MALDI O-antigen glycotyping of Y. pseudotuberculosis using DAN/DHB/K matrix

Yersinia pseudotuberculosis is a zoonotic gram-negative bacterium with a wide range of hosts. Twenty-one classifications of O-antigens of Y. pseudotuberculosis, including their glyco-subtypes, have been reported and used as epidemiological indicators of health damage caused by this bacterium. In this study, we performed rapid identification of Y. pseudotuberculosis O-antigens using a modified MALDI O-antigen glycotyping method. By employing a DAN/DHB/K matrix, the O-antigen repeating units were detected as potassium adduct ions, facilitating the discrimination between hexoses, deoxyhexoses, and dideoxyhexoses, compared to the conventional matrix system with sodium ions. The branched dideoxyhexose of Y. pseudotuberculosis O-antigens was eliminated by the pretreatment or ionization process of MALDI glycotyping, and the repeating unit pattern reflecting the main-chain glycan sequence was given as the main spectrum. Of the four strains investigated, O2b and O3 polysaccharides with the same main-chain glycan sequence gave characteristic repeating unit pattern peaks, reflecting the difference in the position and stereochemistry of the branching dideoxyhexose. This study will accelerate the implementation of intraspecies classification targeting O-antigens in microbial identification techniques using MALDI-TOF MS.