| 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 sepses were caused of 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 endotoxin 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. In past, more attention pays to the 3 main tagets of LPS, but less to other cells or tissues that are waiting for exploiting.Liver is annoter important organ involved in sepsis. It's well known that besides playing a key role in metabolism, Liver carries important immunologic function. It can produces and releases a large number of acute phase proteins in response to inflammatory stress, playing an important role in prompt defense. However, more mechanism about liver acting in sepsis and the changes of itself during development of the disease is under exploit, which attracts us.In this research, we interest in exploiting the changes of liver during the development of the sepsis or septic shock and try to explain its molecular mechanism. So we duplicate the animal model of endotoxemia or endotoxin shock of BALB/c mice in using method of LPS administration, and use their livers as research material.However, a problem we encounter is to choose two strategies of research: one is traditional based on single molecule research scale, and another is innovational, intraducing the principle of "omics". The traditional one is senior in explaining the detail of a single problem, but junior in answering systematic one, while using "omics" method is on the contrast side. In this research, we want to have a systematic view of the problem and form a framework for ongoing studying. Using this strategy may be more effective and easy to graph important aspects underlying the problems. So we start our study with the strategy of proteomics.Initially, the proteomics projects aimed to discover the total proteins of a cell, tissue, or an organ. However when the enormous data of proteins were discovered and stacked without of functional annotation and with less association with biological process, Scientists start to emphasis the biological meaning in the proteomics research and the importance of the functional proteomics. The differential proteomics, an important way of differential proteomics, is to contrast protein groups of different bio-stage and try to discover the specific proteins associated with Special life stage or disease. In this study, we aim to discover the specific proteins of liver which may play important role in the development of the endotoxic shock. However, the enrichmentsof the extracellular matrix proteins and the structural proteins of cells may impair the detecting of low abundant ones, which usually are functional important in disease and special in biological process. We try to enrich low abundant proteins by extracting subcellular proteins, which is less complicated than total proteins of cells or tissues, and benefit for focusing research sights and easy to annotate. So we select cell nuclear as our target.Commonly, the differential proteomics set two groups to contrast, which is a research strategy for reflecting static situation. However, the biological or disease processes are dynamic ones. In order to well understand the molecule mechanism of the procedure of endotoxic shock, we design a dynamic differential proteomics experiments by duplicating multi time point BALB/c endotoxic shock model as 0 min (normal control), 30 min, 1 h, 3 h, and 6 h after peritoneal injection of LPS.In order to acquire reliable results from contrast between multi groups, a key problem is to acquire technical stability and reproducibility in protein separation. Two-dimensional electrophoresis (2-DE) is a traditional technique which was serous affected by operational bias and by the cause of separating proteins in different gel. As an annotation technique, protein chromatographic separation was developed rapidly in recent years, along which, protein fraction 2 dimension (PF2D) system is a good one for the well characters of automation, less affected from operation. PF2D is a 2 dimensional high performance liquid chromatography (HPLC). The firstdimension of PF2D is high performance chromatofocusing (HPCF) , which separates proteins by its property of isoelectric point (pI), and the second is reverse phase high performance liquid chromatography (RP-HPLC), which separates by the one of hydrophobicity. The system supports loading large amount of protein (1-5 mg per time) for the large separation volume of HPCF column. The good performance in reproducibility is fit for us to apply it in dynamic differential proteomics.However seldom experiment was reported in using PF2D separating protein samples extracted from tissue. The success examples are major in separating samples from culture cells. Even there aren't any reports that apply PF2D in researching subcellular fraction except the ones of membrane. The main cause is the incompatible of the technique of protein extraction and the PF2D separating ones. The principle of the fist dimension of PF2D is week anion-exchange chromatograph, which form a changed pH gradient in mobile phase and separate protein by it pI. So the sample buffer can not contain any ion, which will impair the affinity between protein and solid phase medium in very low content. Using non-ion lysis buffer that consists ofhigh content of denaturant and detergent can apply to extract total or membrane protein of culture cells, but not practicable in extracting protein from tissue and applying in subcellular protein extraction. The lysis buffers of extraction protein from tissue or subcellular fraction usually been introduced by high content of salt ion, so the method of desalting is key point in making the extraction and separation technique compatible. The separation effect of PF2D is poor and we nearly failed in detecting protein peaks in pH gradient separating area when we only use one step desalting procedure by using protein desalting-10 (PD-10, a gel-filtration chromatograph column) following the suggestion of Beckman Coulter Company, the producer of PF2D. More effort paid in improving the desalting effect, and after all,we succeed in finding a way to desalt effectively and in separating protein samples extracted from liver by using PF2D. The method is add a desalting procedure by using ultra filtration tech with self created desalting buffer, ultra filtration buffer, before the procedure of using PD-10.Providing by the accurate protein quantification, we use the method we created to separate the 5 groups of liver nuclear protein samples of multi time point BALB/c mice endotoxic shock model. The separation effect is good for it well detecting protein by showing satisfactory peaks, which appears Gaussian distribution and sharp with considerable height. Thus we have mapped the PF2D profile and differential protein ones of the normal and endotoxic shock BALB/c mice liver nuclear proteins. Based on analyzing the profiles, we picked up the fractions of 503 differential peaks and selected 50 fractions of it to identify by ABI4800 MALDI TOF/TOF analyzer. 60 proteins were identified with confident and were observed in the differential protein profile in order to have the dynamic or time-spatial information of its. If a group of differential protein appears in a same time and same space, it infers that they must have relationship between each other. However, how to discover the relationships and set the associations between the relationships and the biological process is major problem eager to be answer. It is useful to apply bioinformatics tools to analyze such problems, to form frame view and to enlight the subsequent research. We predict the subcellular localization of the proteins and make a result that, among the 60 proteins, there are 6 only localized in nuclear, 24 in both nuclear and cytoplasmic, 23 in multi subcellular position besides nuclear, 7 not in nuclear, which validate the effects of the nuclear protein extraction procedure.We also analyzed the protein functions by using gene ontology (GO), protein domain and motif information from the protein database in website. Base on the analysis, we grouped the proteins into 4 kinds: ones of having the potential to bind nucleotide, ones of participating to maintain the stability of proteins and homeostasis of intra-nuclear, ones participating in protein post translation modification, and ones of mediating protein-protein interaction. Further analysis is to discover relationship between proteins. By using String database and software, we find 3 paired of proteins, which may form complex. The one of the pairs consists of Eyes absent homolog 1(Eya1) and pair box protein (Pax1). The two proteins have transcriptional activityand their complex may be involved in the negative regulation of cell apoptosis. The second pair is composed of G-protein signaling modulator 1(Gpsm1) and Gnas, and may be in the pathway of G-protein signaling. The third pair is comsist of enoyl-Coenzyme A hydratase/3-hydroxyacyl Coenzyme A dehydrogenase (Ehhadh) and acyl-Coenzyme A oxidase 2 (Acox) and may has function in protein acetylation modification. By using ingenuity database and its software to analysis relationships between the 60 protein and their biological function, we find two pathways that the proteins identified, which increased after LPS administration, may be involved in. one pathway associates with cytoskeleton reorganization, and another may be involved inimmunologic stress response and cell function maintenance. These analyses suggest that, in responding to the endotoxemia and endotoxic shock stress, there are some events, nuclear skeleton reorganization, transcription activation, and enhancement of DNA repairing and protecting in order to anti-apoptosis of the cell, happened intra nucleus, and thus to make immunological stress response.In analysis of the pI information of the proteins, we find the pI of a group of proteins was changed during in endotoxic shock developing. The proteins are Histl h l t, Histl h2bp, Histone H4 and Hist3h2a, which belong to histone super family, and are basic protein with pI 10-12. but we observed that they have been acidized and their pls were changed to 4.85-5.16, 7.48-7.78, 6.59-6.87 and 6.31-6.61 respectively. The functional analysis also found some proteins may involve in post-translatoin protein modification of acetylation, phosphorylation and methylation. Which suggests that, through the mechanism of "histne code", the transcriptional level haven been increased during the development of the endotoxic shock.Based on the researches above, we have some conclusion:1. We breakthrough the block of the applying PF2D protein separation tech to the field of analyzing protein sample from tissue or organ by implementing the compatible of the protein extraction technique and separation ones. We solve the problem by creating a desalting methods combing of PD-10 desalting and ultra filtration with self made "ultra filtration buffer" which compatible with SB, by enhancing degreasing with combing of washing with centrifugation and filtration by absorbent cotton after organic solvent treating, and by removing the particle with 0.45μm and 0.22μm filter apparatus.2. Providing by the accurate protein quantification, and stability of the experiment procedure, optimizing of the separating model, good reproducibility have been achieved, and satisfied the technique demands of dynamic differential proteomics.3. We have mapped the PF2D profile and differential protein ones of the normal and endotoxic shock BALB/c mice liver nuclear proteins.4. Based on analyzing the profiles, we picked up the fractions of 503 differential peaks and selected 50 fractions of it to identify by ABI4800 MALDI TOF/TOF analyzer. 60 proteins were identified and were observed in the differential protein profile in order to have the dynamic or time-spatial information of them.5. In the 60 differential proteins, we find 3 paired of proteins, which may form complex and mapped 2 signal pathways. Functional events analysis indicating that, in responding to the endotoxemia and endotoxic shock stress, there are some events, nuclear skeleton reorganization, transcription active, and enhancement of DNA repairing and protecting in order to anti-apoptosis of the cell, happened intra nucleus, and thus to make immunological stress response6. We find the pI of a group of histone proteins was changed and has been acidized during in endotoxic shock developing. Also found some proteins may involve in post-translatoin protein modification of acetylation, phosphorylation and methylation. Which suggests that, through the mechanism of"histone code", the transcriptional level haven been increased during the development of the endotoxic shock.Through optimizing the experiment tech, we succeed in establishment a workflow of PF2D-MALDI TOF/TOF MS-bioinformatics in dynamics differential proteomics. The result shows that it is a useful and powerful in exploiting the mechanism of biological process or diseases.
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