Advancing MS spatial resolution and documentation for glycosaminoglycans by sulfate-isotope exchange.

Glycosaminoglycans (GAGs) are carbohydrate polyionic polymers that participate in a host of critically important biological processes. A significant difficulty in the comprehensive structural characterization of GAGs is the determination of specific sulfation position isomers. We chose to circumvent sulfate lability by its liberation followed by specific isotope exchange that makes it amenable to methylation, collisional induced dissociation, and MS disassembly for a detailed structural characterization. A set of chemistries that include sulfate release, isotopic (CD- and CD-CO-) replacement, and methylation have been modified to yield a stable product ideal for sequencing by MS. Disassembly of these samples provides a detailed read-out of sequence inclusive of all sulfation sites. As documenting steps, we applied these chemical modifications to a series of disaccharides and a synthetic GAG pentamer, Arixtra®. Upon disassembly, glycosidic and cross-ring cleavages define the monomer composition including individual sulfation positions. The N- and O-sulfates are differentiated by deuterium-containing mass compositions. The uronic methylesters do not significantly alter the fragmentation patterns. A fragment library of these products is being assembled as an adjunct to our larger fragment library, some 15 years in the making.