Monosaccharide identification as a first step toward de novo carbohydrate sequencing: mass spectrometry strategy for the identification and differentiation of diastereomeric and enantiomeric pentose isomers.

De novo carbohydrate sequencing, including monosaccharide identification, largely remains a tremendous analytical challenge. A first step in the complete structural determination of any large polysaccharide is an accurate and robust method for analysis of the constituent monosaccharides. Herein, the first mass spectrometry-based method for the complete identification and absolute configuration determination of all 12 pentose isomers, including the d and l enantiomers for arabinose, lyxose, ribose, xylose, ribulose, and xylulose, is reported. As compared to earlier work to distinguish hexose isomers, the chiral separation of the pentose isomers was significantly more challenging. Specifically, the 12 pentoses are much more structurally similar to one another, with only the axial or equatorial orientation of two hydroxyl groups differentiating among these isomers in their five-membered ring furanose structure and smaller energetic differences between pentose conformations than between hexose conformations. Despite such inherently minimal energetic differences between the 12 pentoses, two unique fixed ligand kinetic method combinations were discovered to achieve chiral discrimination for this set of isomers. This assay can be readily applied to the identification of any isolated pentose monosaccharide using only microgram quantities and a commercial instrument and complements the method to distinguish hexose isomers. A workflow that incorporates this mass spectrometry-based method and thereby could achieve complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide is proposed.