Anomeric memory of the glycosidic bond upon fragmentation and its consequences for carbohydrate sequencing

Abstract

Deciphering the carbohydrate alphabet is problematic due to its unique complexity among biomolecules. Strikingly, routine sequencing technologies-which are available for proteins and DNA and have revolutionised biology-do not exist for carbohydrates. This lack of structural tools is identified as a crucial bottleneck, limiting the full development of glycosciences and their considerable potential impact for the society. In this context, establishing generic carbohydrate sequencing methods is both a major scientific challenge and a strategic priority. Here we show that a hybrid analytical approach integrating molecular spectroscopy with mass spectrometry provides an adequate metric to resolve carbohydrate isomerisms, i.e the monosaccharide content, anomeric configuration, regiochemistry and stereochemistry of the glycosidic linkage. On the basis of the spectroscopic discrimination of MS fragments, we report the unexpected demonstration of the anomeric memory of the glycosidic bond upon fragmentation. This remarkable property is applied to de novo sequencing of underivatized oligosaccharides.Establishing generic carbohydrate sequencing methods is both a major scientific challenge and a strategic priority. Here the authors show a hybrid analytical approach integrating molecular spectroscopy and mass spectrometry to resolve carbohydrate isomerism, anomeric configuration, regiochemistry and stereochemistry.

Fig. 1

IRMPD signatures of monosaccharide and disaccharide standards. a N-acetylhexosamines, hexosamines and hexoses. b Individual methyl-blocked anomers of N-acetylglucosamine, glucosamine and glucose. c Isomers of GalGlcNAc: Galβ1,4GlcNAc (black); Manβ1,4GlcNAc (blue); Galβ1,3GlcNAc (purple); Galβ1,6GlcNAc (green); Galα1,4GlcNAc (red); GlcNAcα1,4 Gal (yellow). Symbols from ref.

Fig. 2

Spectroscopic analysis of disaccharides fragments. a Nomenclature of carbohydrates MS fragments, after Domon and Costello. b Monosaccharide fragments of Galβ1,4GlcNAc. c Non-reducing fragments of Galβ1,4GlcNAc; Glcβ1,4GlcMe and Manα1,4GlcNAc. Methyl-anomers of Gal, Glc and Man are shown for comparison. Symbols from ref.

Fig. 3

Illustration of the carbohydrate-sequencing procedure in the case of a crude mixture of chito-oligosaccharides: the degree of polymerisation and the fraction of acetylation are resolved by MS; the set of candidate sequences is reduced by MS/MS analysis; the sequence is fully resolved by spectroscopic analysis of the GlcN fragment. Symbols from ref.