Human milk oligosaccharides (HMOs) afford many health benefits to breast-fed infants, such as protection against infection and regulation of the immune system, through the formation of non-covalent interactions with protein receptors. However, the molecular details of these interactions are poorly understood. Here, we describe the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening natural libraries of HMOs against lectins. The HMOs in the libraries were first identified based on molecular weights (MWs), ion mobility separation arrival times (IMS-ATs) and collision-induced dissociation (CID) fingerprints of their deprotonated anions. The libraries were then screened against lectins and the ligands identified from the MWs, IMS-ATs and CID fingerprints of HMOs released from the lectin in the gas phase. To demonstrate the assay, four fractions, extracted from pooled human milk and containing ≥35 different HMOs, were screened against a C-terminal fragment of human galectin-3 (hGal-3C), for which the HMOs specificities have been previously investigated, and a fragment of the blood group antigen-binding adhesin (BabA) from Helicobacter pylori, for which the HMO specificities have not been previously established. The structures of twenty-one ligands, corresponding to both neutral and acidic HMOs, of hGal-3C were identified; all twenty-one were previously shown to be ligands for this lectin. The presence of HMO ligands at six other MWs was also ascertained. Application of the assay to BabA revealed nineteen specific HMO structures that are recognized by the protein and HMO ligands at two other MWs. Notably, it was found that BabA exhibits broad specificity for HMOs, and recognizes both neutral HMOs, including non-fucosylated ones, and acidic HMOs. The results of competitive binding experiments indicate that HMOs can interact with BabA at previously unknown binding sites. The affinities of eight purified HMOs for BabA were measured by ESI-MS and found to be in the 10³ M⁻¹ to 10⁴ M⁻¹ range.

中文翻译：

---

使用CaR-ESI-MS筛选抗凝集素的人乳寡糖天然文库†

人乳寡糖（HMO）通过与蛋白质受体形成非共价相互作用，为母乳喂养的婴儿带来许多健康益处，例如防止感染和免疫系统调节。但是，这些相互作用的分子细节了解甚少。在这里，我们描述了捕获和释放电喷雾电离质谱（CaR-ESI-MS）在筛选抗凝集素的HMO天然文库中的应用。首先根据其去质子化的阴离子的分子量（MWs），离子迁移分离到达时间（IMS-ATs）和碰撞诱导解离（CID）指纹识别文库中的HMO。然后针对凝集素筛选文库，并从MWs中鉴定出配体，气相凝集素释放出的HMO的IMS-AT和CID指纹。为了证明该检测方法，针对人类galectin-3（hGal-3C）的C端片段筛选了从合并的母乳中提取的，含有≥35种不同HMO的四个馏分，此前已对其进行过HMOs特异性研究。血型抗原结合黏附素（BabA）的片段幽门螺杆菌，之前尚未确定HMO的特异性。确定了hGal-3C的21个配体的结构，分别对应于中性和酸性HMO。以前所有21个都是该凝集素的配体。还确定在其他六个MW处存在HMO配体。该测定法对BabA的应用揭示了19种特定的HMO结构，这些结构在另外两个MW处被蛋白质和HMO配体识别。值得注意的是，发现BabA对HMO具有广泛的特异性，并且识别包括非岩藻糖基化的中性HMO和酸性HMO。竞争性结合实验的结果表明，HMOs可以在以前未知的结合位点与BabA相互作用。通过ESI-MS测量了8种纯化的HMO对BabA的亲和力，发现在10种中³ M ^-1至10 ⁴ M ^-1范围。