GLYCOSAMINOGLYCAN GLYCOMICS FROM ORGAN TISSUES

这个子项目是许多利用 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。 糖胺聚糖（GAG）是与核心蛋白共价结合的糖链。它们在细胞表面和细胞外基质中丰富，具有广泛的功能。然而，GAG的结构不受直接基因调控，在组成、链长、硫酸化和差向异构化模式方面表现出很大程度的多分散性。糖胺聚糖在不同物种不同组织中的糖组学研究旨在了解糖胺聚糖的结构和数量与衰老、发育、突变和特定疾病的关系。质谱是分析糖胺聚糖结构的有力工具。其使用取决于从组织中提取GAG的通用方法的可用性。我们的目标是开发这样的MS兼容的方法。 用链霉蛋白酶和benzonase消化来自大鼠肝脏、脑和肌肉的干燥脱脂组织（mg）。使用二乙基氨基乙基（DEAE）阴离子交换柱回收所有GAG。在对其他形式的GAG进行彻底酶消化后，通过额外的DEAE程序回收靶向GAG。这些GAG被部分或完全消化，然后被2-邻氨基苯甲酸还原胺化。 这些样品通过酰胺-80正相毛细管柱进行色谱分离，使用Esquire 3000 QIT质谱仪进行在线检测。MS和MS 2均用于二糖和寡糖的分析。用2-氨基吖啶酮还原胺化二糖，采用聚丙烯酰胺涂层毛细管电泳激光诱导荧光检测。

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. Glycosaminoglycans (GAGs) are sugar chains covalently bound to a core protein. They are abundant on the cell surface and in the extracellular matrix, serving a wide range of functions. The structure of GAGs, however, is not subject to direct gene regulation, exhibiting a great degree of polydispersity in composition, chain length, sulfation and epimerization pattern. Glycomic studies of GAGs in different tissues of different species aim to understand how the structures and quantities of GAGs are related to aging, development, mutation and specific diseases. Mass spectrometry is a powerful tool for structural analysis of GAGs. Its use depends on the availability of a general method for extraction of GAGs from tissue. Our goal is to develop such an MS-compatible method. Milligrams of dry, defatted tissues from liver, brain, and muscle of rats were digested with pronase and benzonase. All GAGs were recovered using diethylaminoethyl (DEAE) anion exchange cartridges. The targeted GAGs were recovered by an additional DEAE procedure after exhaustive enzymatic digestion of other forms of GAGs. These GAGs are partially or completely digested, followed by reductive amination by 2-anthranilic acid. These samples were chromatographically separated by an amide-80 normal phase capillary column with on-line detection using an Esquire 3000 QIT mass spectrometer. Both MS and MS2 were acquired to analyze disaccharides and oligosaccharides. Disaccharides reductively aminated with 2-aminoacridone were identified using capillary electrophoresis on a polyacrylamide coated capillary with laser-induced fluorescence detection.