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

) 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.