用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.     

Proteomic Analysis of Osteoblasts Exposed to Fluoride In Vitro

Proteomical analysis is defined as the characterization of the entire set of protein encoded a genome. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) are main techniques used in proteomic analysis to achieve information about protein expression profiles. Knowledge about the mechanism of skeletal fluorosis can be gained by recognizing changes in protein expression. To better understand the skeletal fluorosis process, osteoblasts isolated from calvarial of neonatal mouse were cultured and treated with 2 ppm fluoride for 72 h, and proteins of the osteoblast were profiled by 2-DE. With the analysis of Image-Master 2D analysis software, we detected a total number of 493 matching spots on 2-DE images. Among them, 28 protein spots showed twofold significant alteration (P < 0.05) in fluoride-exposed groups. Moreover, 12 proteins were identified by MALDI-TOF MS. These identified proteins in fluoride-exposed group were associated with cell proliferation, metabolism, and oxidative folding. Thus, our study provides useful information on fluoride-related changes of proteome and shows that proteomical analysis is a powerful methodology for the better understanding of skeletal fluorosis.     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Comparison of PDQuest and Progenesis software packages in the analysis of two-dimensional electrophoresis gels

Efficient analysis of protein expression by using two-dimensional electrophoresis (2-DE) data relies on the use of automated image processing techniques. The overall success of this research depends critically on the accuracy and the reliability of the analysis software. In addition, the software has a profound effect on the interpretation of the results obtained, and the amount of user intervention demanded during the analysis. The choice of analysis software that best meets specific needs is therefore of interest to the research laboratory. In this paper we compare two advanced analysis software packages, PDQuest and Progenesis. Their evaluation is based on quantitative tests at three different levels of standard 2-DE analysis: spot detection, gel matching and spot quantitation. As test materials we use three gel sets previously used in a similar comparison of Z3 and Melanie, and three sets of gels from our own research. It was observed that the quality of the test gels critically influences the spot detection and gel matching results. Both packages were sensitive to the parameter or filter settings with respect to the tendency of finding true positive and false positive spots. Quantitation results were very accurate for both analysis software packages.     

Challenges related to analysis of protein spot volumes from two-dimensional gel electrophoresis as revealed by replicate gels

Assumptions that need to be considered prior to statistical analysis of protein spot volumes from two-dimensional gel electrophoresis (2-DE) data are studied using replicate gels of the same sample. The most important observation is that the data tables of protein spot volumes from 2-DE images contain a large number of missing values, which are not consistent with the presence or absence of the proteins. This implies both loss of information and problems for the subsequent statistical analysis. Challenges with 2-DE protein spot volumes are viewed in light of multiple gel comparisons and multivariate data analysis.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

Pixel-based analysis of multiple images for the identification of changes: a novel approach applied to unravel proteome patterns [corrected] of 2-D electrophoresis gel images

A novel approach for revealing patterns of proteome variation among series of 2-DE gel images is presented. The approach utilises image alignment to ensure that each pixel represents the same information across all gels. Gel images are normalised, and background corrected, followed by unfolding of the images to 1-D pixel vectors and analysing pixel vectors by multivariate data modelling. Information resulting from the data analysis is refolded back to the image domain for visualisation and interpretation. The method is rapid and suitable for automatic routines applied after the gel alignment. The approach is compared with spot volume analysis to illustrate how this approach can solve persistent problems like mismatch of protein spots, erroneous missing values and failure to detect variation in overlapping proteins. The method may also detect variation in the border area of saturated proteins. The approach is given the name pixel-based analysis of multiple images for the identification of changes (PMC). The method can be used for multiple images in general. Effects of pretreatment of the images are discussed.     

Development and optimization of two-dimensional-electrophoresis protocol ofLeptospirillum ferriphilum

Leptospirillum ferriphilum is important in bioleaching, in which process it is often under heavy stresses of heavy metal ions and high oxidation reduction potential (ORP). Two-dimensional-electrophoresis (2-DE) and comparative proteomic analysis are useful to investigate the responses ofL. ferriphilum to environmental stresses. But, 2-DE analysis forL. ferriphilum is not successful as the samples ofL. ferriphilum contain low protein concentration, complex composition, high salt concentration, and many other interfering components, which make it difficult for 2-DE analysis. In this research, optimizations on the sample preparation and purification methods, sample volume, sample loading methods for isoelectric focusing (IEF), and gel visualization methods were made. More than 629 Coomassie stained spots in single gel were obtained. The image quality and protein concentration in most of the spots met the requirements for both differential spots analysis and mass-spectrum analysis. The 2-DE protocol forL. ferriphilum was successfully developed for the first time.     

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.     

一个未知的子宫雌激素反应蛋白ULF-250的鉴定研究

摘要 鉴定一个未知的子宫雌激素反应蛋白（ULF-250）。去卵巢大鼠补充给予雌二醇造成子宫产生大量宫腔液（ULF）,受雌激素调节的蛋白分泌其中。收集ULF分别进行SDS-PAGE和双向电泳（2-DE）分离;通过Western 和2D-Western确认抗ULF-250抗体识别的蛋白成分并与凝胶上的条带或斑点相对应;对目标蛋白分别用MALDI-TOF-MS和HPLC-ESI-MS/MS两种方法进行质谱分析并获得肽段序列数据;与蛋白数据库比对及文献检索鉴定未知蛋白。2D-Western显示抗ULF-250抗体特异识别的蛋白成分。从2D胶上切下对应的蛋白斑点进行MALDI-TOF-MS。 结果提示：ULF-250是ebnerin/DMBT1。另外,经SDS-PAGE分离的250 kD蛋白条带进行液-质联用分析。结果同样提示：ULF-250是ebnerin/DMBT1。与文献报道比较,ULF-250与ebnerin/DMBT1在子宫的组织定位和调节是相同的。因此,我们认为ULF-250是子宫表达的ebnerin/DMBT1。通过2-DE结合质谱分析以及查阅文献确认未知的雌激素反应蛋白ULF-250是ebnerin/DMBT1。此外,我们的研究提示,该蛋白不仅在子宫上皮细胞表达而且分泌到子宫腔液中,其功能值得进一步研究。     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

西花蓟马蛋白的提取及双向电泳体系的建立

【目的】蛋白样品的制备是获得良好双向凝胶电泳(2-DE)图谱的前提,建立合理的西花蓟马蛋白的双向电泳体系,获得分辨率较高、重复性较好的图谱,能够为后续的研究提供有力支撑。【方法】实验以西花蓟马成虫为实验材料,对比了饱和酚法、TCA/丙酮法和直接裂解法3种蛋白提取方法,从中选出最适宜双向电泳分析的一种蛋白提取方法。【结果】3种方法蛋白提取率差异显著,直接裂解法蛋白提取率最高,饱和酚法的蛋白提取率最低;3种方法的SDS-PAGE条带数差异不明显;TCA/丙酮法的双向凝胶图谱效果最好,蛋白点最多。【结论】TCA/丙酮法能够有效去除西花蓟马蛋白中的干扰物质,是最适合西花蓟马双向凝胶电泳的蛋白提取方法,为后续西花蓟马在蛋白组学方面的研究奠定了基础。     

A high-efficiency,two-dimensional gel electrophoresis platform for mature leaves of grass pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Grass pea (Lathyrus sativus L.) is the most drought-tolerant legume crop rich in dietary protein. However, little is known about the molecular mechanisms of its drought resistance. Two-dimensional gel electrophoresis (2-DE) is an important experiment technique in proteomics, which has been applied extensively in studies on plant resistance to abiotic stress. To establish an effective 2-DE platform and further study the drought-resistance mechanisms of grass pea using proteomic approaches, three protein extraction methods, different isoelectric focusing (IEF) conditions and various types of gel strips were evaluated using mature leaves. The results showed that the trichloroacetic acid (TCA)/acetone protein extraction method, extending time at low voltage for IEF and using 18 cm gel strip with pH 4.0–7.0 were optimum conditions for 2-DE analysis of grass pea leaves. Applying these optimized 2-DE conditions, 1,481 total protein spots were detected in control leaves and 1,346 spots in polyethylene glycol -treated leaves, of which 67 differentially expressed protein spots were obtained relative to the control. These data suggested that an efficient 2-DE platform with high repeatability and resolution for grass pea mature leaves had been established for the first time here, which could be further used to investigate the drought-resistance molecular mechanisms of grass pea.     

Preliminary proteome analysis of rabbit serum with hepatic failure

The administration of dimethylnitrosamine (DMN) into rabbit induced liver fibrosis/cirrhosis and finally caused a lethal hepatic failure. Blood collected from the rabbit was centrifuged and the supernatant was analyzed by two-dimensional gel electrophoresis (2-DE) for the study of proteome in serum. Compared with 2-DE gel of serum from healthy rabbit, a significant reduction in the number of protein spots having molecular weights (MWs) below 21 kDa was observed in the gels of the serum from the rabbit treated with DMN, while the secretion of albumin was kept at a high level. Separated spots in the two-dimensional gel were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Serum amyloid A-3 protein precursor (SAA3) and other serum amyloid A (SAA) protein precursors were identified by matching the peptide masses with those in database. In the SAA family of acute-phase/inflammatory response proteins, SAA3 is mainly synthesized in the liver. The SAA3 secreted level in the serum decreased with time after DMN administration as the result of hepatic dysfunctions.     

Evaluation of 2-DE protein patterns from pre- and postnatal stages of the mouse brain

Brains of the mouse from three developmental stages, embryo day 16 (Ed16), postnatal stage one week (1W) and eight weeks (8W), were distributed to different laboratories for a collaborative proteome analysis (The Human Brain Proteome Project). As one of the laboratories involved in this project, we separated total protein extracts of the brains by large gel 2-DE. From the 2-DE protein patterns a section was evaluated for each of the three stages according to resolution, reproducibility and quantitative changes using an image analysis software. The evaluated pattern section was selected to allow comparisons of 2-DE patterns between different laboratories on the basis of optimum separation. Changes in protein expression were analysed within two phases of development: Stage Ed16 versus stage 1W and stage 1W versus stage 8W. Out of the 200 protein spots evaluated 5-6% showed quantitative changes in the range of > or = 30% between two stages. The relationship in the frequency of up- and down-regulated protein spots differed between the two investigated phases. Most of the protein spots which showed altered expression between two stages were identified by MS. High quality in protein separation and evaluation is demonstrated.     

Polar electrophoresis: shape of two-dimensional maps is as important as size

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.     

Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.