目的 通过构建靶蛋白固定化磁珠，从威廉环毛蚓Pheretima guillemi中垂钓出与靶蛋白（纤维蛋白原、纤溶酶原）直接作用的抗血栓活性大分子成分，并经液相色谱-质谱联用（LC-MS/MS）技术鉴定。方法 优化并合成纤维蛋白原、纤溶酶原固定化羧基磁珠，分别通过固定化量、透射电镜、傅里叶红外光谱仪、激光纳米粒度分析仪表征功能化磁珠的靶蛋白固定量、微观形态、官能团及表面带电性；利用功能化磁珠从威廉环毛蚓抗血栓部位DPf3中垂钓与靶蛋白直接作用的抗血栓大分子活性成分，洗脱后经LC-MS/MS技术分析，并结合威廉环毛蚓物种蛋白质数据库比对鉴定。结果 靶蛋白固定化磁珠的最佳制备方案为将纤维蛋白原（0.4 mg/mL）或纤溶酶原（1 mg/mL）与磁珠于4 ℃混旋孵育8 h，纤维蛋白原和纤溶酶原的最大偶联量分别为（9.84±2.17）μg/mg和（7.24±0.91）μg/mg；经表征，靶蛋白均已成功固定于磁珠表面。DPf3经纤维蛋白原、纤溶酶原固定化磁珠垂钓所得洗脱液经扫描比对，分别鉴定到20种和27种蛋白质，多属于丝氨酸蛋白酶类成分。结论 通过磁珠配体垂钓策略和LC-MS/MS技术可从威廉环毛蚓中快速筛选和鉴定目标生物活性大分子，可为动物类中药大分子的分离鉴定提供参考。

Objective By constructing the immobilized magnetic beads of target proteins, the antithrombotic macromolecular components directly acting with the target proteins (fibrinogen and plasminogen) were fished from Pheretima guillemi, and identified by liquid chromatogram-mass spectrometry (LC-MS/MS). Methods Fibrinogen and plasminogen-immobilized carboxyl magnetic beads were optimized and synthesized. The immobilized amount of target protein, microscopic morphology, functional groups and surface electrification of functional magnetic beads were characterized by immobilization amount, transmission electron microscope, Fourier infrared spectrometer and laser nanoparticle particle size analyzer, respectively. Functional magnetic beads were used to fish for antithrombotic macromolecular ingredients that directly interact with target proteins in DPf3, which were prepared from P. guillemi, and the elution was analyzed by LC-MS/MS technique and compared and identified via specific species protein database. Results The optimal preparation scheme of immobilized magnetic beads for the target protein was as follows: fibrinogen (0.4 mg/mL) or plasminogen (1 mg/mL) was mixed and incubated with magnetic beads at 4 ℃ for 8 h, and the maximum coupling amounts of fibrinogen and plasminogen were (9.84±2.17) μg/mg and (7.24±0.91) μg/mg, respectively. The target proteins were successfully fixed on the surface of magnetic beads. By scanning and comparison, 20 and 27 kinds of proteins in DPf3 were identified in the elution obtained from the fibrinogen and plasminogen immobilized magnetic beads, and most of them belonged to serine proteases. Conclusion The target bioactive macromolecules can be rapidly screened and identified from P. guillemi by using the magnetic beads fishing strategy and LC-MS/MS technique, which can provide a reference for the isolation and identification of macromolecules in the medicinal animal.

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国家自然科学基金资助项目（82104352）；河南省高校科技创新团队（23IRTSTHN026）；河南省中医药科学研究专项课题（重点课题）（2023ZY1003）；中医药高质量发展协同创新转化工程自主立项项目（CXZH03）