VALIDATION OF IDAWG IN MESC AND HESC

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 稳健的量化是比较经济学战略的重要组成部分。在这方面，糖组学落后于蛋白质组学。尽管我们和其他人最近开发的各种同位素标记和直接定量方法增强了比较多糖分析，但还没有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的发展，即谷氨酰胺对氨基糖的同位素检测，用于将差异质量标记整合到培养细胞的糖链中。在该方法中，使用含有酰胺-15N-Gln的培养基来代谢标记重氮的细胞氨基糖。由于谷氨酰胺侧链是生物合成GlcNAc、GalNAc和唾液酸的唯一氮源，我们已经证明，在酰胺-15N-Gln介质中培养小鼠或人类胚胎干细胞72小时，15N几乎完全掺入N-连接和O-连接的糖链中。重同位素单糖残基为解释糖链断裂提供了额外的信息，也允许在全MS和MS/MS模式下进行定量。因此，IDAWG很容易实现，但却是糖组学工具箱的一个强大的新定量工具。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.