Development of Isotopically Labeled Glycoproteins for use as Internal Standards

项目总结/摘要 准确定量连接至糖蛋白的聚糖链的能力具有广泛的应用前景。 影响过去40年的大量研究表明， 糖基化发生在几乎所有类型的人类癌症中，并证明了使用 聚糖标志物的诊断或预后的方式。重组蛋白的糖基化 蛋白质疗法也被认为具有深远的影响，其中一个较知名的例子是 是由糖工程引起的促红细胞生成素（EPO）的血清半衰期增加。因此，我们认为， 糖蛋白聚糖的定量在发现新的 诊断/预后标志物对各种治疗药物的开发。 目前进行定量糖组学的障碍是缺乏广泛可用的 标准糖蛋白和同位素标记的试剂，以实现精确定量。问题 由人类研究所进行的实验室间研究强调了聚糖定量 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，在分析中， 在参与实验室之间比较了中低水平聚糖。无法准确 定量低水平聚糖尤其令人担忧，因为通常是低丰度的聚糖， 具有最大的影响，如治疗性人静脉注射免疫球蛋白G所见。 （免疫球蛋白）。 这项建议的重点是开发一个收集良好的特点标准同位素标记 糖蛋白，使准确，稳健，和可重现的分析N-和O-连接的聚糖， 相对和潜在的绝对水平。选择一组糖蛋白以覆盖 生物制药和学术研究人员的需求。在这里，已知量的 同位素标记的糖蛋白可以直接加入到样品中，通过任何标准进行分析 将通过比较提供包括质谱和聚糖定量的程序 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.