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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 鲁棒定量是比较 - 摩西策略的重要组成部分。 在这方面，糖基因落后于蛋白质组学。 尽管我们自己和其他人开发的各种同位素标记和直接的定量方法最近增强了比较聚糖分析，但一种细胞培养标记策略，可以为Silac提供的蛋白质组学提供的优势提供糖基因，但尚未描述。 因此，我们报告了IDAWG的发展，与谷氨酰胺对氨基糖的同位素检测，以将差分标签掺入培养细胞的聚糖中。 在这种方法中，含有酰胺-15N-GLN的培养基用于代谢标记具有重氮的细胞氨基糖。 由于GLN的酰胺侧链是GlcNAC，GalNAC和唾液酸生物合成的唯一来源，因此我们已经证明，在酰胺15N-GLN培养基存在下，在几乎完全融入N-GLN培养基的结果中，将小鼠或人类胚胎干细胞培养72小时。 同位素沉重的单糖残基提供了解释聚糖碎片的其他信息，还允许在完整的MS和MS/MS模式下进行定量。 因此，IDAWG易于实现，但它是Glycomics Toolbox的功能强大的新定量工具。