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.

中文翻译：

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用于二糖分析和异构体区分的组成依赖性 MRM 通道和基于结构指示性洗脱片段 （CMTSES） 的 LC-MS 策略

 背景

碳水化合物表现出多种功能和广泛的生物活性，在生命科学领域引人注目。然而，它们固有的多样性和复杂性（由异构体单体、糖苷键、构型等的变化产生）给结构分析带来了相当大的挑战。考虑到这些挑战，具有更简单结构的二糖构建单元可以提供更多的结构信息。尽管已经探索了各种方法，但需要足够的标准品或专用设备来区分和表征异构体。因此，迫切需要一种应对这些挑战的策略。

 结果

开发了一种基于组成依赖性 MRM 通道和结构指示性洗脱片段 （CMTSES） 的液相色谱-三重四极杆质谱 （LC-QQQ-MS） 策略，以全面分析二糖单元并区分异构体。首先，通过 QQQ-MS 生成二糖的组成相关前体和结构特异性子离子。因此，提出了 MRM 通道，以便能够全面分析二糖并在两个末端快速注释其组成和糖类型。接下来，有效区分二糖的键、组成和构型异构体，并呈现特征性 LC 洗脱。此外，寻求低成本和可用的“位置参考”（甘露糖、半乳糖和异麦芽糖）来定义结构指示性洗脱片段，以鉴定异构己糖二糖。在此基础上，设计了基于 CMTSES 的新型 LC-MS 策略，并通过成功区分和鉴定实际样品中的混合均相和/或异质二糖异构体进一步验证了其可行性。即使对于由多种单体类型组成的 RNA，也获得了足够的结构信息。

 意义和新颖性

该策略全面分析了主要和次要二糖，并有效地区分了多种类型的异构体。使用现成的“位置参考”有助于鉴定异构己糖二糖，减少了对标准品的依赖性，从而扩大了该策略的适用性。然而，用其他组合物表征二糖是具有挑战性的。对分子内氢键模拟的进一步深入研究应该会提供解决方案。此外，基于 CMTSES 的 LC-MS 策略有望分析复杂的结构和样品。