Method for sequence determination using NMR
The invention relates to methods for analyzing polysaccharides. In particular, compositional and sequence information about the polysaccharides are derived. Some methods use NMR in conjunction with another experimental method, such as, capillary electrophoretic techniques for the analysis.
1. A method for analyzing an oligosaccharide, comprising:
a) performing nuclear magnetic resonance (NMR) spectroscopy and a second experimental method on an oligosaccharide, and
(b) identifying a set of properties of the oligosaccharide based on the result of (a), wherein the set of properties includes the reducing or non-reducing end of the oligosaccharide, and wherein the combination of NMR and the second experimental method provides a measurement of the set of properties.
2. The method of claim 1 , wherein the reducing or non-reducing end is identified by determining a signature of the reducing or non-reducing end.
3. The method of claim 2 , wherein the signature of the reducing end is methylation.
4. The method of any of claims 1 - 3 , wherein the second experimental method includes chemical degradation, end-labeling, the use of a mass tag or capillary electrophoresis.
5. The method of claim 4 , wherein the chemical degradation involves the use of nitrous acid.
6. The method of claim 4 , wherein the mass tag is 2-aminobenzoic acid, semicarbazide, Girard's P reagent, Girard's T reagent, p-aminobenzoic ethyl ester, biotin-x-hydrazide, 2-aminobenzamide, 2-aminopyridine, anthranilic acid, 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein or 8-aminonaphthalene-1,3,6-trisulfonic acid or 2-aminoacridone.
7. The method of claim 1 , wherein the method includes the use of an enzyme.
8. The method of claim 7 , wherein the enzyme is a sulfotransferase or sulfatase, glucuronidase, iduronidase or heparinase.
9. The method of claim 8 , wherein the sulfotransferase or sulfatase is 2-O sulfatase or sulfotransferase, 3-O sulfatase or sulfotransferase, 6-O sulfatase or sulfotransferase or N-deacetylase-N-sulfotransferase.
10. The method of claim 8 , wherein the glucuronidase is D-glucuronidase.
11. The method of claim 8 , wherein the iduronidase is L-iduronidase.
12. The method of claim 8 , wherein the heparinase is heparinase-I, heparinase-II or heparinase-III.
13. The method of claim 1 , wherein the NMR spectroscopy identifies both the reducing and non-reducing end.
14. The method of claim 1 , wherein the oligosaccharide is an HSGAG.