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.     

Geometric algorithms for the analysis of 2D-electrophoresis gels.

In proteomics, two-dimensional gel electrophoresis (2-DE) is a separation technique for proteins. The resulting protein spots can be identified either by using picking robots and subsequent mass spectrometry or by visual cross inspection of a new gel image with an already analyzed master gel. Difficulties especially arise from inherent noise and irregular geometric distortions in 2-DE images. Aiming at the automated analysis of large series of 2-DE images, or at the even more difficult interlaboratory gel comparisons, the bottleneck is to solve the two most basic algorithmic problems with high quality: Identifying protein spots and computing a matching between two images. For the development of the analysis software CAROl at Freie Universit?t Berlin, we have reconsidered these two problems and obtained new solutions which rely on methods from computational geometry. Their novelties are: 1. Spot detection is also possible for complex regions formed by several "merged" (usually saturated) spots; 2. User-defined landmarks are not necessary for the matching. Furthermore, images for comparison are allowed to represent different parts of the entire protein pattern, which only partially "overlap." The implementation is done in a client server architecture to allow queries via the internet. We also discuss and point at related theoretical questions in computational geometry.     

Age-related proteome analysis of the mouse brain: a 2-DE study

2-DE remains the most popular and versatile protein separation method among a rapidly growing array of various proteomics technologies. However, variability in sample processing, experimental design and data analyses results in a limited cross-validation between studies performed in different laboratories. One of the goals of the Human Proteome Organization (HUPO) is to establish standards and guidelines for proteomics studies. We contributed to the HUPO Brain Proteome Project by analyzing brains from neonatal and adult mice using 2-DE. Here we propose a standard workflow to analyze 2-DE images and extract statistically significant differences. After differential analysis and identification by MALDI-TOF/TOF, dihydropyrimidinase-related proteins, brain FABP, stathmin, isocitrate dehydrogenase, gamma enolase, annexin V, glutamine synthetase, creatine kinase B chain, triosephosphate dehydrogenase, and malate dehydrogenase were found differentially expressed between the two groups. The functions and potential mechanisms underlying the variation observed for these proteins are discussed.     

Proteomic analysis of high-density lipoprotein

Plasma lipoproteins, such as high-density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL-associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1-DE and 2-DE MALDI-TOF, isotope-coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL-associated proteins during isolation, a one-step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high-performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL-associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone-associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.     

Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells

Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty.     

Proteomic analysis of Rac1 transgenic mice displaying dilated cardiomyopathy reveals an increase in creatine kinase M-chain protein abundance

Here, we demonstrate the application of the proteomic approach to the study of a transgenic mouse model of heart failure and provide an example of a disease-associated protein alteration that can be observed using this approach. Specifically, we applied the proteomic approach to the analysis of a mouse model of dilated cardiomyopathy in which the small GTPase, Rac1, was constitutively expressed specifically in the myocardium. We utilized the methods of two-dimensional gel electrophoresis (2-DE) for protein separation, silver-staining for protein visualization and mass spectrometry (MALDI-TOF and MS/MS) for protein spot identification. Computer-generated composite images were created which represent a normalized average of four 2-DE gel images derived from analysis of either Rac1 transgenic (n = 4) or non-transgenic (n = 4) mice. Analysis of composite images derived from NTG and Rac1 experimental groups revealed numerous statistically significant differences in mean protein spot intensities. Here, we report a statistically significant increase, of approximately 1.6-fold, in the mean protein spot intensity for creatine kinase M-chain in the composite image of Rac1 transgenic mice compared to control. This protein alteration may be consistent with an end-stage heart failure phenotype in which maximal myocardial reserve is employed to sustain survival.     

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。     

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.     

Protein spot detection and quantification in 2-DE gel images using machine-learning methods

Two-dimensional gel electrophoresis (2-DE) is the most established protein separation method used in expression proteomics. Despite the existence of sophisticated software tools, 2-DE gel image analysis still remains a serious bottleneck. The low accuracies of commercial software packages and the extensive manual calibration that they often require for acceptable results show that we are far from achieving the goal of a fully automated and reliable, high-throughput gel processing system. We present a novel spot detection and quantification methodology which draws heavily from unsupervised machine-learning methods. Using the proposed hierarchical machine learning-based segmentation methodology reduces both the number of faint spots missed (improves sensitivity) and the number of extraneous spots introduced (improves precision). The detection and quantification performance has been thoroughly evaluated and is shown to compare favorably (higher F-measure) to a commercially available software package (PDQuest). The whole image analysis pipeline that we have developed is fully automated and can be used for high-throughput proteomics analysis since it does not require any manual intervention for recalibration every time a new 2-DE gel image is to be analyzed. Furthermore, it can be easily parallelized for high performance and also applied without any modification to prealigned group average gels.     

Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry are being used as proteomic tools in an integrated functional genomics program focused on the model legume Medicago truncatula. Due to the perceived high levels of indeterminate error associated with 2-DE we deemed it necessary to quantify the coefficient of variance (or relative standard deviation) for both analytical and biological sources associated with 2-DE of Medicago truncatula leaf protein extracts. Leaf protein extracts were chosen because of their biological significance and due to the more challenging nature of green tissues. Analytical variance was calculated for fifty proteins from ten replicate 2-DE gels of the same protein extract. Biological variance was calculated for the same fifty proteins from ten independent 2-DE gel analyses of ten independent but similar plants grown under identical conditions. Average analytical and biological variances were calculated for both data sets and represent the average variance of approximately 500 independent measurements of protein concentration. Analytical variance was determined to be 16.2% and biological variance was determined to be 24.2%. These average variances provide a quantified and statistical basis for evaluation of protein expression changes in future comparative proteomic investigations. It is proposed that 2-DE measured protein expression levels should differ by a minimum of 3.92sigma (i.e. /+/-2sigma/ and sigma = standard deviation), or 94.7% based on our measured variances, for the difference to be significant at the 95% confidence level.     

肺鳞癌患者与健康人血清的差异蛋白质组学研究

为筛选肺鳞癌的血清标志物,采用二维凝胶电泳(2-DE)技术分离I期肺鳞癌患者和健康人的血清蛋白质,PDquest图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白,然后应用蛋白质印迹和免疫组化方法分别检测差异蛋白——结合珠蛋白-2(haptoglobin-2,HP-2)在肺鳞癌患者血清和健康人血清以及肺鳞癌组织和癌旁正常支气管上皮组织中的表达.建立了肺鳞癌患者和健康人血清的2-DE图谱,图像分析软件识别了1O个差异蛋白质点,质谱鉴定了4种差异蛋白;蛋白质印迹分析显示,HP-2在肺鳞癌血清中的表达水平显著高于健康人(P<0.05),但其表达水平与肺鳞癌的临床分期无明显相关性;免疫组化结果显示,HP-2在肺鳞癌组织中的表达水平高于癌旁正常支气管上皮组织(P<0.05).研究结果提示:HP-2是候选的肺鳞癌血清分子标志物,血清中HP-2水平对肺鳞癌诊断可能具有一定的参考价值;肺鳞癌组织中HP-2表达上调可能是患者血清中HP-2表达升高的原因之一.     

An integrated proteome database for two-dimensional electrophoresis data analysis and laboratory information management system

We describe an integrated proteome database, termed Yonsei Proteome Research Center Proteome Database (YPRC-PDB) which can store, retrieve and analyze various information including two-dimensional electrophoresis (2-DE) images and associated spot information that were obtained during studies of hepatocellular carcinoma (HCC). YPRC-PDB is also designed to perform as a laboratory information management system that manages sample information, clinical background, conditions of both sample preparation and 2-DE, and entire sets of experimental results. It also features query system and data-mining applications, which are amenable to automatically analyze expression level changes of a specific protein and directly link to clinical information. The user interface is web-based, so that the results from other laboratories can be shared effectively. In particular, the master gel image query is equipped with a graphic tool that can easily identify the relationship between the specific pathological stage of HCC and expression levels of a potential marker protein on the master gel image. Thus, YPRC-PDB is a versatile integrated database suitable for subsequent analyses. The information in YPRC-PDB is updated easily and it is available to authorized users on the World Wide Web (http://yprcpdb.proteomix.org/ approximately damduck/).     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

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.     

Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry

The large individual variation in meat quality seen both within and between animals is not fully understood. Consequently, our long-term goal is to identify reliable proteins which control or determine bovine meat quality. Using a proteomic approach, bovine skeletal muscle samples were analyzed by two-dimensional gel electrophoresis (2-DE) using an immobilized pH 4-7 gradient in the first dimension and mass spectrometry. We first tested the reproducibility of the method. These experiments showed slightly greater intersample than intrasample variability. In order to evaluate the type of visualized proteins in 2-DE, we initiated the construction of a protein reference map of bovine Semitendinosus muscle. In total, 129 protein spots corresponding to 75 different gene products were identified. Of these proteins, the largest portion is involved in metabolism (25.5%), cell structure (17%), cell defense (16%) and contractile apparatus (14.5%). One quarter of the identified proteins are represented by two or several protein spots and multiple isoforms of troponin T are present. Peptide mass fingerprint results indicate that these isoforms are partly generated by alternative splicing. The data presented here are an important step for further proteome analyses on bovine muscle. This may lead to progress in understanding the mechanisms controlling postmortem muscle metabolism and meat quality.     

Resolution of Escherichia coli O157:H7 That Contaminated Radish Sprouts in Two Outbreaks by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) was performed to examine exoproteins and periplasmic proteins of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated from cases associated with radish sprouts in two outbreaks. We found that STEC O157:H7 released a large number of proteins into the medium during the stationary phase of growth, as observed with 2-DE. Although pulsed-field gel electrophoresis (PFGE) patterns of STECs NGY9 (RIMD0509894), a Sakai isolate; NGY33, a Gamagoori isolate; and NGY120, a Kanagawa isolate, were all the same, comparison of 2-DE patterns of exoproteins and periplasmic proteins clarified that NGY9 was distinct from NGY33, whereas NGY33 and NGY120 were of close lineage. We therefore suggest that 2-DE analysis of exoproteins and periplasmic proteins is a powerful epidemiological method with high resolution. Received: 21 August 2000 / Accepted: 9 March 2001     

Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl beta-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.