El-Hawiet Amr, Chen Yajie, Shams-Ud-Doha Km, Kitova Elena N, St-Pierre Yves, Klassen John S
Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta Canada T6G 2G2.
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University , Alexandria, Egypt.
Anal Chem. 2017 Sep 5;89(17):8713-8722. doi: 10.1021/acs.analchem.7b00542. Epub 2017 Aug 16.
The intense interest in the mechanisms responsible for the beneficial effects of breast-feeding on infant health has created a significant need for analytical methods capable of rapidly identifying interactions between human milk oligosaccharides (HMOs) and their protein receptors. Currently, there are no established, high-throughput assays for the screening libraries of free (unmodified) HMOs against lectins. The present work describes a rapid and label- and immobilization-free assay, based on catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), capable of simultaneously screening mixtures of free HMOs of known concentration for binding to lectins in vitro. Ligand identification relies on the molecular weights (MWs), ion mobility separation arrival times, and collision-induced dissociation fingerprints of HMO anions released from the target protein in the gas phase. To establish the reliability of the assay, a library of 31 free HMOs, ranging in size from tri- to octasaccharide, was screened against three human galectin (hGal) proteins (a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C) and hGal-7), with known HMO affinities. When implemented using an equimolar concentration library, the CaR-ESI-MS assay identified 100% of ligands with affinities >500 M and ≥93% of all HMO ligands (hGal-1-31 of 31 ligands; hGal-3C-25 of 25; hGal-7-28 of 30); no false positives were detected. The assay also successfully identified the majority of the highest affinity HMO ligands (or isomer sets that contain the highest affinity ligands) in the library for each of the three hGal. Notably, for each lectin, CaR-ESI-MS screening required <1 h to complete and consumed <5 ng of each HMO and <0.5 μg of protein.
人们对母乳喂养对婴儿健康有益影响的机制有着浓厚兴趣，这使得迫切需要能够快速识别母乳低聚糖（HMOs）与其蛋白质受体之间相互作用的分析方法。目前，尚无针对游离（未修饰）HMOs文库与凝集素进行筛选的成熟高通量检测方法。本研究描述了一种基于捕获与释放电喷雾电离质谱（CaR-ESI-MS）的快速、无标记且无需固定化的检测方法，该方法能够在体外同时筛选已知浓度的游离HMOs混合物与凝集素的结合情况。配体鉴定依赖于气相中从目标蛋白释放的HMO阴离子的分子量（MWs）、离子淌度分离到达时间以及碰撞诱导解离指纹图谱。为了确定该检测方法的可靠性，针对三种已知HMO亲和力的人半乳糖凝集素（hGal）蛋白（hGal1的稳定突变体（hGal-1）、hGal-3的C端片段（hGal-3C）和hGal-7），筛选了一个包含31种游离HMOs的文库，其大小从三糖到八糖不等。当使用等摩尔浓度文库实施时，CaR-ESI-MS检测方法鉴定出了所有亲和力>500 M的配体的100%以及所有HMO配体的≥93%（hGal-1的31个配体中的hGal-1-31；hGal-3C的25个配体中的hGal-3C-25；hGal-7的30个配体中的hGal-7-28）；未检测到假阳性。该检测方法还成功鉴定出了文库中针对三种hGal中每一种的大多数最高亲和力HMO配体（或包含最高亲和力配体的异构体组）。值得注意的是，对于每种凝集素，CaR-ESI-MS筛选完成所需时间<1小时，每种HMO消耗<5 ng，蛋白质消耗<0.5 μg。
