VALIDATION OF IDAWG IN MESC AND HESC

IDAWG 在 MESC 和 HESC 中的验证

• 批准号: 8363032
• 负责人: Lance Wells
• 金额: $ 14.79万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363032
• 关键词: Amides; Amino Sugars; Anabolism; Cell Culture Techniques; Culture Media; Cultured Cells; Detection; Development; Funding; Glutamine; Grant; Hour; Isotopes; Label; Link; Methods; Monosaccharides; Mus; National Center for Research Resources; Nitrogen; Polysaccharides; Principal Investigator; Proteomics; Reporting; Research; Research Infrastructure; Resources; Sialic Acids; Side; Source; Technology; United States National Institutes of Health; Validation; comparative; cost; human embryonic stem cell; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Robust quantification is an essential component of comparative -omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods developed by ourselves and others have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, had not been described. Thus, we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we have demonstrated that culturing mouse or human embryonic stem cells for 72 hours in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is simple to implement, yet a powerful new quantitative tool for the glycomics toolbox.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 稳健的量化是比较组学策略的重要组成部分。 在这方面，糖组学落后于蛋白质组学。 尽管我们和其他人开发的各种同位素标记和直接定量方法最近增强了比较聚糖分析，但尚未描述一种细胞培养标记策略，该策略可以为糖组学提供 SILAC 为蛋白质组学提供的优势。 因此，我们报告了 IDAWG（谷氨酰胺氨基糖同位素检测）的开发，用于将差异质量标签掺入培养细胞的聚糖中。 在此方法中，含有酰胺-15N-Gln 的培养基用于用重氮代谢标记细胞氨基糖。 由于 Gln 的酰胺侧链是 GlcNAc、GalNAc 和唾液酸生物合成的唯一氮源，因此我们已经证明，在 amide-15N-Gln 培养基存在下培养小鼠或人类胚胎干细胞 72 小时可产生15N 几乎完全融入 N 连接和 O 连接聚糖中。 同位素重的单糖残基为解释聚糖断裂提供了额外的信息，并且还允许在完整 MS 和 MS/MS 模式下进行定量。 因此，IDAWG 易于实施，但却是糖组学工具箱的一个强大的新定量工具。