Identification Of Differential Proteome Of Mitochondria From Liver Of Mice With Endotoxic Shock

Sepsis is a kind of systemic inflammatory response syndrome induced by infection,and the sepsis shock,a severe complication of sepsis,is one of the major causes of death for patients suffered from trauma,burn,and post operation.Statistics indicate that half of sepsis in clinic is caused by Gram-negative bacteria infection,and that lipopolysaccharide(LPS),or called endotoxin,which is one of composing components of Gram-negative bacteria cell wall,is considered to be the major molecule responsible for sepsis caused by Gram-negative bacteria.LPS can widely act on human multiple organs and tissues,among which endotheliocyte,macrophagus and neutrophil are the main effector cells.Stimulated by LPS,they produce a large number of cytokines,namely "cytokine storm",and then lead to excess inflammatory cascade,and eventually result in endotoxin shock,tissue damage and multiple organ dysfunction.Over the last decade,many researchers have devoted to investigating the pathogenesis of sepsis,and few satisfactory results were obtained,though they have carried out amounts of animal experiments and clinical trials using different inflammatory factor antagonists,such as endotoxin antibodies,tumor necrosis factor etc.Up to now,the mortality of sepsis is still high.All above suggest that sepsis, especially the endotoxic shock,still need further research.In addition,more and more evidence indicates that the mitochondria injury of vital organs played an important role in the progression of sepsis,but its molecular mechanism remains unclear.Mitochondria are important organelles in eukaryotic cells as an energy convert place,,which usually appear in granular or bacillar shapes,sometimes also could be annular,dumbbell-shaped,or other shapes.The main chemical constituents of them are proteins and lipids.Mitochondria are enclosed by the inner and outer layer membranes,which consist of four parts,include the outer membrane,the inner membrane,the intermembrance space and matrix respectively.Additionally, mitochondria are not equally distributed in cells,and they can move,transform and proliferate in the cytoplasm according to the needs of cell metabolism.The main functions of mitochondria are synthesizing ATP by carrying out oxidative phosphorylation to provide direct energy for life activities of cells.In conclusion, mitochondria not only provide energy for cells,but also play an important role in regulation of generation of oxygen free radical,cell apoptosis,cell signal transduction, transporting intracellular ion through membrane and regulating electrolyte homeostasis in cells.Lots of experiments proved that changes of protein structures and functions in mitochondria are associated with many human diseases,such as degenerative diseases, heart disease,aging and cancer.Therefore,the research on mitochondrial protein absorbed more and more attention,especially in the area of neurodegenerative diseases.Very limited knowledge is known on what functional events had happened and their specific molecular mechanisms at the early stage of endotoxic shock in mitochondria.But answers to this are significantly meaningful to increase the understanding of the pathogenesis of sepsis on molecular level.Whereas,functional events happened in mitochondria during endotoxic shock are finally executed by proteins,therefore,the answer to the question "which proteins have changed?" is the core of the whole project.Fortunately,the differential proteomics method could unveil this real mystery.Through comparing the protein expression level between differences samples, differential expression proteins were identified,which can be used to solve various biological problems.With the development of proteomics technology,it is feasible to study whole changes of mitochondrial proteome in the physiological and pathological process now.At present there are many novel differential proteomics analysis methods,among which the difference gel electrophoresis(difference gel electrophoresis,DIGE) technology becomes one of the most popular one,because it's not only have the high-resolution feature,inherited from two-dimensional gel electrophoresis(2-DE),but also have high reproducibility,high sensitivity,high throughput and high dynamic range.DIGE is a method which label protein samples with different fluorescent dyes before 2-D electrophoresis,and then separate up to three different protein samples at the same time in one two-dimensional gel,The application of the internal standard could further increase the credibility of the experiment,and ensure the results could reflect the real biological differences,while avoid influence of systematic errors on experimental results.Since The most obvious advantage of DIGE system is integrating the advantages of both CyDye DIGE dye multiple labeling method and DeCyder difference 2-D analysis software.DeCyder software takes the advantage of the spots co-detecting algorithm,which can automatically detected fluorescence images,eliminate background,quantifiy,normalize and match spots in gel,thus, systematic errors caused by different operators can be eliminated.Based on the advantages of DIGE technology platform,great opportunities comes to us to discover the information of differential proteome contained in mice liver mitochondria with endotoxic shock and provide novel theories for sepsis research.A technology platform based on difference gel electrophoresis,MALDI TOF/TOF mass spectrometry and bioinformatics analysis was established first,and then some assays on its sensitivity and reproducibility was done in our research.After duplicating the endotoxic shock model of BALB/c mice after anesthesia,we collected mice liver,applied differential centrifugation and density gradient centrifugation method to extract purified liver mitochondria,and extracted the entire mitochondrial proteins by lysis buffer.Then we got the expression patterns of liver mitochondrial differential proteome of endotoxic shock mice by using DIGE technology.Through analysis by use of DeCyder difference 2-D analysis software,26 differential protein spots were found.The numbers of up-regulated and down-regulated proteins are both 13.Finally,a total of 22 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF).The co-expression of these differential proteins indicates that they must have direct or indirect relations.Therefore,how to find out and discover the pathophysiology meaning of these relations in endotoxic shock liver mitochondria is the key for further research.Bioinformatics is a useful tool to solve this problem. Firstly the identified differential proteins were processed by subcellular localization analysis with subcellular localization software WoLF PSORT.The results indicated that two proteins predicted only localized in the mitochondria;eight proteins predicted localized in mitochondria;eight proteins predicted localized in cytoplasm; six proteins predicted localized in mitochondria or other cell organelles;10 proteins predicted only localized in other subcellular structures but not in mitochondria.These results prove that the mitochondrial extraction method is successful.After subcellular localization analysis,the protein functional analysis had been done.Through analyzing protein domain and motif database,the identified proteins were roughly categorized into five groups based on functions,which include protein and lipid metabolism,energy metabolism,regulation of generation of oxygen free radicals,apoptosis,and cellular signal transduction.However,this is not enough to know the relationship among proteins,and predicting proteins interactions were also needed.Using the "String" protein interaction databases,we had analyzed the primary interaction of the 22 kinds of proteins(direct effect) and the result showed that two groups of the proteins were of direct interaction.Among these groups,Non-specific lipid transfer protein(Scp-2) and Phytanoyl-CoA dioxygenase(Ln1) are two proteins located in the mitochondria, the expression of which were both down-regulated 2.0 folds in mitochondrial after being stimulated by LPS for 1 hours.The complex of these two proteins may be involved in the regulation of fatty acid metabolism.Another group,albumin(Alb),α-1-anti-trypsin 1-1(Aat2) and thyroxine transporter binding protein(Ttr),after LPS stimulation for an hour,were all up-regulated by twice.According to software prediction,these three proteins located in the plasma,while not in mitochondria. Through consulted the literature,the expression of Ttr is reduced in plasma in inflammatory stimulation,nevertheless,we found these three proteins were all up-regulated,which is a meaningful discovery.We speculate that these three proteins may transfer from plasma into the mitochondria in the form of functional complex, involved in the stress response to endotoxin in cells.Through the research,five conclusions are drawn as follows:1.Established DIGE technology platform which providing a new system with high reliability,high reproducibility for differential proteomics research in our laboratory.2.Extracted and purified mitochondria from mice liver successfully and confirmed by transmission electron microscopy.3.Identified difference gel electrophoresis patterns of mice liver mitochondrial differential proteome between the early stage with endotoxic shock and the normal BALB/c mice.4.Found 26 different express proteins by differential software analysis,then identified 22 different proteins using mass spectrometry identification. 5.Predicted two direct protein-protein interaction complexes by applying bioinformatics analysis on 22 different proteins.Through the experimental techniques exploration,we have established a set of differential proteomics analysis techniques based on the difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and Bioinformatics.This set of techniques is successfully applied to the analysis of differential proteomics of mice liver mitochondria with endotoxic shock.In conclusion,The research not only provide us a novel technology system on revealing the molecular mechanism of life process,but also provide us a new chance for further studying the pathogenesis of sepsis and its control strategy.