VALIDATION OF IDAWG IN MESC AND HESC

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 稳健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，但尚未描述可以为糖组学提供SILAC为蛋白质组学提供的优势的细胞培养标记策略。 因此，我们报告的IDAWG，同位素检测氨基糖与谷氨酰胺的发展，为纳入差异质量标签到培养细胞的聚糖。 在该方法中，含有酰胺-15 N-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，我们已经证明在酰胺-15 N-Gln培养基存在下培养小鼠或人胚胎干细胞72小时导致15 N几乎完全掺入N-连接和O-连接聚糖中。 同位素重单糖残基为解释聚糖片段提供了额外的信息，也允许在全MS和MS/MS模式下进行定量。 因此，IDAWG易于实现，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.