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，糖链公司落后了。 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量化方法 最近增强的比较多糖分析，一种细胞培养标记策略，可以为糖组分提供 SILAC为蛋白质组学提供的优势尚未被描述。因此，我们报告了IDAWG的发展， 谷氨酰胺对氨基糖的同位素检测，用于将差示质量标记结合到糖链中 培养的细胞。在这种方法中，含有酰胺-15N-Gln的培养基用来代谢性标记细胞氨基糖 用重氮气。因为Gln的酰胺侧链是GlcNAc生物合成的唯一氮源， GalNAc和唾液酸，我们已经证明了在小鼠或人类胚胎干细胞培养72小时 在酰胺-15N-Gln介质的存在下，15N几乎完全结合到N-连接和O-连接的糖链中。这个 同位素重的单糖残基为解释糖链断裂提供了额外的信息，还 允许在全MS和MS/MS模式下进行量化。因此，IDAWG很容易实现，但却是一个功能强大的新 糖组学工具箱的定量工具。

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.