Microwave assisted trypsin digestion with cavity type resonator reactor as an innovative proteomics technology

摘要

Sumamry form only given. The word “omics” indicates the study of a total information in biological cell, such as the genome, which is all DNA in a living cell, or the proteome which is all the proteins. Omics technologies of genomics and proteomics, such as tailor-made treatment, it has been positioned in the center of the future of medical technology. The efficiency of this technology is also expected by microwave technology. Microwave technology, for the time shortening of omics pretreatment reaction, has been fully achieved. The corresponding to the small-scale is also strongly desired to high-throughput technology such as a well plate. The corresponding to the small-scale is strongly desired to high-throughput technology such as a well plate. In this study, we tried the microwave assisted proteomics for enzymatic hydrolysis of protein molecules with a protease tripsin. Chicken egg white-derived lysozyme having a molecular weight 14,300 as a model protein was used in the enzymatic hydrolysis. The molar ratio of trypsin and lysozyme was adjusted to 1 : 50, reductive alkylation lysozyme was digested with 37°C, and analyzed by MALDI-TOF MS after the reaction. An irradiation reactor of cavity resonator type was applied to enzyme reaction microwaves continuously irradiated with microwaves of 16 W, and maintained at 37°C, of the reaction temperature. The conventional method was used with a water bath. As a result, reaction time was reduced from 20 hours to 10 minutes. It was found that the reaction had proceeded completely, from Mass spectrum. In the enzymatic hydrolysis, there is the problems to digest for oneself. However, the autolysis of trypsin under microwave irradiation was not detected. Next, we had checked by MASCOT searching after TOFMS analysis, it was confirmed which part of the protein is cleaved from the protein fragments. Enzyme-substrate concentration, time of 1 to 10, the sequence coverage of 84% by microwave irradiation was 57% under normal conditions. Trypsin recognized amino acid sequence, which means that 84% is broken, it was considered to cleave amino acid residues inside the protein.