Analysis Method Study Of Kunitz Trypsin Inhibitor By Malditof In Soybean

The safety of the transgenic food was more and more to be pay attention for the utility of GM soybean increasing in our country. Soybean trypsin inhibitor is the main substance affecting the soybean food safety. It is still unknown whether the exogenous gene would lead to change of the composition of soybean trypsin and whether this change would bring new safety issues? At present no methods are suitable to evaluate the differences between GM soybean and soybean.In this paper, the chymotrypsin was used as the main hydrolysis enzyme and the basic hydrolysis conditions of kunitz trypsin inhibitor of soybean was37℃and16h, according to the results, a MALDI-TOF-MS analysis method determinating under positive ion reflection mode was established to analyse the enzymatic hydrolysis peptides of kunitz trypsin inhibitor, which was accelerating voltage ion of source1and source2was respectively25kv and22kv, energy was85ev, N2laser wavelength was337nm, pulse width was5ns, ion delay extract was500ns, vacuum degree was5x10"9Pa, mass spectrum signal accumulated50times in a single scan. Matrix peak was used for internal calibration and peptides of standard samples were used for external calibration. The improved double drying method (the first layer is HCCA, the second is kunitz trypsin inhibitor) was used for kunitz trypsin inhibitor by chymotrypsin hydrolyzed and sample amount was1μL. Peptide mass fingerprint by MALDI-TOF-MS detected was compared with the conservative domain sequences of the family of kunitz trypsin inhibitor in NCBI database, the results showed that kunitz trypsin inhibitor sequence coverage of the non-GM and GM soybean were respecticely21%and8%. Peptide mass fingerprint of the family of kunitz trypsin inhibitor was improved by peptide mass fingerprint and secondary quality score ion spectra of kunitz trypsin inhibitor of the non-GM and GM soybean, and tried to establish a building method of peptide mass fingerprint of soybean trypsin inhibitor, which could provide a new measure to compare and evaluate the safety of GM soybean by peptide mass fingerprint of kunitz trypsin inhibitor.