摘要
目的使用磁固相萃取、转录组文库及液相色谱-质谱联用(LC-MS/MS)鉴定技术,筛选和鉴定日本医蛭中的抗凝活性成分。方法采用通用合成方法制备凝血酶固定化磁纳米粒,日本医蛭粗提液经磁固相萃取后,洗脱溶液使用高效液相串联三重四级杆飞行时间质谱联用(TripleTOF LC-MS/MS)技术并结合日本医蛭唾液腺转录组文库,进行抗凝多肽的筛选。结果经傅立叶变换红外分光光度计及透射电镜表征,凝血酶成功固定于磁珠上,凝血酶磁珠的酶活性稳定性良好(37℃,19 d,RSD为4.74%);洗脱液经扫描比对含有5种水蛭素变体,均为凝血酶抑制剂,洗脱液冻干粉抗凝活性为70 U·g^(-1)。结论将磁固相萃取、转录组文库以及LC-MS/MS技术相结合成功开发了一种能快速筛选和鉴定日本医蛭中抗凝多肽分子的方法,该方法方便易行,可为动物药活性成分发掘提供参考。
Objective To establish a combination approach of magnetic solid-phase extraction,the transcriptomic preparation and liquid chromatography coupled with triple quadrupole and time-of-flight mass spectrometry(triple TOF LC-MS/MS)technology,and to identify anti-thrombosis biomolecules from H.nipponica.Methods Thrombin-immobilized nanobeads were synthesized and employed to screen anticoagulants from leech saliva.A customized transcriptomic database was constructed to search against the peptide MS/MS spectrum for antithrombin biomolecule identifications.Results The Fourier transform infrared(FT-IR)spectrum and transmission electron microscope(TEM)image indicated that all modifications were successfully immobilized on the surface of materials.Within 19 days at 37℃,the storage stability of immobilized thrombin was satisfactory with the relative standard deviation 4.74%.Five direct thrombin inhibitors,all referring to hirudin variants,were successfully enriched and identified.The antithrombin activity was 70 U·g^(-1).Conclusion By incorporating magnetic solid-phase extraction,the transcriptomic preparation with triple TOF LC-MS/MS technology,a convenient and easily-handled method is successfully developed to screen and identify the antithrombin biomolecules from H.nipponica.This efficacious application also show its practical and bright future in biomolecules discovery from animal medicines.
作者
黄小清
汪波
肖凌
聂晶
HUANG Xiaoqing;WANG Bo;XIAO Ling;NIE Jing(Key Laboratory of Combinatorial Biosynthesis and Drug Discovery,Ministry of Education,School of Pharmaceutical Sciences,Wuhan University,Wuhan 430071,China;Key Laboratory of Traditional Chinese Medicine Quality Control of State Drug Administration,Hubei Institute for Drug Control,Wuhan 430074,China)
出处
《医药导报》
CAS
北大核心
2021年第5期651-657,共7页
Herald of Medicine
基金
湖北省科技计划项目(2016CFB404)。
关键词
日本医蛭
抗凝活性成分
磁固相萃取
液相色谱-质谱联用
凝血酶固定化磁珠
Hirudo nipponica
Anticoagulant biomolecules
Magnetic solid-phase extraction
Liquid chromatography-tandem mass spectrometry
Thrombin-immobilized magnetic nanobeads
