双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。     

Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS-MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS-MS and 1-DE/MS-MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.     

猪卵母细胞蛋白质组双向电泳体系的建立及初步分析

建立了猪(Sus scrofa)卵母细胞蛋白质双向电泳平台,并对裂解液的组成、样品处理、双向电泳程序等相关技术进行优化,得到清晰的微量卵母细胞蛋白质的电泳图谱.利用上述优化后的体系分别对未成熟和成熟的猪卵母细胞进行双向电泳分析,并用ImageMaser软件对图谱进行比对分析.结果表明,电泳图谱上大约有800个左右的蛋白点,其中差异蛋白35个,包括上调蛋白22个及下调蛋白13个.说明基于双向电泳的蛋白质组学可以用于卵母细胞成熟的蛋白表达差异的研究.     

Protein extraction from mature rice leaves for two-dimensional gel electrophoresis and its application in proteome analysis

Sample preparation is crucial for extraction and higher resolution of proteins by two-dimensional gel electrophoresis (2-DE). In this study, we present an efficient protocol to extract proteins from mature rice leaves by minimizing the presence of nonprotein contaminants and by maximizing contact between the sample and extraction buffer. A combination of chemical and physical processes remarkably improved protein extraction for 2-DE. The efficiency of this protocol was demonstrated by comparison of the rice proteome at two developmental stages.     

Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis

Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.     

红色红曲菌蛋白质组双向凝胶电泳体系的建立

红曲菌是一种具有较高食用和药用价值的丝状真菌,能够产生红曲色素、莫纳可林K等多种生理活性物质。通过分析比较培养基、蛋白质裂解液组成以及水化上样条件对双向电泳结果的影响,建立了红色红曲菌蛋白质组的双向凝胶电泳体系,为从蛋白质水平研究红曲菌及其次级代谢产物的生物合成提供依据。结果表明：用YES培养基培养红色红曲菌6 d,TCA 丙酮法提取菌体总蛋白质,蛋白质裂解液组分为8mol/L尿素,2mol/L硫脲,4 % CHAPS,1 % DTT和2 % Bio-lyte,可获得蛋白质样点数量多,清晰度高的双向电泳图像,为进一步研究红曲菌蛋白质组奠定了基础。     

应用优化的双向电泳技术建立纤维堆囊菌So0157-2蛋白质组数据库

堆囊菌丰富的次级代谢产物是新药的重要来源,而蛋白质组学分析是研究代谢调控的有效方法．然而堆囊菌含有大量的胞外多糖以及黏液,干扰了蛋白质组学分析中蛋白质的溶解度、分辨率及重现性．为了高通量地筛选Sorangium cellulosum So0157-2表达的特异性蛋白,实验优化了S. cellulosum So0157-2双向电泳方法．首先,S. cellulosum So0157-2蛋白在裂解液中有更好的溶解度．pH 3～10非线性胶条和1 mg的蛋白上样量适用于第一向等电聚焦,分别提高了蛋白质点的分辨率和低丰度蛋白质的表达．15% SDS-PAGE 改善了S. cellulosum So0157-2蛋白分离的分辨率和重现性．最终,通过优化的双向电泳方法获得了S. cellulosum So0157-2 在M26培养基中培养3天的全蛋白质表达谱,并检测到552个蛋白质点．进而对表达蛋白通过MALDI-TOF-MS进行质谱鉴定,其中474个蛋白质得到鉴定,鉴定率85.9%．得到鉴定的蛋白质包括细胞结构和功能组分,以及细胞代谢合成酶类,其中8个蛋白质与糖类的转化和代谢相关,这有助于糖基化埃博霉素A的深入研究．该优化方法为进一步建立纤维堆囊菌So0157-2在各种培养条件下的蛋白质组表达数据库打下基础．     

箭毒木种子蛋白质样品制备及双向电泳改良方法

建立箭毒木（Antiaris toxicaria）种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris—HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris—HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris—HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。     

Expressed proteome analysis of human hepatocellular carcinoma in nude mice (LCI-D20) with high metastasis potential

We report for the first time an expressed proteome for human hepatocellular carcinoma (HCC) in nude mice model. Most cases of human liver cancer are HCC with highly metastatic ability. Therefore, the early prediction or diagnosis and effective treatment are the key points of research. We have previously successfully established a human HCC nude mice model (LCI-D20) with high metastasis potential. To understand better the tumor biology of HCC it is worth to explore the relativity of all expressed protein profiles in the LCI-D20 HCC nude mice model. With advanced proteomics technologies, we have carried out a proteomic analysis with following stages: protein sample preparation of cancer tissue, including total cellular extraction and sequential fractionation, 2-DE and 2-D LC separation, ESI/MALDI-MS/MS identification, as well as data-dependent bioinformatics. The identified proteins were classified bioinformatically respective to their function, biological process and intracellular localization. Some important proteins found in HCC, e.g. metabolism enzymes, proteins regulating cell motility, signaling proteins, and heat shock proteins, are discussed in terms of their metastasis.     

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.     

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease

We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.     

结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦,12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描,PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法,用裂解液8mol／L尿素结合2mol／L硫脲,140mmol／LDTT,0．5％biolyte,4％CHAPs,400mg／m1lOG处理,成功提取了蛋白,并通过结核分枝杆菌双向电泳技术体系的优化,建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点,占所检测到的蛋白总数的86％,绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。     

双向凝胶电泳比较三种常用蛋白质提取方法

组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(2-DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF-7蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF-7细胞总蛋白的提取效率.MCF-7细胞经培养后,分别采用M-PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF-7细胞蛋白质提取的相对最佳方法.结果显示,M-PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15~70kD,pH4.7~6.3的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M-PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF-7细胞的蛋白质,并与双向凝胶电泳条件更兼容.     

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.