Duan Jicheng, Liang Zhen, Yang Chun, Zhang Jie, Zhang Lihua, Zhang Weibing, Zhang Yukui
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, PR China.
Proteomics. 2006 Jan;6(2):412-9. doi: 10.1002/pmic.200500234.
A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.
通过在十二烷醇和环己醇的二元致孔剂混合物存在下原位聚合丙烯酰胺、甲基丙烯酸缩水甘油酯（GMA）和二甲基丙烯酸乙烯酯（EDMA），随后进行氨水溶液处理、戊二醛活化和胰蛋白酶修饰，在熔融石英毛细管中制备了整体式酶微反应器。发现选择丙烯酰胺作为共聚单体有助于提高胰蛋白酶修饰的效率，从而提高酶活性。优化后的微反应器背压非常低，能够快速消化流经反应器的蛋白质。通过在高流速下消化细胞色素c证明了整体式微反应器的性能。然后通过CE和HPLC-MS/MS对消化产物进行表征，序列覆盖率为57.7%。发现消化效率比传统方法高230倍以上。此外，首次使用MS/MS检测将在水和乙腈混合物中进行的蛋白质消化与传统的水相反应进行比较，发现前一种溶液对蛋白质消化更具兼容性且更高效。
