GLYCOSAMINOGLYCAN GLYCOMICS FROM ORGAN TISSUES

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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.