Danone Nutricia Research, Uppsalalaan 12, 3584 CT, Utrecht, the Netherlands.
Biomolecular Mass Spectrometry and Proteomics Division, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, the Netherlands.
Anal Bioanal Chem. 2019 Jan;411(1):231-250. doi: 10.1007/s00216-018-1434-7. Epub 2018 Nov 16.
Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.
人乳（HM）支持新生儿的健康发育，并通过所含的游离人乳低聚糖（HMOS）发挥许多有益作用。这些 HMOS 表现出复杂性和结构多样性，这对分析构成了重大挑战。对 HMOS 进行详细表征是必不可少的，因为每个单独的结构都可能具有不同的功能/活性。某些 HMOS 异构体甚至在生物学功能上可能存在根本差异，特别是通过 LC 或 LC-MS 进行的表征常常受到共洗脱现象的影响。因此，需要更有效的具有增强结构选择性的分析方法。因此，我们开发了一种负离子模式 LC-ESI-MS 方法，该方法具有简单的样品制备、环保的 EtOH 梯度洗脱以及通过多重反应监测（MRM）增强的、半定量的特征，可用于鉴定不同的天然 HMOS。我们的 MRM-LC-MS 装置利用高度选择性的、依赖于聚糖构型的碰撞诱导解离（CID）片段来鉴定单个中性和酸性 HMOS。值得注意的是，许多人乳低聚糖异构体可以以与保留时间无关的方式区分。这与其他依赖于相当非特异性 MRM 跃迁的当代 MRM 方法形成对比。我们的方法用于在单个 LC-MS 测定中半定量地确定最丰富的人乳三糖、四糖、五糖和六糖。检测到的 HMO 结构包括岩藻糖乳糖（例如 2'-FL）、乳-N-二岩藻四糖（LDFT）、乳-N-四糖（LNTs）、乳-N-岩藻五糖（例如 LNFP I、LNFP II 和 III）、乳-N-二岩藻六糖（LNDFHs）以及唾液乳糖（SL），并推测分配了血型 A 和 B 四糖，从中可以证明正确的人乳类型分配。通过高压阴离子交换色谱（HPAEC）与脉冲安培检测（HPAEC-PAD）偶联，通过验证 I-IV 乳组来验证乳型的正确性。
