Study On The Multi-dimensional Liquid Chrmaography Coupled With Tanderm Mass Spectrometry For Proteomics Of Rice Leaves

Rice,as an important food crop,is a food source for surviving more than half of the world’s population.It is of great significance to have a deep understanding of the rice growth and development process and regulation.Combining with the development trend of multi-dimensional liquid chromatography for the analysis of plant proteomics,two-dimensional liquid chromatography and three-dimensional liquid chromatography tandem mass spectrometry were established in this paper.Rice leaves in the filling stage were used as experimental materials to optimize the flow of multidimensional liquid chromatography.Parameters such as phase pH and number of collected fractions were used to establish high orthogonality and good resolution of multi-dimensional liquid chromatography tandem mass spectrometry to analyze rice leaf proteomics.The main research results of the experiment include the following:1.This experiment established an off-line hydrophilic interaction-two-dimensional liquid chromatography combined with fraction collection technology to analyze rice leaf protein.By examining the influence of pH on the orthogonality of the system to the mobile phase of hydrophilic interaction chromatography and reversed phase chromatography,the following conclusions were obtained:the combination of hydrophilic interaction chromatography(pH 9.3)and reversed phase chromatography(pH 3.3)can achieve better results.Orthogonality and the method also showed better orthogonality and separation ability in actual rice leaf protein analysis.2.The experiment also ptimized the number of fractions collected in two-dimensional liquid chromatography system and one-dimensional liquid chromatography crude peptide peptides and other parameters.The following conclusions are obtained:The combination of hydrophilic interaction chromatography(pH 9.3)and reversed phase chromatography(pH 3.3)can achieve better orthogonality R~2=0.34113.The number of fractions collected and the amount of crude peptides extracted from one-dimensional liquid chromatography were used to determine the number of proteins identified in rice leaves.The number of fractions collected and the one-dimensional liquid chromatogram were obtained.When the amount of enzymatic peptides was 22 and 4 mg respectively,two-dimensional liquid chromatography showed high-efficiency and high-throughput for analysis.3.Based on two-dimensional liquid chromatography,the orthogonality of the system was investigated by optimizing the ion-pair reversed-phase chromatography ion pairs and other parameters to establish a three-dimensional liquid chromatography tandem mass spectrometry analysis of rice leaf protein.The experimental results show that when HFBA-RP is used as a one-dimensional liquid chromatography,the orthogonality of the three-dimensional liquid chromatography system is best:R~2=0.43139 and R~2=0.66473,respectively.This method,which applied to the analysis of actual rice leaf protein showed higher separation ability and resolution.Meanwhile,by this method,46404 peptides that belonged to 6976 proteins were identified.