A mouse serum two-dimensional gel map: application to profiling burn injury and infection

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.     

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in‐gel electrophoresis

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor‐specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non‐cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in‐gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine‐rich α2 glycoprotein‐1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.     

Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatment with our spot detection program. The resulting two-dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/ humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two-dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post-translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two-dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (greater than 30%) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (less than 20%) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1% vs. 0.2%; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3%) than in brains (0.0%), and higher in solubilized nuclear proteins (5.3%) than in structure-bound nuclear proteins (2.1%).     

Proteome analysis of UV-B-induced anti-apoptotic regulatory factors

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low-dose UV-B irradiation (approximately 0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low-dose UV-B-irradiated NIH3T3 cells, using two-dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI-TOF/TOF-MS, some as structural proteins. Although typical apoptosis-related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

大鼠骨髓间充质干细胞分化过程的比较蛋白质组学研究

从蛋白质组学角度分析大鼠骨髓间充质干细胞(MSCs)体外定向分化为心肌细胞过程中蛋白表达情况, 采用二维电泳分离蛋白, 用PDQuest软件分析蛋白表达差异, 并采用质谱(MALDI-TOF-MS)进行鉴定, 得到了54个蛋白点, 对蛋白的生物功能分析表明, 部分蛋白通过不同的信号途径参与了MSCs的分化过程.     

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.     

Change in serum proteome during allogeneic hematopoietic stem cell transplantation and clinical significance of serum C-reactive protein and haptoglobin

Successful hematopoietic stem cell transplantation (HSCT) involves the restoration of hematopoietic function after engraftment, arising from the differentiation and proliferation of hematopoietic stem cells. Several factors could influence the course of allogeneic-HSCT (allo-HSCT). Therefore, knowledge of serum proteome changes during the allo-HSCT period might increase the efficacy of diagnosis and disease prevention efforts. This study conducted proteomic analyses to find proteins that were significantly altered in response to allo-HSCT. Sera from five representative patients who underwent allo-HSCT were analyzed by 2-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, and were measured on a weekly basis before and after allo-HSCT in additional 78 patients. Fourteen protein spots showing changes in expression were further examined, and most proteins were identified as acute phase proteins (APPs). Studies of 78 additional patients confirmed that C-reactive protein (CRP) and haptoglobin undergo expression changes during allo-HSCT and thus may have the potential to serve as representative markers of clinical events after allo-HSCT. Maximal CRP level affected the development of major transplant-related complications (MTCs) and other problems such as fever of unknown origin. Particularly, an increase in CRP level 21 days after allo-HSCT was found to be an independent risk factor for MTC. Maximal haptoglobin and haptoglobin level 14 days after allo-HSCT were predictive of relapses in underlying hematologic disease. Our results indicated that CRP and haptoglobin were significantly expressed during allo-HSCT, and suggest that their level can be monitored after allo-HSCT to assess the risks of early transplant-related complications and relapse.     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

4-(2-苄氧基乙氧基羰基)氧杂环丁-2-酮的合成及表征

通过16例人肺鳞癌和小细胞肺癌组织中表达蛋白的二维电泳分离和质谱分析,经数据库检索鉴定了53个蛋白,其中24个蛋白与肺癌发病机制相关,4个蛋白在其它癌症中有报道,表达呈现差异的蛋白点有44个,其中34个在表达量上有差异,lO个蛋白在鳞癌和小细胞癌间表现为有和无的关系,蛋白功能分析提示人肺鳞癌与小细胞癌的蛋白质组表达存在差异,分析这些差异蛋白有利于肺癌分型及其生物标志物研究。     

Proteomic analysis of the small intestine and colon epithelia of adenomatous polyposis coli gene-mutant mice by two-dimensional gel electrophoresis

Mutations of the adenomatous polyposis coli gene (APC) have been implicated in the occurrence of sporadic colon cancer. Various APC mutant strains of mice have been created to better understand the function of this gene. Previously, we had mice express a mutant form of mRNA of the APC protein that encoded 474 amino acids instead of the 2845 amino acids due to exon duplication. These APC mutant mice (APC delta 474) developed intestinal and mammary tumors, as have other APC mutant mice previously reported (Sasai, H., et al. Carcinogenesis, in press). To elucidate the mechanism of the tumor development, we prepared protein samples from both normal and tumor tissues from APC delta 474 mutant mice, as well as tissues from normal mice, and used them for proteomic analysis. After two-dimensional electrophoresis, the gels were silver stained and the protein spots were analyzed. We analyzed about 1000 protein spots per sample and found several protein spots that are specific for normal or tumor samples from APC delta 474 mutant mice, as well as proteins with altered expression levels. Among the identified protein spots, truncated beta-tubulins were specific to APC delta 474 mutant mice polyp samples. The apparent molecular mass of these proteins suggested that these beta-tubulins may be truncated very close to the binding site of the anti-tumor drug taxol.     

肺鳞癌和小细胞肺癌组织比较蛋白质组学研究

通过16例人肺鳞癌和小细胞肺癌组织中表达蛋白的二维电泳分离和质谱分析,经数据库检索鉴定了53个蛋白,其中24个蛋白与肺癌发病机制相关,4个蛋白在其它癌症中有报道.表达呈现差异的蛋白点有44个,其中34个在表达量上有差异,10个蛋白在鳞癌和小细胞癌间表现为有和无的关系.蛋白功能分析提示人肺鳞癌与小细胞癌的蛋白质组表达存在差异,分析这些差异蛋白有利于肺癌分型及其生物标志物研究.     

用于识别不同细胞蛋白质组的噬菌体抗体芯片

将4个鼠源噬菌体抗体克隆和1个人源噬菌体抗体克隆偶联到羧基终止的硅片表面,制成分析型模型芯片.挑选健康人体淋巴细胞为正常细胞的代表, HeLa细胞为肿瘤细胞的代表,提取细胞的全部蛋白质并用荧光染料Cy3标记,与制成的分析芯片反应,得到了不同的结合图谱.实验结果表明,以噬菌体抗体为分子感受器的分析芯片可用于识别不同细胞的蛋白质组.     

Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growth

The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth.     

嗅觉记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了C57BL/6J小鼠在嗅觉训练和记忆测试后嗅球蛋白表达的差异, 探讨了与嗅觉记忆相关的蛋白质. C57BL/6J小鼠经嗅觉训练后, 可对相应的气味保持记忆能力, 其嗅球蛋白表达存在明显差异, 5种蛋白与嗅觉记忆形成显著相关. 上述蛋白功能涉及神经发育, 信号转导及细胞骨架和核苷酸代谢, 其中神经发育和信号转导相关蛋白表达上调, 而细胞骨架和核苷酸代谢相关蛋白表达水平降低. 这些与嗅觉记忆形成相关的蛋白深化了对嗅觉记忆机制的认识, 为研究和治疗认知相关疾病提供了新靶标.