Itraq Quantitative Methods To Analyze And Non-standard Steel Lily Plant Chloroplast Differences In Protein

Research has shown that about2%of disease is correlative with gene sequence, and98%of disease is closely correlative with the expression of protein. As a result of that, the research about protein will clarify the mechanism of the life changes under physiological or pathological condition. However, protein research technique is very difficult because of diversity and variability of the protein itself, and complexity of the conformation of the protein. Thankfully, differential proteomics, which focuses on meaningful differences to identify proteins, does not require us to capture all proteins, and thus is very high technically achievable. With quantitative techniques, changes in expression of proteins in different states, different environment, and under different processing conditions can be detected. What is more, it simply requires us to quantify the relative content in order to find differential proteins.We use both the iTRAQ quantitation technique and label free quantitation technique to study the differences in protein expression of Liliopsida’s chloroplast under different intension of treatment by Sodium carbonate (different concentration and processing time), based on Triple TOFTM5600, which is a performance liquid chromatographic tandem mass spectrometry from AB SCIEX.The whole study would provide the basis for analyzing the related genes and the functions of proteins encoded by caries-related genes.In the first chapter, we illustrate research actuality of proteomics and differential proteomics, highlight the related technology used in differential proteomics and discuss the quantitative technology in detail.The work in the second chapter mainly focuses on the usage of iTRAQ technology to find differential proteins of chloroplast. After the treatment of reduction, alkylation, enzymolysis, labeling by iTRAQ reagents, mix, fraction by SCX and desalting, the sample is analysed by LC-MS/MS. The raw data from the LC-MS/MS system is searched and analysed by ProteinPilot software from AB SCIEX, combined with Liliposida fasta database from Uniprot. The iTRAQ experiment is replicated by twice. The final result shows106and80proteins, which could be used for quantification, are identified by the two experiments respectively, with30proteins overlapping between them. With1.5and0.666as the up-regulation and down-regulation cutoff point for differential proteins in iTRAQ experiment, we identify71and58proteins which express differently as oppose to control group in two experiments.14of the30overlapping proteins show the same changing trends, which provide important information for differential proteomics research.Chapter3illustrates the usage of label free method to find differential proteins. As opposed to iTRAQ technique, after the treatment of reduction, alkylation and enzymolysis, the samples are analysed by LC-MS/MS respectively, with3technique replicate. After the raw data is searched by ProteinPilot, we identify253proteins used for quantification using PeakView, MarkerView and Protein Quantitation MicroApp software. Because of the differences between label free technique and iTRAQ technique, the paper set2.5and0.4as the up-regulation and down-regulation cutoff point for differential proteins in label free experiment. Finally, we identify142differential proteins.Afterwards, the iTRAQ technique and label free technique are compared based on their different quantification results.36proteins overlap between label free experiment and the first iTRAQ experiment, and33proteins overlap between the label free experiment and the second iTRAQ experiment, which indicate the complementarity between two methods.14proteins overlap among the three experiments, and one of them is called "2-Cys peroxiredoxin BAS1, chloroplastic-like", which shows the same changing trends and is worth further studying.By the comparison between the two techniques, we find each of their advantages and drawbacks. For iTRAQ, the accuracy is better, but iTRAQ protein ratio accuracy is underestimated especially in high ratio areas. As for label free, though more protein could be identified for quantification, it is limited to the number of samples that can be compared because of the high reproducibility required over the LC-MS runs to avoid variance issues. Consequently, the proteomics approach should be selected according to the biological problems and the experimental design.