erythropoietin (EPO) resulting from glycoengineering. Hence, the quantification of glycoprotein glycans plays important roles from the discovery of new diagnostic/prognostic markers to the development of various therapeutic agents. A current impediment for performing quantitative glycomics is the shortage of widely available standard glycoproteins and isotopically labeled reagents to enable accurate quantitation. The issue with glycan quantitation was highlighted by inter-laboratory studies conducted by the Human Proteome Organization (HUPO) and the Association of Biomolecular Resource Facilities (ABRF). Both of these studies demonstrated errors greater than several hundred percent in the analysis of mid-to-low level glycans were compared across participating laboratories. The inability to accurately quantitate low-level glycans is particularly worrisome since it is often glycans of low abundance that have the greatest impact, as is seen with the therapeutic human intravenous immunoglobulin G (IVIg). The focus of this proposal is to develop a collection of well-characterized standard isotopically labeled glycoproteins to enable the accurate, robust, and reproducible analysis of N- and O-linked glycans at the relative and potentially absolute level. A collection of glycoproteins was selected to cover the needs of both biopharmaceutical and academic researchers. Here, a known quantity of the isotopically labeled glycoprotein can be added directly to the sample, analyzed by any standard procedure that includes mass spectrometry, and glycan quantitation will be provided by comparing the ratios of the native to isotopically labeled ions.