Pilot Study Of Mitochondrial Phosphoproteome From Liver Of Mice With Endotoxic Shock

Sepsis is a syndrome of systemic inflammatorome induced by infection,of which the severe complication,sepsis shock,is the major cause of death of the patients suffered from wound,burn,and post operation.Statistic indicates that half of the sepsis were caused by Gram-negative bacteria,and that lipopolysaccharide（LPS）, which release from the cytoderm of Gram-negative bacteria,was considered to be the major molecule who responsible for the endotoxic sepsis.LPS can stimulates endothelium,macrophage and neutrophil,the 3 main targets of its,producing a large number of cytokine,which leads to inflammatory cascade reaction out of control,and leads to endotoxin shock,tissue damage and multiple organ dysfunction syndrome at last.Liver is the body of material and energy metabolism in center as well as most vulnerable to septic shock.Many evidences have showed that:the losses of mitochondrial function of liver cells are one of the main causes of liver damage in sepsis.During the development of endotoxin shock,the mitochondrial matrix calcium overload,substantial release of oxygen free radicals,NO generation increased,and a variety of cytokines,directly or indirectly,damage its function,so that mitochondrial structural of liver changes,metabolic dysfunction,leading to hepatocyte apoptosis, further leading to liver dysfunction and liver failure.Endotoxin induced signal transduction pathway is the important component in the process of endotoxin pathopoiesis.The key research of cellular signal transduction is to feel environmental stimuli,transduct signals to regulate the metabolism of physiological responses and gene expression of the molecular pathway. Phosphorylation modification itself with the simple,flexible,reversible characteristics,as well as phosphate donor ATP’s easy to get,has been selected to become one of the most common means of regulation in eukaryotic cells.Protein phosphorylation,which plays an irreplaceable role in signal transduction,is a well-known truth.Protein phosphorylation and dephosphorylation almost regulates all life activities,such as cell proliferation,development,differentiation,signal transduction,apoptosis,neural activity,muscle contraction,the process of tumor and so on.Now,lots of human diseases have reported to be caused because of unusual modification by phosphorylation,and some unnormal modification of phosphorylation is caused by certain disease.So,what endotoxic shock early mitochondrial function case,what happened to the function of mitochondria in early endotoxic shock? What about their specific molecular mechanism? We know very little about it.Thus,it is important to study mitochondrial phosphoproteins during endotoxic shock by large-scale proteomics analysis,which is useful to further understand the mechanism of the occurrence and development of endotoxic shock.Most previous studies are focus on several moleculars to analyze and find out few moleculars which properly have great significance to the occurrence and development of diseases,so as to further study its function.This is a traditional research strategy.Its merit is that the research questions can be focused and studied deeply,and the question can be answered thoroughly on a point.But if put this question in a system level,its function has become blurred trickle.It is caused by the limitations of research tools.Only accumulate enough results of such a "point",the question can be clear,but this process usually takes a long time.The proteome is highly dynamic,even if the same cell in different physiological or pathological environment,its protein expression are different,and such different proteins are often a sign of certain diseases happened.Through comparing the protein expression level between normal and disease cell（organization）,differential expression proteins were identified,which can be used to screen and find out potential drug target from the scale on the overall level,as well as for early diagnosis,intervention and treatment protein markers of disease.However,The quantitative analysis of Changes of proteins often limit our research perspective.Because a lot of important activity of the protein is regulated by post-translational modification,Sometimes protein level does not show significant changes while the level of post-translational modification has been significant change.Therefore,it is very useful to analysis of mitochondrial phosphoproteome before and after endotoxin stimulation by using quantitative techniques,which can help us to understand molecular mechanism of mitochondria damaged by endotoxin,and to find out more specific and sensitive protein markers which are closely related to the development of liver damage during endotoxic shock.Difference in gel electrophoresis（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.In this experiment,we use statistical analysis software,which was developed as part of Ettan DIGE system,to exploit the multiplexing,for DeCyder 2D analysis software for statistical analysis.DeCyder 2D^TM 2D software is an automated image analysis software suite which enables detection,quantitation,matching and analysis of Ettan DIGE system gels.Our experiment uses the DIA and BVA modules to analyze gel images.The Cy2 as the internal standard is used to calculate and standardize the ratio and we choose P＜0.05 as significant difference.In order to understand the molecule mechanism of mitochondria of endotoxic shock,we design a differential proteomics experiments by duplicating BALB/c endotoxic shock model as 0 min（control） and 60 min after peritoneal injection of LPS.We fractionated highly purified two groups BALB/c mice liver mitochondria by differential centrifugation combination with density-gradient centfifugation.Then mitochondrial proteins were extracted with lysis buffer and mitochondrial phosphoproteome were extracted by the phosphate metal affinity chromatography resin（PMAC）.The result of western blot showed that the enrichment of mitochondrial phosphoproteome was efficient.Finally,the differential expression of protein profile of two groups has been established by using DIGE technology. Through analysis by use of DeCyder difference 2-D analysis software,105 differential protein spots were found.The numbers of up-regulated proteins is 49; while the numbers of down-regulated proteins is 56.At last,a total of 42 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry（MALDI-TOF/TOF）.26 of them are had confirmed to be phosphorylated and 24 of them are closely related to functions of mitochondria.We predicted the subcellular localization of the differential proteins and made a result that,among 42 proteins,there are only 5 proteins localized in mitochondria; 34 proteins in both mitochondrial and other subcellular position;3 proteins in other subcellular organelle,but not in mitochondria.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,we also predicting proteins interactions using the "String" protein interaction databases.We found that 27 of identified proteins are interacted with each other and they constitute an interaction network.Based on the researches above,we have some conclusion:1.By transmission electron microscopy and immunoblotting,we found that differential centrifugation and dense centrifugation is an effective approach to isolate mitochondia.2.Mitochondrial phosphoproteins were enriched through the phosphate metal affinity chromatography resin（PMAC）.Subsequently,the phosphoproteins of normal control and endotoxic shock（LPS 1hr） BALB/c liver were separated by DIGE.105 differential protein spots with statistical significance between normal control and LPS1 hr were detected.3.105 differential protein spots were analyzed by mass spectrometry.42 proteins were identified after deleting keratin and identical proteins.26 of them are had confirmed to be phosphorylated.4.We predicted the subcellular localization of the differential proteins and made a result that,among 42 proteins,there are only 5 proteins localized in mitochondria;34 proteins in both mitochondrial and other subcellular position;3 proteins in other subcellular organelle,but not in mitochondria.5.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.27 of identified proteins are interacted with each other and they constitute an interaction network.