基于核酸适配体微流控芯片酶反应器用于蛋白质分析.pdfVIP

2012年11月 Vol.30 No.11 November2012 ChineseJournal of Chromatography 1127 ~1132 研究快报 DOI:10.3724/ SP.J.1123.2012.07019 基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析 肖　 鹏,　 李大雷,　 满　 燕,　 耿利娜,　 吕雪飞,　 邓玉林∗ (北京理工大学生命学院,北京 100081) 摘要:将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联 质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进 行了初步评价。 结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平 行分析,肽段覆盖率及相对标准偏差分别为44．3%、6．5% (牛血清白蛋白),65．0%、2．7% (肌红蛋白)和62．0%、 5．6% (细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白 质组学分析中发挥重要作用。 关键词:高效液相色谱-串联质谱;胰蛋白酶;蛋白质;核酸适配体;芯片酶反应器 中图分类号:O658　 　 　 文献标识码:A　 　 　 文章编号:1000-8713(2012)11-1127-06 An enzyme reactor based on aptamer modified microfluidic chip for protein analysis XIAO Peng,LI Dalei,MAN Yan,GENG Lina,LÜ Xuefei,DENGYulin∗ (School of LifeScience,Beijing Institute of Technology,Beijing 100081,China) Abstract:As a kind of recognition molecule,aptamer has been studied and applied widely in numerous sciencefields in recent years. Immobilized enzymaticreactor has drawn much atten- tion because of its striking advantages,such as high digestion efficiency and ease in coupling with the separation and detection systems. In this study,a novel microfluidic enzymatic chip, which immobilizedtrypsin based on aptamer,was prepared and proposed. An online analysis platform,which consisted of an aptamer-based chip and high performance liquid chromatogra- phy tandem mass spectrometry,was establishedby using a6-port valve and applied to protein analysis. The enzymatic capacity and stability performance of chip reactor were characterized by using mixedprotein sample,which co