Department of Chemistry, University of Utah, 315 South 1400 East, Room 2020, Salt Lake City, Utah, 84112, USA.
Department of Chemistry, Clemson University, Clemson, South Carolina, 29625, USA.
J Mass Spectrom. 2024 Aug;59(8):e5076. doi: 10.1002/jms.5076.
Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/β anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.
人乳寡糖 (HMOs) 是一类重要的生物分子，负责婴儿脑肠轴的健康发育。不幸的是，由于各种原因，它们的准确特征在很大程度上受到限制 - 可能有 200 多种 HMO 结构，而可用的真实分析标准只有 10 种左右。此外，由于它们许多可能的糖苷键位置和相应的α/β端基异构性，导致它们的异构异质性进一步复杂化了它们的分析。尽管液相色谱串联质谱法仍然是 HMO 分析的金标准，但它常常无法分辨所有可能的异构体，因此需要开发其他正交方法。高分辨离子淌度质谱与质谱联用已成为凝聚相分离的快速替代方法，但在很大程度上仍然仅限于与异构体分辨率相关的定性信息。在这项工作中，我们评估了使用全甲基化来改善环状离子淌度分离与质谱联用分析 HMO 的分辨率和灵敏度。除此之外，我们还使用我们之前建立的校准方案，为全甲基化 HMOs 开发了第一个高分辨碰撞截面数据库。我们设想，这个来自高分辨环状离子淌度质谱 - 质谱的内部参考数据库将极大地帮助 HMO 的准确特征描述，并为现有的基于液相色谱 - 串联质谱的方法提供有价值的正交方法。
