Study Of The Protein Digestion By Using Enzyme Reactor Based On The Open Tubular Capillary Column And Its Application In Proteomics

Capillary liquid chromatography was normally used in proteomics research owing to the small amount of biological samples, so the capillary chromatographic columns played a very important role in proteomics study. Open capillary columns are prepared by coating the organic modifiers in the inner wall of capillary, and they have the advantages of easy preparation, low column back-pressure and so on.The research work in this thesis is mainly about the new technology and its application in proteomics based on the open capillary columns. Considering that the open tubular columns have excellent application in nano-HPLC coupled to mass spectrometry, we choose them as our carrier and successfully prepare the immobilized trypsin reactors. The experimental results demonstrate that the newly prepared immobilized trypsin reactors can increase the efficiency of the protein digestion, compared with the traditional in-solution digestion technology, and shorten the digestion time from 16 h to 1 min. The whole research work in this thesis is mainly focused on new technology of enzyme-immobilized capillary columns and included four chapters.In chapter 1, the background and methods of proteomics are reviewed including the advance of biological mass spectrometry, the methods of protein identification and protein digestion technologies. Recently, new achievements of protein digestion methods have been made, such as immobilized enzyme technology. Using the immobilized enzyme in replace of enzyme solution for protein digestion is a trend. And protein digestion using enzyme immobilized on the inner wall of capillary column is also attracting more and more attention due to its advantages, such as its easy preparation, on-line manipulation and high efficiency.In chapter 2, we developed the new method for preparing a multi-layer open-tubular capillary column based on the micro-immobilized enzyme reactor for tryptic digestion of proteins. Combined the preparation of multi-layer open-tubular capillary column and the immobilization of trypsin, and with the glutaraldehyde as the coupling agent, we prepared the multi-layer open tubular capillary enzyme-immobilized reactor. After digestion, the peptides were identified by MALDI-TOF MS. The results demonstrate that our enzyme-immobilized reactor has high digestion efficiency.In chapter 3, we prepared the immobilized enzyme reactor based on atom transfer radical polymerization (ATRP) reaction successfully, and the application of this enzyme reactor in digestion of proteins was also studied. First the initiator inducing the atom transfer radical polymerization reaction was synthesized, and then it was bonded to the inner wall of the capillary. After above steps, we introduced the epoxy group, amino group, aldehyde group. At last the trypsin was immobilized in the inner wall of the capillary. For evaluating the digestion performance, standard proteins were digested by using the newly prepared enzyme immobilized capillary columns, and the peptides were identified by MALDI-TOF MS and FT LTQ-MS/MS, and the good results were obtained. In order to further check the performance of this enzyme reactor, we analyzed the digest of the extracts from an Hep G2 cell lysate in the same condition, and the results suggest that the enzyme-immobilized capillary columns have good digestion performance.In chapter 4, a reversed-phase open tubular capillary column was prepared and applied in the separation of peptides. First, the capillary is activated to expose the Si-OH of inner wall, then polymerization reaction of monomer solution containing lauryl methacrylate (LMA) occurs to attach the C12 functional group onto the inner wall of the capillary column. Finally, this reversed-phase open tubular capillary column was applied in the separation of myoglobin digests with trypsin in the nano-HPLC and the good results have been obtained.