Study On Absolute Quantification Of Functional Proteins In Rice Based On MRM Mass Spectrometry

Quantitative analysis of protein expression levels in plant populations,tissues,cells,as well as sub-cells,is an important theoretical basis for comprehensive understanding and revealing the laws of plant life activities and the research in various fields of plants.However,traditional protein quantification techniques face many problems such as low analytical sensitivity,narrow linear range,and severe matrix interference.In this study,rice was used as the research object,and a series of key technologies such as rice protein extraction,enzymatic hydrolysis,purification and isotope labeling were systematically studied.The MRM mass spectrometry technique of LC-MS/MS was used to establish an absolute quantitative method for rice protein.The related techniques were successfully applied for the absolute quantification of Glb33 in rice grains.The main results of this study are as follows:1.An MRM analysis method for 16 typical rice proteins was developed.The 16 typical rice proteins actually monitored were researched,and the selection of their tryptic peptides were selected and mass spectrometry conditions were optimized.The results showed that the 16 peptides were well separated on the Poroshell 120 EC-C18 column with a gradient of acetonitrile and 0.1%(v/v)formic acid in mobile phase.16 peptides showed a good linear relationship with the in the range of 0.02~500 nM,and the correlation coefficient was greater than 0.99.The detection limits and quantitation limits of the instrument reached 0.007~0.5 nM and 0.02~1.5 nM,respectively.2.A highly efficient SOD protein extraction method was established.16 target proteins were used as research objects by optimizing extraction buffer,protein precipitation reagent and enzymatic hydrolysis conditions.The results showed that the buffer containing 1% SDS was used as the protein extraction reagent,and the total protein obtained by acetone precipitation was trypsinized by 1:50(enzyme: protein,v:v),and the optimum enzymatic hydrolysis efficiency can be achieved at a pH of 8.0 and a temperature of 48 ℃ for 4-8 hours.Compared with the traditional phenol method and TCA-acetone method,the method established in this study was conducted with short enzymatic hydrolysis time and simple operation,which can not only effectively improve the analytical sensitivity of 16 target proteins,but also effectively avoid the loss of target protein.3.The best solid phase extraction purification technology was established.5 kinds of solid phase extraction cartridges,named C18,MAX,MCX,HLB and SD-C18,were selected to systematically study the adsorption capacity and interference removal performance for different peptides.It was found that the SD-C18 column has strong retention ability for different characteristic peptides with the recovery of 80.1%~105.7% eluted by 70% acetonitrile solution.When the enzymatic peptide was directly injected into the sample,most of the peptides showed a moderate-intensity matrix inhibitory effect(ME value ranged from-50% to-20%),while the the matrix effect was reduced to within ±20% when enzymatic hydrolysate passed through the SD-C18 column,which basically meets the analytical requirements.4.The internal standard peptides of different length flanks affected the accuracy of the quantitative results.The characteristic peptides of OsGSTF14 and Glb33 were used as research objects,and the effects of number of flanking amino acids on the absolute quantitative results were discussed by artificially synthesizing isotopically labeled peptides of flanking sequences of different lengths.The results showed that when the number of flanking amino acids was 6,the enzymatic hydrolysis and solubility characteristics of endogenous proteins in the samples could be simulated,and the corrected recovery rate was over 92%,which effectively improved the quantitative accuracy of the corresponding proteins.5.The MRM mass spectrometry of LC-MS/MS was successfully applied to the absolute quantification of the allergenic protein Glb33 in rice grains.The results showed that the content of allergenic protein Glb33 in 24 rice cultivars varied greatly,ranging from 11.7 to 682.0 μg/g.This method provides important technical support for screening and analysis of allergenic proteins in rice and products.