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. The goal of this work is to develop a strategy for derivatization of CS oligosaccharides to facilitate their sequencing and quantification using tandem mass spectrometry. A series of CID MS/MS spectra were acquired for CS oligosaccharides corresponding to degree of polymerization 2-8. CS-A is composed primarily of GlcA(1-3)3GalNAc-4-sulfate(1-4) and CS-C of GlcA(1-3)GalNAc-6-sulfate(1-4). CSB is composed of IdoA(1-3)GalNAc-4-sulfate(1-4). This project investigates the effects of reducing terminal derivatives on the information content of tandem mass spectra generated from these molecules. The cleanup step is of primary importance for these studies. Because glycosaminoglycans are sulfated, they have different chromatographic properties than do neutral oligosaccharides. In the past year, we have developed a workup scheme using microcrystalline cellulose spin filters that is proving to be extremely effective for cleaning up the derivatization mixtures. Although particulate microcrystalline cellulose is widely available, it has not been applied to the problem of reductive amination cleanup. CID MS/MS spectra for CS oligosaccharides reductively aminated with 2-anthranilic acid produced tandem mass spectra that were similar in general terms to those of native structures. The product ion patterns of labeled oligosaccharides derived from each of the three main classes of chondroitin sulfate generated from CS were then compared. The abundances of ions that differentiate the isomeric forms were found to be more abundant than those for the native structures. Thus, there appear to be analytical value in the derivatization scheme in terms of the product ion abundances.
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