Abstract: The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks such as long incubation time, enzyme autodigestion, and non-reusability. Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor. The digestion efficiency, repeatability and recovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by using standard proteins and subsequent mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 minutes. Standard proteins could be almost completely digested with sequence coverage up to 93%, which is higher than that of 79% sequence coverage obtained by in-solution digestion for 16 h. BSA was digested 8 times within a month by using the SW-Trypsin. The result of sequence coverage was between 89% to 97%, with an average sequence coverage of 94%, which showed that SW-Trypsin has good stability. In addition, The recovery test by using MYO showed that the recovery rate was 87.67%. At last, the extract from Tengchong thermophilic bacteria was digested by SW-Trypsin in 20 min and in-solution trypsin in 16 h, respectively. The results of sequence coverage and the number of identified proteins were similar. Moreover, the ratio of the number of peptides with zero missed cleavages to the number of all identified peptides by using SW-Trypsin is higher than that by in-solution digestion. Also, the SW-Trypsin is easily removed from digestion solution. Good performances of SW-Trypsin imply that it has a good prospect in the application in future proteomics research.
ZHOU Lian-Qi,ZHANG Jiao,TIAN Fang et al. Preparation of a Trypsin Immobilizd Reactor on Silver Wire Modified by Atom Transfer Radical Polymer and its Application in Proteome Identification[J]. Chinese Journal of Chromatography, 0, (): 0-0.