Stable Isotope Dimethyl Labeling-Based Animal And Plant Tissue Quantitative Proteomic Studies

Stable isotope dimethyl labeling is a chemical labeling method, which can label the N-terminus and the side chain of lysine residues. By introducing stable isotope deuterium and 13C, it can form differentially labeled peptide isotope in MSI. Stable isotope dimethyl labeling is a simple, fast and economic strategy which can be applied to any sample due to its peptide-labeling feature, especially suitable to tissue sample analysis.In the first part of this dissertation, we used stable isotope dimethyl labeling combined with mass spectrometry to investigate the difference in mitochondria of Honglian rice sterile line YtA and fertile line YtB. We found a reduced quantity of specific proteins in mitochodrial complexes in YtA compared with YtB while the total amount of mitochondrial proteins was similar. By enzyme activity assay, we discovered that the activity of oxidative phosphorylation complexes was reduced in the sterile line YtA. By transmission electron microscopy, altered mitochondrial morphology was found in the sterile line YtA. All these results indicated that despite of the unchanged total amount of mitochondrial proteins, assembled mitochondrial protein complexes were reduce in YtA, suggested there may be a complex assembly defect in the sterile line YtA. Besides, we also found jasmonic acid biosynthesis was abnormal in the sterile line YtA.In the second part of this dissertation, we used stable isotope dimethyl labeling combined with mass spectrometry to study the proteome changes in ischemia and reperfusion (I/R) model of rat retinas. Based on the relative quantitation data of 1088 proteins,234 proteins (-22%) showed a greater than 1.5-fold change following I/R injury,194 of which were up-regulated and 40 were down-regulated. Gene ontology and bioinformatics analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. These data suggest that following I/R injury, essential metabolic activities that are critical for cell survival may be elevated, but functional activities may be suppressed. Through STRING protein-protein interaction nertwork analysis, a ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. In the down-regulated proteins, we found a synaptic-related protein network was significantly down-regulated. Combined with Western blots and immunohistochemistry (IHC) studies, the data suggested a functional reduction of neurons following a retinal I/R injury.In the third part of this dissertation, we used stable isotope dimethyl labeling combined with mass spectrometry to study the protein level differences between the normal and live-specific gp78 knock out mice. In two biological replicates of male mice, a total of 3193 protein were quantified with 2088 proteins shared by both replicates. In these 2088 quantified proteins,42 proteins showed higher than 1.5 fold change. By bioinformatics analysis, we found these 42 proteins are mostly metabolic process related enzymes, especially lipid metabolism related enzymes. All lipid metabolism-related enzymes showed some degree of reduction, indicating that the lipid biosynthesis in gp78 knock out mice was suppressed.