内毒素休克小鼠肝脏核蛋白质组学研究
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摘要
脓毒症(sepsis)是感染引起的全身炎症反应征候群，脓毒症的严重并发症-感染性休克(septic shock)是创伤、烧伤和手术后病人最主要的死亡原因之一。统计表明，临床半数的脓毒症是由于革兰氏阴性菌(Gram-negative bacteria)引起的，在革兰氏阴性菌的细胞壁上的脂多糖(lipopolysaccharide，LPS)，或称为内毒素(endotoxin)，被认为是革兰氏阴性菌引起脓毒症的主要作用分子。LPS广泛作用于机体多组织器官，其中内皮细胞、巨噬细胞和中性粒细胞是最主要的效应细胞，在LPS的刺激下，它们产生大量的细胞因子，即“细胞因子风暴”(cytokine storm)，进而引起失控性的炎症级联反应，最终引起内毒素休克、组织损伤和多器官功能障碍(mutiple organ dysfunction)。因此，脓毒症(sepsis)或内毒素休克(endotoxic shock)的分子机制的研究大多聚焦在内皮、巨噬细胞和中性粒细胞上，而在其它组织和器官上的相对研究较少。
其实，脓毒症是多组织器官和系统的疾病，疾病从菌血症(bacteremia)、毒血症(toxicemia)演进到休克、多器官功能障碍的过程中，不但病原微生物及其毒性产物会与机体组织器官发生相互作用，而且各组织器官之间也会发生错综复杂联系，共同促进疾病的发生发展和转归。肝脏是人体代谢的枢纽，也是重要的免疫器官，血液中大量蛋白也是由肝脏合成分泌的。在脓毒症的炎性应激刺激下，肝脏大量合成分泌急性期蛋白(acute phase protein，AP)，发挥一种启动迅速的机体防御机制，然而肝脏对病原微生物或其毒性产物产生什么样的反应，与及脓毒症或感染性休克发生发展进程中肝脏自身变化如何?肝脏通过什么样的分子调控机制上调多种炎症相关反应蛋白的表达?对于这些问题，目前所知有限。这些问题的解答，对于了解错综复杂的脓毒症及其感染性休克的发病机制无疑是有益的。
针对肝脏在感染性休克时介导产生多种炎症相关蛋白这一复杂过程，本课题通过检查脓毒症及感染性休克发生发展进程中肝脏组织的变化，从分子机制上了解肝脏在这种疾病条件下所做出的反应。本研究选用了BALB／c小鼠的肝脏作为研究对象，利用LPS作为刺激因子，制备内毒素血症(脓毒症主要类型)和内毒素休克(感染性休克的主要类型)的动物模型，从而获取这种疾病状态下的肝脏组织。然而，选择什么样的研究策略对组织器官进行有效的分子机制的研究成为本课题的关键。传统的研究方式是单分子或几个分子的研究，其优点是研究问题集中，容易深入，可以比较透彻地回答一个点上的问题，但是将这个结果放到一个体系层面上，其功能又变得扑簌迷离，而由点到面的形成过程又需要较长时间。随着人类基因组计划(human genomic prolect，HGP)的完成、蛋白质组学的方法技术和理论系统已成为后基因组时代的主旋律。这为我们用系统的观点来研究问题提供了机遇。本课题是从一定的体系上来提出问题，因此可以探索用蛋白质组学的策略来开始研究。
早期的蛋白质组学主要是解决结构学的问题，即解析特定细胞、组织或器官所有蛋白质的集合。随着工作的积累，结果数据的急剧膨胀，人们发现要解析庞大的蛋白质数据是一个巨大的挑战。因此提出了功能蛋白质组学(functionalproteomics)的概念，通过差异蛋白质组学(differential proteomics)技术，寻找不同生命过程或疾病过程状态下出现的差异蛋白，从而找到与这些生命过程或病理过程功能相关的蛋白质集群。本研究拟通过差异蛋白质组学技术来寻找肝脏组织内与内毒素休克相关的蛋白质。然而，在肝脏组织里，蛋白质的种类相当复杂，而且细胞外基质(extracellular matrix)蛋白相当丰富，这些蛋白与及细胞内的结构性蛋白的含量占据了肝脏蛋白质组的主要部分，而一些功能性蛋白往往表达丰度很低，在蛋白质组分离过程中，由于上样量有限，高丰度蛋白的存在会使低丰度蛋白很难被检出。所以我们希望通过提取亚细胞(subcellular)的蛋白质组来富集低丰度蛋白。亚细胞蛋白质组相对于全细胞或组织蛋白质组相对单纯得多，解析起来相对容易，而且可以使我们研究的问题更加集中，所以我们首先选择了细胞核来进行研究。另外，疾病的发生发展是一个动态的过程，仅仅进行一个对照组和一个实验组的差异比较很难解释疾病发生发展的机制。因此我们需要选择疾病发生发展过程中的多个时间点来进行比较分析，观察蛋白质的动态差异。根据上述思考，我们利用LPS来制作内毒素休克BALB／c小鼠模型，分别提取LPS刺激后30 min、1h、3h和6h的小鼠及正常对照组小鼠肝脏的核蛋白来进行研究。
要实现多组差异图谱的比较，分离技术的稳定性和重复性是获得可靠结果的关键性问题，传统的双向凝胶电泳技术(two-dimensional electrophoresis，2-DE)由于在不同的胶上分离蛋白、过程复杂和人为影响因素太大等原因而使重复性相对较差，不太适应于动态差异蛋白质组学。而其近年来的2-DE的新发展，荧光差异显示双向凝胶电泳(fluorescence 2D differential in-gel electrophoresis，F-2D-DIGE)虽然具有很好组间可比性，但是在同一块胶内最多也只能分离3组样品，同样不能达到本实验比较5组样品的要求。二维高效液相色谱(twodimensional high performance liquid chromatograph，2D-HPLC)是近年发展起来的蛋白分离新技术，其主要的特点是分离过程实现自动化，人为的因素影响较少，分离的效果主要取决于色谱柱的柱效。其中，二维蛋白分离系统(protein fraction2 dimension，PF2D)是一套新开发的2D-HPLC，其第一维色谱为高效色谱聚焦(high performance chromatofocusing，HPCF)，按蛋白质的等电点将样品进行粗分离，第二维色谱为反相高效液相色谱(reverse phase-high performance liquidchromatography，RP-HPLC)，按蛋白质的疏水性对蛋白质进行分离。这套系统的特点是其第一维HPCF色谱柱的柱体较大，上样量高，一次可以上样1～5 mg(2-DE技术最多只能上样1mg)，这对我们找到低丰度(low abundance)蛋白十分有利。另外，在保证柱效的前提下，PF2D可以分离多组样品而保持较好的重复性。可以解决我们对重复性的要求。
然而，目前在国际上，能够利用PF2D进行组织来源的样品分离的报道尚未见到，成功分离的基本上是培养细胞来源的和体液的样品。同时，该系统在亚细胞蛋白质组学领域的成功应用也仅局限于培养细胞膜蛋白的分离。其主要原因在于蛋白提取技术与PF2D分离技术的未能实现良好的兼容。PF2D第一维的分离是弱阴离子交换色谱(weak anion exchange chromatography)技术，利用流动相(liquid phase)形成动态的pH梯度实现不同等电点蛋白质的分离。因此，样品溶液内不能含有任何的盐离子(ion)。在提取培养细胞的全蛋白(tatal protein)或膜蛋白(membrane protein)时，我们可以用不含盐离子的高含量变性剂(denaturant)、去污剂(detergent)的裂解液(lysis buffer)来实现。但是对于提取组织样品和提取亚细胞蛋白是不可行的，往往要介入高含量的盐离子，因此需要进行除盐的处理。然而PF2D的第一维分离对于样品除盐的要求远远超出传统的2-DE技术。一点点的盐离子存在都可能导致蛋白质与色谱柱固相结合能力的下降而无法实现分离。我们在提取肝脏组织细胞核时介入了高浓度的盐离子。用PF2D系统配套的除盐方案进行除盐后，样品分离的效果很差，pH梯度分离范围内几乎检测不到蛋白峰。为此，我们增加了除盐强度，自己研制与公司提供的第一维流动相试剂兼容的超滤缓冲液(ultrafiltration buffer)，利用超滤浓缩除盐的方法，将含盐的核蛋白提取液更换为不含盐的超滤缓冲液，再通过PD-10进一步除盐。通过不断地对超滤缓冲液进行优化，终于使得PF2D分离组织来源蛋白达到了较好的效果。
用这种组织蛋白分离的方法，在确保组问样品定量准确一致的条件下，通过优化PF2D分离工作模式，我们分离了上述几个LPS刺激时间点和正常对照组的核蛋白样品，蛋白分离表现出良好的重现性，蛋白峰基本呈正态分布(normal distribution)，不少峰呈比较满意的尖锐峰(sharp peak)，峰高也比较理想。各组间既有良好的能够重现的峰出现，也有差异峰出现。结果，我们建立了LPS刺激不同时间点和正常的BALB／c小鼠肝脏核蛋白PF2D图谱和组间比较的差异蛋白图谱(dfferential protein profile)。
根据差异图谱，在5个组间我们找到了503个差异蛋白峰。将50个差异蛋白峰所在的二维组分(fraction)取出，进行质谱(mass spectrum)酶解样品制备，使用ABI 4800 MALDI TOF／TOF质谱仪鉴定得到了60个差异蛋白(有的峰鉴定出2～3个差异蛋白)。我们分别观察这60个蛋白所在的差异峰在正常组和LPS刺激不同时间点的丰度，即观察其在核内的量的动态变化，从而获得了这些蛋白的时空变化的信息。
另外，这些差异蛋白的同时出现，必定意味着它们之间有着直接和间接的联系，如何找到它们之间的联系，并且挖掘出这些联系在内毒素休克肝脏中的病理生理学意义，组织出一个框架性的认识，为进一步深入的研究指明道路。这也是目前功能蛋白质组学十分关注和急待解决的问题。而生物信息学(bioinformatics)可以作为解决这个问题的有用工具。我们首先通过亚细胞定位(subcellular localization)的生物信息学方法对60个蛋白质进行了定位的分析，结果完全定位于核内的有6个；既定位于核内，又可以定位于胞浆的有24个；既定位于核内，又可以定位于其它多个亚细胞结构的有23个；在其它亚细胞结构定位，但是没有定位于胞核内的有7个。说明我们的核蛋白提取是有效的。
通过定位分析后，我们分别对蛋白质进行功能分析，利用蛋白质数据库的GO(gene ontology)功能注释、蛋白质结构域(domain)和模体(motif)的分析，粗略地对鉴定的蛋白质进行功能分组，归纳下来有4类：能与核酸结合的、参与氨基酸残基修饰的、参与维持蛋白质或核内理化性质稳定的及参与介导蛋白质-蛋白质相互作用(protein-protein interaction)的。通过这些分析，我们对这群蛋白质的认识有了一定的条理。然而仅仅以此，还不能达到了解蛋白质间相互关系的目的，需要对蛋白质间的相互作用进行预测和分析。我们利用String蛋白相互作用数据库对这60个蛋白进行了一级相互作用(直接作用)的分析，结果发现这些蛋白质中，有3对蛋白质是发生直接的相互作用的，其中缺眼基因同源物1(Eyes absent homolog 1，Eyal)与配位盒蛋白1(pair box protein 1，Pax1)是一对具有转录活性的蛋白质，它们形成的复合物(complex)可能参与细胞凋亡(apoptosis)的负向调节转录(tranpcripton)机制；另外一对蛋白质是G-蛋白调节因子1(G-protein signaling modulator 1，Gpsml)和Gnas，二者形成的复合物可能参与G-蛋白信号通路(G-protein signal pathway)。乙酰辅酶A水合酶／3-羟氨基辅酶A脱氢酶(enoyl-Coenzyme A，hydratase／3-hydroxyacyl Coenzyrne Adehvdrogenase，Ehhadh)和乙酰辅酶A氧化酶2(acyl-Coenzyme A oxidase 2，Acox2)是一对参与乙酰化反应(acetylation)的蛋白质，能对赖氨酸(lysine)进行乙酰基化反应。另外我们还通过Ingenuity信号通路数据库对LPS刺激后丰度增加的蛋白质进行信号通路分析，结果2个通路与这群蛋白质的比较匹配度较高，一个是参与细胞骨架重构(reorganization of cytoskeleton)的，另外一个是参与免疫应激(immunological stress)和细胞功能维持的。上述分析结果提示我们，在内毒素休克进程中，肝脏核内有可能发生核骨架重构、免疫应激；做出这些反应的同时，还加强了核内环境稳定性的维护，防止毒性金属离子和过氧化物损伤，在增加转录调节的同时，加强对DNA的保护和修补，抑制凋亡。
另外，通过观察鉴定的蛋白质在PF2D等电点中的信息，我们发现一类蛋白质的等电点发生了变化，它们分别是睾丸H1组蛋白(Histlhlt)、组蛋白H2B型1-p(Histlh2bp)、组蛋白H4(Histone H4)、组蛋白H2A型3(Hist3h2a)、这类蛋白质是组蛋白家族成员，均为碱性蛋白质，pI在10-12之间，在核内环境里带正电荷。然而，我们分别从pI 4.85-5.16、7.48-7.78、6.59-6.87、6.31-6.61等组分中找到这些蛋白质，同时通过蛋白质功能信息的检索中，还在60个鉴定的蛋白质中找到了一些蛋白质是参与乙酰化、磷酸化(phosphorylation)及甲基化(methylation)反应的。这些提示我们，内毒素休克进程中，可能通过组蛋白密码机制，肝脏细胞的转录加强，对刺激做出适应性的反应。
通过研究，我们得到以下结论：
1．研制了与SB相兼容的超滤缓冲液，使用超滤浓缩除盐与PD-10除盐相结合的方法，加强样品除盐处理；联合使用0.45μm和0.22μm滤器对蛋白样品进行除颗粒处理，使样品制备得以优化。实现蛋白提取技术与离子交换色谱分离技术的兼容，突破了PF2D难以应用于组织样品的技术难关。
2．在PF2D分离操作中，加强蛋白质定量的准确性，强化实验操作的稳定性，优化PF2D分离工作模式，从而成功地实现多组蛋白质样品分离的良好可比性，达到了动态差异蛋白质组学分离技术要求。
3．建立了正常和内毒素休克BALB／c小鼠多时间点(30min、1 h、3 h、6h)肝脏核蛋白PF2D图谱和两两比较的DeltaVue差异图谱及5组比较的线形差异图谱。
4．在正常和内毒素休克BALB／c小鼠肝脏核蛋白差异图谱中选取了503个差异蛋白峰，选取其中的50个进行质谱鉴定，结果鉴定出60个差异蛋白。
5．对60个差异蛋白进行生物信息学分析，预测出3个直接的蛋白质-蛋白质相互作用和绘制了2个细胞信号转导通路图。功能事件分析表明内毒素休克小鼠肝脏细胞核内发生了核骨架重构，加强了核内环境稳定性的维护，在增加转录调节的同时，加强了对DNA的保护和修补，抑制凋亡，参与免疫应激反应。
6．发现内毒素休克BALB／c肝脏细胞核内组蛋白等电点发生了酸化改变，找到了一些具有对蛋白质进行乙酰化、磷酸化、甲基化修饰功能的蛋白质和复合物。提示组蛋白密码可能是内毒素休克小鼠肝脏细胞转录调节机制之一。
通过实验技术方法的探索，我们建立了一套基于二维高效液相色谱分离技术的PF2D-MALDI TOF／TOF质谱-生物信息学的蛋白质组学分析技术。并将这套技术成功地应用于动态差异蛋白质组学分析。为揭示生命过程和疾病机制提供了新方法。
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 response
6. 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.
其实，脓毒症是多组织器官和系统的疾病，疾病从菌血症(bacteremia)、毒血症(toxicemia)演进到休克、多器官功能障碍的过程中，不但病原微生物及其毒性产物会与机体组织器官发生相互作用，而且各组织器官之间也会发生错综复杂联系，共同促进疾病的发生发展和转归。肝脏是人体代谢的枢纽，也是重要的免疫器官，血液中大量蛋白也是由肝脏合成分泌的。在脓毒症的炎性应激刺激下，肝脏大量合成分泌急性期蛋白(acute phase protein，AP)，发挥一种启动迅速的机体防御机制，然而肝脏对病原微生物或其毒性产物产生什么样的反应，与及脓毒症或感染性休克发生发展进程中肝脏自身变化如何?肝脏通过什么样的分子调控机制上调多种炎症相关反应蛋白的表达?对于这些问题，目前所知有限。这些问题的解答，对于了解错综复杂的脓毒症及其感染性休克的发病机制无疑是有益的。
针对肝脏在感染性休克时介导产生多种炎症相关蛋白这一复杂过程，本课题通过检查脓毒症及感染性休克发生发展进程中肝脏组织的变化，从分子机制上了解肝脏在这种疾病条件下所做出的反应。本研究选用了BALB／c小鼠的肝脏作为研究对象，利用LPS作为刺激因子，制备内毒素血症(脓毒症主要类型)和内毒素休克(感染性休克的主要类型)的动物模型，从而获取这种疾病状态下的肝脏组织。然而，选择什么样的研究策略对组织器官进行有效的分子机制的研究成为本课题的关键。传统的研究方式是单分子或几个分子的研究，其优点是研究问题集中，容易深入，可以比较透彻地回答一个点上的问题，但是将这个结果放到一个体系层面上，其功能又变得扑簌迷离，而由点到面的形成过程又需要较长时间。随着人类基因组计划(human genomic prolect，HGP)的完成、蛋白质组学的方法技术和理论系统已成为后基因组时代的主旋律。这为我们用系统的观点来研究问题提供了机遇。本课题是从一定的体系上来提出问题，因此可以探索用蛋白质组学的策略来开始研究。
早期的蛋白质组学主要是解决结构学的问题，即解析特定细胞、组织或器官所有蛋白质的集合。随着工作的积累，结果数据的急剧膨胀，人们发现要解析庞大的蛋白质数据是一个巨大的挑战。因此提出了功能蛋白质组学(functionalproteomics)的概念，通过差异蛋白质组学(differential proteomics)技术，寻找不同生命过程或疾病过程状态下出现的差异蛋白，从而找到与这些生命过程或病理过程功能相关的蛋白质集群。本研究拟通过差异蛋白质组学技术来寻找肝脏组织内与内毒素休克相关的蛋白质。然而，在肝脏组织里，蛋白质的种类相当复杂，而且细胞外基质(extracellular matrix)蛋白相当丰富，这些蛋白与及细胞内的结构性蛋白的含量占据了肝脏蛋白质组的主要部分，而一些功能性蛋白往往表达丰度很低，在蛋白质组分离过程中，由于上样量有限，高丰度蛋白的存在会使低丰度蛋白很难被检出。所以我们希望通过提取亚细胞(subcellular)的蛋白质组来富集低丰度蛋白。亚细胞蛋白质组相对于全细胞或组织蛋白质组相对单纯得多，解析起来相对容易，而且可以使我们研究的问题更加集中，所以我们首先选择了细胞核来进行研究。另外，疾病的发生发展是一个动态的过程，仅仅进行一个对照组和一个实验组的差异比较很难解释疾病发生发展的机制。因此我们需要选择疾病发生发展过程中的多个时间点来进行比较分析，观察蛋白质的动态差异。根据上述思考，我们利用LPS来制作内毒素休克BALB／c小鼠模型，分别提取LPS刺激后30 min、1h、3h和6h的小鼠及正常对照组小鼠肝脏的核蛋白来进行研究。
要实现多组差异图谱的比较，分离技术的稳定性和重复性是获得可靠结果的关键性问题，传统的双向凝胶电泳技术(two-dimensional electrophoresis，2-DE)由于在不同的胶上分离蛋白、过程复杂和人为影响因素太大等原因而使重复性相对较差，不太适应于动态差异蛋白质组学。而其近年来的2-DE的新发展，荧光差异显示双向凝胶电泳(fluorescence 2D differential in-gel electrophoresis，F-2D-DIGE)虽然具有很好组间可比性，但是在同一块胶内最多也只能分离3组样品，同样不能达到本实验比较5组样品的要求。二维高效液相色谱(twodimensional high performance liquid chromatograph，2D-HPLC)是近年发展起来的蛋白分离新技术，其主要的特点是分离过程实现自动化，人为的因素影响较少，分离的效果主要取决于色谱柱的柱效。其中，二维蛋白分离系统(protein fraction2 dimension，PF2D)是一套新开发的2D-HPLC，其第一维色谱为高效色谱聚焦(high performance chromatofocusing，HPCF)，按蛋白质的等电点将样品进行粗分离，第二维色谱为反相高效液相色谱(reverse phase-high performance liquidchromatography，RP-HPLC)，按蛋白质的疏水性对蛋白质进行分离。这套系统的特点是其第一维HPCF色谱柱的柱体较大，上样量高，一次可以上样1～5 mg(2-DE技术最多只能上样1mg)，这对我们找到低丰度(low abundance)蛋白十分有利。另外，在保证柱效的前提下，PF2D可以分离多组样品而保持较好的重复性。可以解决我们对重复性的要求。
然而，目前在国际上，能够利用PF2D进行组织来源的样品分离的报道尚未见到，成功分离的基本上是培养细胞来源的和体液的样品。同时，该系统在亚细胞蛋白质组学领域的成功应用也仅局限于培养细胞膜蛋白的分离。其主要原因在于蛋白提取技术与PF2D分离技术的未能实现良好的兼容。PF2D第一维的分离是弱阴离子交换色谱(weak anion exchange chromatography)技术，利用流动相(liquid phase)形成动态的pH梯度实现不同等电点蛋白质的分离。因此，样品溶液内不能含有任何的盐离子(ion)。在提取培养细胞的全蛋白(tatal protein)或膜蛋白(membrane protein)时，我们可以用不含盐离子的高含量变性剂(denaturant)、去污剂(detergent)的裂解液(lysis buffer)来实现。但是对于提取组织样品和提取亚细胞蛋白是不可行的，往往要介入高含量的盐离子，因此需要进行除盐的处理。然而PF2D的第一维分离对于样品除盐的要求远远超出传统的2-DE技术。一点点的盐离子存在都可能导致蛋白质与色谱柱固相结合能力的下降而无法实现分离。我们在提取肝脏组织细胞核时介入了高浓度的盐离子。用PF2D系统配套的除盐方案进行除盐后，样品分离的效果很差，pH梯度分离范围内几乎检测不到蛋白峰。为此，我们增加了除盐强度，自己研制与公司提供的第一维流动相试剂兼容的超滤缓冲液(ultrafiltration buffer)，利用超滤浓缩除盐的方法，将含盐的核蛋白提取液更换为不含盐的超滤缓冲液，再通过PD-10进一步除盐。通过不断地对超滤缓冲液进行优化，终于使得PF2D分离组织来源蛋白达到了较好的效果。
用这种组织蛋白分离的方法，在确保组问样品定量准确一致的条件下，通过优化PF2D分离工作模式，我们分离了上述几个LPS刺激时间点和正常对照组的核蛋白样品，蛋白分离表现出良好的重现性，蛋白峰基本呈正态分布(normal distribution)，不少峰呈比较满意的尖锐峰(sharp peak)，峰高也比较理想。各组间既有良好的能够重现的峰出现，也有差异峰出现。结果，我们建立了LPS刺激不同时间点和正常的BALB／c小鼠肝脏核蛋白PF2D图谱和组间比较的差异蛋白图谱(dfferential protein profile)。
根据差异图谱，在5个组间我们找到了503个差异蛋白峰。将50个差异蛋白峰所在的二维组分(fraction)取出，进行质谱(mass spectrum)酶解样品制备，使用ABI 4800 MALDI TOF／TOF质谱仪鉴定得到了60个差异蛋白(有的峰鉴定出2～3个差异蛋白)。我们分别观察这60个蛋白所在的差异峰在正常组和LPS刺激不同时间点的丰度，即观察其在核内的量的动态变化，从而获得了这些蛋白的时空变化的信息。
另外，这些差异蛋白的同时出现，必定意味着它们之间有着直接和间接的联系，如何找到它们之间的联系，并且挖掘出这些联系在内毒素休克肝脏中的病理生理学意义，组织出一个框架性的认识，为进一步深入的研究指明道路。这也是目前功能蛋白质组学十分关注和急待解决的问题。而生物信息学(bioinformatics)可以作为解决这个问题的有用工具。我们首先通过亚细胞定位(subcellular localization)的生物信息学方法对60个蛋白质进行了定位的分析，结果完全定位于核内的有6个；既定位于核内，又可以定位于胞浆的有24个；既定位于核内，又可以定位于其它多个亚细胞结构的有23个；在其它亚细胞结构定位，但是没有定位于胞核内的有7个。说明我们的核蛋白提取是有效的。
通过定位分析后，我们分别对蛋白质进行功能分析，利用蛋白质数据库的GO(gene ontology)功能注释、蛋白质结构域(domain)和模体(motif)的分析，粗略地对鉴定的蛋白质进行功能分组，归纳下来有4类：能与核酸结合的、参与氨基酸残基修饰的、参与维持蛋白质或核内理化性质稳定的及参与介导蛋白质-蛋白质相互作用(protein-protein interaction)的。通过这些分析，我们对这群蛋白质的认识有了一定的条理。然而仅仅以此，还不能达到了解蛋白质间相互关系的目的，需要对蛋白质间的相互作用进行预测和分析。我们利用String蛋白相互作用数据库对这60个蛋白进行了一级相互作用(直接作用)的分析，结果发现这些蛋白质中，有3对蛋白质是发生直接的相互作用的，其中缺眼基因同源物1(Eyes absent homolog 1，Eyal)与配位盒蛋白1(pair box protein 1，Pax1)是一对具有转录活性的蛋白质，它们形成的复合物(complex)可能参与细胞凋亡(apoptosis)的负向调节转录(tranpcripton)机制；另外一对蛋白质是G-蛋白调节因子1(G-protein signaling modulator 1，Gpsml)和Gnas，二者形成的复合物可能参与G-蛋白信号通路(G-protein signal pathway)。乙酰辅酶A水合酶／3-羟氨基辅酶A脱氢酶(enoyl-Coenzyme A，hydratase／3-hydroxyacyl Coenzyrne Adehvdrogenase，Ehhadh)和乙酰辅酶A氧化酶2(acyl-Coenzyme A oxidase 2，Acox2)是一对参与乙酰化反应(acetylation)的蛋白质，能对赖氨酸(lysine)进行乙酰基化反应。另外我们还通过Ingenuity信号通路数据库对LPS刺激后丰度增加的蛋白质进行信号通路分析，结果2个通路与这群蛋白质的比较匹配度较高，一个是参与细胞骨架重构(reorganization of cytoskeleton)的，另外一个是参与免疫应激(immunological stress)和细胞功能维持的。上述分析结果提示我们，在内毒素休克进程中，肝脏核内有可能发生核骨架重构、免疫应激；做出这些反应的同时，还加强了核内环境稳定性的维护，防止毒性金属离子和过氧化物损伤，在增加转录调节的同时，加强对DNA的保护和修补，抑制凋亡。
另外，通过观察鉴定的蛋白质在PF2D等电点中的信息，我们发现一类蛋白质的等电点发生了变化，它们分别是睾丸H1组蛋白(Histlhlt)、组蛋白H2B型1-p(Histlh2bp)、组蛋白H4(Histone H4)、组蛋白H2A型3(Hist3h2a)、这类蛋白质是组蛋白家族成员，均为碱性蛋白质，pI在10-12之间，在核内环境里带正电荷。然而，我们分别从pI 4.85-5.16、7.48-7.78、6.59-6.87、6.31-6.61等组分中找到这些蛋白质，同时通过蛋白质功能信息的检索中，还在60个鉴定的蛋白质中找到了一些蛋白质是参与乙酰化、磷酸化(phosphorylation)及甲基化(methylation)反应的。这些提示我们，内毒素休克进程中，可能通过组蛋白密码机制，肝脏细胞的转录加强，对刺激做出适应性的反应。
通过研究，我们得到以下结论：
1．研制了与SB相兼容的超滤缓冲液，使用超滤浓缩除盐与PD-10除盐相结合的方法，加强样品除盐处理；联合使用0.45μm和0.22μm滤器对蛋白样品进行除颗粒处理，使样品制备得以优化。实现蛋白提取技术与离子交换色谱分离技术的兼容，突破了PF2D难以应用于组织样品的技术难关。
2．在PF2D分离操作中，加强蛋白质定量的准确性，强化实验操作的稳定性，优化PF2D分离工作模式，从而成功地实现多组蛋白质样品分离的良好可比性，达到了动态差异蛋白质组学分离技术要求。
3．建立了正常和内毒素休克BALB／c小鼠多时间点(30min、1 h、3 h、6h)肝脏核蛋白PF2D图谱和两两比较的DeltaVue差异图谱及5组比较的线形差异图谱。
4．在正常和内毒素休克BALB／c小鼠肝脏核蛋白差异图谱中选取了503个差异蛋白峰，选取其中的50个进行质谱鉴定，结果鉴定出60个差异蛋白。
5．对60个差异蛋白进行生物信息学分析，预测出3个直接的蛋白质-蛋白质相互作用和绘制了2个细胞信号转导通路图。功能事件分析表明内毒素休克小鼠肝脏细胞核内发生了核骨架重构，加强了核内环境稳定性的维护，在增加转录调节的同时，加强了对DNA的保护和修补，抑制凋亡，参与免疫应激反应。
6．发现内毒素休克BALB／c肝脏细胞核内组蛋白等电点发生了酸化改变，找到了一些具有对蛋白质进行乙酰化、磷酸化、甲基化修饰功能的蛋白质和复合物。提示组蛋白密码可能是内毒素休克小鼠肝脏细胞转录调节机制之一。
通过实验技术方法的探索，我们建立了一套基于二维高效液相色谱分离技术的PF2D-MALDI TOF／TOF质谱-生物信息学的蛋白质组学分析技术。并将这套技术成功地应用于动态差异蛋白质组学分析。为揭示生命过程和疾病机制提供了新方法。
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 response
6. 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.
引文
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8 夏其昌，曾嵘．蛋白质化学与蛋白质组学．第1版．北京：科学出版社，2004．
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2 郭爱华，姜勇．从全身炎症反应综合征到脓毒性休克．中国危重病急救医学，2002，14(8)：500-503．
3 Sat Sharma, FRCPC, FCCP, et al. Septic Shock, Multiple Organ Failure, and Acute Respiratory Distress Syndrome. Curr Opin Pulm Med, 2003, 9(3): 199-209.
4 Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet, 2005, 365 (9453):63-78.
5 赵克森，金丽娟．休克的细胞和分子基础．第1版．北京：科学出版社，2002．
6 Cusick ME, Klitgord N, Vidal M, et al. Interactome: gateway into systems biology. Hum Mol Genet, 2005, 14 (2):171-181.
7 Li Ym, Chen TX, Jiang Y, et al. Two dimensional liquid phase chromatographic fractionation of phosphoproteome of mouse liver. Med J Chin PLA. 2005, 30 (12): 1033-1037.
8 夏其昌，曾嵘．蛋白质化学与蛋白质组学．第1版．北京：科学出版社，2004．
9 Shin YK, Lee H J, Lee JS, et al. Proteomic analysis of mammalian basic proteins by liquid-based two-dimensional column chromatography. Proteomics, 2006, 6 (4): 1143-1150.
10 Levreri I, Musante L, Petretto A, et al. Separation of human serum proteins using the Beckman-Coulter PF2D system: analysis of ion exchange-based first dimension chromatography. Clin Chem Lab Med, 2005, 43(12): 1327-1333.
11 Sheng S, Chen D, Van Eyk JE. Multidimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phase of the human serum proteome: optimization and protein database. Mol Cell Proteomics. 2006, 5(1):26-34.
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