Composition-dependent MRM transitions and structure-indicative elution segments (CMTSES)-based LC-MS strategy for disaccharide profiling and isomer differentiation.

Background: Carbohydrates exhibit diverse functions and extensive biological activities and are notable in the field of life sciences. However, their inherent diversity and complexity-steaming from variations in isomeric monomers, glycosidic bonds, configurations, etc.-present considerable challenges in structural analysis. Considering these challenges, the disaccharide building blocks with simpler structures could provide more structural information. Although various approaches have been explored, sufficient standards or specialized equipment are required to differentiate and characterize isomers. Therefore, a strategy that addresses these challenges is urgently needed.

Results: A Composition-dependent MRM Transitions and Structure-indicative Elution Segments (CMTSES)-based liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) strategy was developed to comprehensively profile disaccharide units and differentiate isomers. First, the composition-related precursor and structure-specific product ions of disaccharides were generated by QQQ-MS. Thereout, MRM transitions were proposed to enable the comprehensive profiling of disaccharides and rapid annotation of their compositions and saccharide types at both termini. Next, the linkage, composition, and configuration isomers of disaccharides were effectively differentiated and presented characteristic LC elution. Furthermore, low-cost and available "location references" (mannose, galactose, and isomaltose) were sought to define structure-indicative elution segments for the identification of isomeric hexose disaccharides. Building on this foundation, the novel CMTSES-based LC-MS strategy was designed, and its feasibility was further verified by successfully differentiating and identifying mixed homogenous and/or heterogenous disaccharide isomers in real samples. Sufficient structural information was obtained even for those consisting of diversified monomer types.

Significance And Novelty: This strategy comprehensively profiles both major and minor disaccharides and effectively differentiates multiple types of isomers. The use of readily available "location references" facilitated the identification of isomeric hexose disaccharide with reduced dependence on standards, thereby broadening the applicability of this strategy. However, the characterization of disaccharides with other compositions is challenging. Further in-depth investigations into intramolecular hydrogen bond simulation should provide solutions. Additionally, CMTSES-based LC-MS strategy is promising to analyze complex structures and samples.