Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Am Chem Soc. 2024 Aug 7;146(31):21700-21709. doi: 10.1021/jacs.4c05805. Epub 2024 Jul 25.
Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces mbrane chor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.
糖基结合蛋白 (GBP) 与细胞膜中糖脂 (GSL) 的相互作用涉及广泛的生物学过程。然而，由于纯化的 GSL 数量有限，以及单价 GBP-GSL 相互作用的亲和力较弱，因此研究 GSL 结合受到了阻碍。使用可溶性模型膜进行的天然质谱 (nMS) 是发现 GBP 配体的一种很有前途的方法，但检测弱相互作用仍然具有挑战性。本工作引入了膜辅助 nMS（MEAN-nMS）来检测低亲和力 GBP-GSL 复合物。该测定法利用膜锚定，通过 GBP 和膜中的脂质的共价交联产生，将 GBP 定位在表面并促进 GSL 结合。通过 nMS 检测完整的 GBP-GSL 复合物（MEAN-nMS）或使用捕获和释放（CaR）策略来鉴定配体，其中 GSL 在碰撞激活时从 GBP-GSL 复合物中释放出来并被检测到（MEAN-CaR-nMS）。为了建立可靠性，将包含在纳米盘中的纯化神经节苷脂文库与人类免疫凝集素进行筛选，并将结果与相应的神经节苷脂寡糖的亲和力进行比较。没有膜锚定，nMS 分析主要产生假阴性。相比之下，通过 MEAN-(CaR)-nMS 可以鉴定出所有的配体，没有假阳性。为了突出 MEAN-CaR-nMS 在配体发现方面的潜力，将天然 GSL 文库纳入纳米盘，并与人类和病毒蛋白进行筛选，以发现难以捉摸的配体。最后，直接从细胞中进行基于 nMS 的 GSL 配体检测。这一突破为使用完整细胞进行鸟枪法糖组学筛选铺平了道路。
