New Frontiers in Nuclear Magnetic Resonance Spectroscopy: Use of the Nano*NMR Probe for the Analysis of Microgram Quantities of Complex Carbohydrates
We describe here the use of a Nano«NMR probe for obtaining one-and two-dimensional1H nuclear magnetic resonance (NMR) spectra of complex oligosaccharides available in only very small quantities. These results were obtained on glycosaminoglycan (GAG)-type chains isolated from human melanoma cells in culture; however, because there are no intrinsic limita tions to the approach, it is applicable to any situation where an NMR analysis of a limited amount of a carbohydrate is required. The GAG-type oligosaccharides used in this study were obtained by adding /3-xylosides to the cell culture to prime GAG biosynthesis [1,2]. These acceptors diffuse into the Golgi apparatus, where they compete with endogenous core pro teins to make protein-free GAG chains. Freeze and coworkers  had characterized several of the GAG chains produced in this manner by human melanoma cells; however, about 10-15% of the anionic oligosaccharides proved to be resistant to the enzymatic digestions used to successfully ana lyze the majority of oligosaccharides. The use of NMR spectroscopy allowed us to explain the resistance of the minor oligosaccharides to enzy matic digestions and demonstrated the existence of a previously unidenti fied core-related structure.