以靶找药：SPR配体垂钓技术在中草药活性成分的发现中的应用

研究人员基于SPR配体垂钓技术，将携带膜蛋白CXCR4的慢病毒颗粒固定在CM5芯片上，对川芎、穿心莲、苦楝子的草药提取物进行SPR分子垂钓，垂钓回收产物利用UPLC−QTOF-MS技术鉴定，鉴定到来源于川芎的洋川芎内酯I，并且通过后续实验证明蛋白与小分子配体间的相互作用。

Preparation of sample extracts:The crude drugs were smashed into powders and then sieved through a 40-mesh sieve. Powder samples in 1 g were extracted by supersonic extraction with 10 mL ethanol: H2O (80: 20) for 30 mins. Subsequently, the extracts were centrifuged and filtered through a 0.22-μm filter. The samples were stored at room temperature.

LVPs immobilization condition scouting:SPR assays were performed on Biacore T200 system (GE Healthcare, Sweden). The physical absorption response signal of LVPs on CM5 chips with four different pH buffers (4.0, 4.5, 5.0, and 5.5) was tested to find the optimal buffer. Then the physical absorption response signal of LVPs on CM5 chips at four different concentrations (200, 300, 400 and 500 μg/mL, use total protein concentration of LVPs as reference) was detected to find the optimal concentration of LVPs. Then LVPs were diluted in10mM sodium acetate at the optimal condition (pH = 4.0, 500 μg/mL) and then immobilized on detection cells by applying EDC/NHS cross-linking reaction. For affinity detection module, flow cell (FC) 1 was immobilized with LVPs with low CXCR4 expression (CXCR4- -LVPs) and FC2 was immobilized with LVPs with high CXCR4 expression (CXCR4+ -LVPs). For ligand screening module, all the four FCs on activity chip were immobilized with CXCR4+ -LVPs and all the four FCs on reference chip were immobilized with CXCR4- -LVPs

Recovery of CXCR4-bound ingredients:Herbal extract was injected over the sensor surface for 180 s at 5 μL/min. Phosphate buffer saline (PBS) with 5% DMSO was selected as running buffer. The flow system was washed with 0.5% formic acid. A small volume of 2 μL sample was injected into the flow cells and incubated for 20 s. Then, the flow direction over the sensor surface was reversed and the sample containing CXCR4-bound ingredients was deposited in 10 μL ammonium bicarbonate (50 mM). In a cycle, the recovery procedure was replicated for 5 times.