Center for Biomedical Engineering, and ‡Department of Chemical and Biological Engineering, University of New Mexico , Albuquerque, New Mexico 87131, United States.
Anal Chem. 2017 Jun 20;89(12):6440-6447. doi: 10.1021/acs.analchem.7b00296. Epub 2017 Jun 9.
Most druggable targets are membrane components, including membrane proteins and soluble proteins that interact with ligands or receptors embedded in membranes. Current target-based screening and intermolecular interaction assays generally do not include the lipid membrane environment in presenting these targets, possibly altering their native structure and leading to misleading or incorrect results. To address this issue, an ideal assay involving membrane components would (1) mimic the natural membrane environment, (2) be amenable to high-throughput implementation, and (3) be easily multiplexed. In a step toward developing such an ideal target-based analytical assay for membrane components, we present fluorescently indexed multiplexed biomimetic membrane assays amenable to high-throughput flow cytometric detection. We build fluorescently multiplexed biomimetic membrane assays by using varying amounts of a fluorescently labeled lipid, NBD-DOPE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)], incorporated into a phospholipid membrane bilayer supported on 3 μm silica microspheres. Using flow cytometry, we demonstrate this multiplexed approach by measuring specific affinity of two well-characterized systems, the fluorescently labeled soluble proteins cholera toxin B subunit-Alexa 647 and streptavidin-PE/Cy5, to membranes containing different amounts of ligand targets of these proteins, GM1 and biotin-DOPE, respectively. This work will enable future efforts in developing highly efficient biomimetic assays for interaction analysis and drug screening involving membrane components.
大多数可成药的靶点是膜成分，包括膜蛋白和可溶性蛋白，它们与嵌入膜中的配体或受体相互作用。当前基于靶点的筛选和分子间相互作用测定通常不包括脂质膜环境来呈现这些靶点，这可能会改变它们的天然结构，并导致误导或不正确的结果。为了解决这个问题，一个理想的涉及膜成分的测定方法将（1）模拟自然的膜环境，（2）易于高通量实施，以及（3）易于多重检测。为了朝着开发这种理想的基于膜成分的靶点分析测定方法迈出一步，我们提出了荧光索引的多重仿生膜测定方法，该方法适用于高通量流式细胞术检测。我们通过使用不同量的荧光标记脂质 NBD-DOPE [1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-（7-硝基-2-1,3-苯并恶二唑-4-基）] 构建荧光多重仿生膜测定方法，该脂质被掺入到磷脂双层膜中，该双层膜支撑在 3μm 的二氧化硅微球上。我们使用流式细胞术通过测量两个经过充分表征的系统的特定亲和力来证明这种多重方法，这两个系统是荧光标记的可溶性蛋白霍乱毒素 B 亚单位-Alexa 647 和链霉亲和素-PE/Cy5，它们与分别含有这些蛋白的配体靶 GM1 和生物素-DOPE 的不同量的膜相互作用。这项工作将为未来开发涉及膜成分的高效仿生测定方法用于相互作用分析和药物筛选提供支持。
