Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.

中文翻译：

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母乳低聚糖分析和表征的新策略

母乳是一种极其丰富且复杂的生物流体，由蛋白质、脂质和复杂碳水化合物组成，其中包括多种游离母乳低聚糖 (HMO)。引人注目的是，HMO 不会被婴儿消化，但可以作为细菌菌株的益生元，具有多种益处。考虑到 HMO 的多种有益作用以及影响母乳成分的大量因素，HMO 多样性和复杂性的分析至关重要。使用参与儿童营养不良和发育迟缓研究的一群孟加拉国母亲的母乳样本，我们通过全甲基化、随后液相色谱结合高分辨率串联质谱 (LC-MS/MS) 表征了母乳寡糖的组成。 ） 分析。该方法鉴定了 100 多种不同的糖型，并显示出乳汁成分的广泛多样性，其中岩藻糖基化和唾液酸化 HMO 比未修饰的 HMO 占主导地位。我们观察到这些样品平均含有 80 个 HMO，检测到的最高全甲基化质量超过 5000 个质量单位。在此，我们报告了一种易于实施的方法，用于对大量 HMO（包括较高分子量的唾液酸化 HMO）进行分离、表征和相对定量。我们的最终目标是创建一种简单、高通量的方法，可用于唾液酸化和/或岩藻糖基化 HMO 的全面表征。这些结果证明了如何应用当前的分析技术来表征母乳成分，