Differentiation of disaccharide isomers via diagnostic-ion-weighted spectral library searching using data from tandem MS analysis of chloride adducts

Abstract

Rare sugars, comprising over 50 known saccharide isomers, have broad significance in food science, pharmaceuticals, and biomedical applications. However, the inherent structural complexity (due to isomerization) and low natural abundance of these saccharides pose substantial analytical challenges to their characterization. Herein, we developed a spectral library searching platform capable of distinguishing disaccharide isomers based on chloride-adducted (M + Cl⁻) tandem MS (MS/MS) data. A set of unique diagnostic fragment ions was generated from each of the 15 disaccharide isomers, allowing the development of a spectral similarity algorithm that incorporates diagnostic-ion weighting to improve isomer differentiation. The resulting diagnostic-ion-weighted scores enabled discrimination among stereoisomers, linkage isomers, and compositional isomers. As a direct infusion MS/MS method, we tested spectral library searching for binary mixtures using both a traditional algorithm without diagnostic-ion weighting and our method based on diagnostic-ion weighting. With our platform incorporating diagnostic-ion weighting, false-positive assignments were reduced from 74 to 21, and true negatives increased from 555 to 608. Finally, we applied the method to analyze complex mixtures such as apple juice, Coca-Cola, and Rhinegeist beer. In all cases, the known disaccharide isomers were confirmed by our diagnostic-ion-weighted library-searching workflow. Collectively, these results suggest that negative-ion MS/MS data derived from conventional collision-induced dissociation can be a powerful method for saccharide isomer characterization, especially when paired with automated spectral library searching algorithms.