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. Reductive amination is derivatization procedure that is widely used in glycoconjugate chemistry. The addition of a hydrophobic group improves signal response, regardless of ionization method, and for this reason has been of considerable interest to the mass spectral community. The addition of a chromophore or fluorophore also carries the advantage of improving the detectability of oligosaccharides. A number of core and collaborative projects at the Mass Spectrometry Resource use this chemistry to facilitate the analyses of different glycan classes. Disaccharides produced from enzymatic depolymerization of GAGs may be derivatized with fluorescent tags to facilitate their detection using capillary electrophoresis. Reductive amination is also being used to isotopically label oligosaccharides for the purposes of mass spectrometric quantification for glycomics applications. All uses of reductive amination hinge on the ability to recover the material after the reaction. The reaction is typically carried out in an organic solvent such as DMSO in the presence of excess amine and sodium cyanoborohydride. After the reaction, the derivatized oligosaccharide must be recovered. Despite the wide use of this chemistry, methods for the recovery of small quantities of derivatized oligosaccharides are lacking. Traditional methods involve applying the reaction mixture to filter paper and washing the excess organic reagents away with organic solvent. The oligosaccharide are then eluted with water. We sought to maximize oligosaccharide recovery using microspin columns filled with commercially available microcrystalline cellulose. The results showed recovery of low picomol quantities of oligosaccharides after the reductive amination step. Oligosaccharides purified in this way produced superior capillary electropherograms. In addition, such mixtures could be directly applied to mass spectrometry.
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