A bioanalytical research program to unravel the human milk glycome

项目总结 虽然人类乳寡糖(HMOS)参与了各种生物过程，使 婴儿脑肠轴的健康发育，真正了解他们的分子结构和 决定这些功能的成分在很大程度上仍然是一个谜。医疗保健组织在很大程度上兼具这两种特征 结构的复杂性和异构体的异质性，因为它们有许多可能的连锁定位排列 和单糖组成排列。因此，改进的分析进步对于 解开这个异构性之谜，更好地理解它们在婴儿中的特定作用。质谱学 (MS)与液相色谱(LC)分离联用已被广泛用于HMO分析。 不幸的是，在许多情况下，基于LC-MS的技术存在色谱分辨率不足的问题 导致共洗脱物种的MS/MS谱曲折，从而限制了准确的鉴定和 HMOS的结构阐明。我们研究计划的总体目标是开发一种生物分析 工作流程，以便更有效地确定HMO的特征。为了实现这一目标，我们将实施 利用循环液相色谱联用的多维分离平台 结合环离子迁移率分离和质谱学的发展 包括溶液和气相化学探针以及自上而下的测序方法。我们的多维 分色平台不仅可以提高峰值容量，还可以实现更高分辨率的分色 便于表征以前无法区分的HMO异构体。此外，我们的环状离子迁移率- 基于分隔将允许确定高度精确的碰撞横截面值 传播到糖生物界的其他实验室。我们的化学探测器将有助于 改进了异构体HMOS中糖苷链定位的识别，特别是在没有可靠标准的情况下 是存在的。最后，我们的自上而下的测序策略将使单糖的排列成为可能 成分有待更好地确定。总体而言，我们设想我们提议的生物分析工具箱将使 迈向HMO的从头测序，并帮助实现我们的首要目标--解开人类 奶糖，以确定必要的HMO纳入婴儿配方奶粉。

PROJECT SUMMARY While human milk oligosaccharides (HMOs) are involved in a variety of biological processes that enable the healthy development of the brain-gut axis of infants, a true understanding of how their molecular structures and composition dictate such functions has largely remained a mystery. HMOs possess a tremendous degree of both structural complexity and isomeric heterogeneity due to their many possible permutations of linkage positioning and monosaccharide constituent arrangements. Therefore, improved analytical advancements are necessary to unravel this isomerism puzzle and gain a better understanding of their infant-specific roles. Mass spectrometry (MS) in conjunction with liquid chromatographic (LC) separations has been broadly used for HMO analyses. Unfortunately, in many instances, LC-MS-based techniques suffer from insufficient chromatographic resolution resulting in convoluted MS/MS spectra for co-eluting species, and thus limiting the accurate identification and structural elucidation of HMOs. The overall goal of our research program is to develop a bioanalytical workflow to enable more effective characterization of HMOs. To achieve this, we will implement a multidimensional separations platform using recycling liquid chromatography coupled to high- resolution cyclic ion mobility separations and mass spectrometry in conjunction with the development of solution and gas-phase chemical probes and top-down sequencing approaches. Our multidimensional separations platform will not only enable improved peak capacity, but also higher resolution separations to facilitate the characterization of previously indistinguishable HMO isomers. Additionally, our cyclic ion mobility- based separations will allow the determination of highly precise collision cross section values that will be disseminated to other laboratories in the glycobiology community. Our chemical probes will facilitate the improved identification of glycosidic linkage positioning in isomeric HMOs, especially when no authentic standard is present. Lastly, our top-down sequencing strategies will enable the arrangement of monosaccharide constituents to be better determined. Overall, we envision that our proposed bioanalytical toolbox will make strides toward the de novo sequencing of HMOs and help achieve our overarching goal of unraveling the human milk glycome to identify essential HMOs to be incorporated into infant formula.