Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

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.     

小鼠黑质双向凝胶电泳技术的优化

为了优化小鼠黑质双向凝胶电泳(2-DE)技术,比较了不同样品预处理方法、不同胶条和不同上样量对凝胶电泳结果的影响。研究发现,两种样品预处理方法均可顺利升至最高等电聚焦(IEF)电压(10000V)。与Clean-upkit法相比,TAC/丙酮沉淀法预处理后蛋白得率较低,同时丢失了样品中的部分中、小分子蛋白质。pH3-10L胶条中蛋白点主要分布在中间区域;pH3-10NL胶条对中间区域的蛋白点的分离有所改善;pH4-7胶条中蛋白点分布均匀,横条纹较少。80μg上样量的图谱背景干净,但点数最少(411);180μg上样量的图谱蛋白点较多(883),且圆润、清晰,背景干净,分辨率高;300μg上样量的图谱蛋白点虽然最多(904),但背景较深,部分蛋白点融合,横条纹也较多。研究表明,对于小鼠黑质蛋白质,使用Clean-upkit法对样品进行预处理,选取pH4-7的胶条,采用180μg的上样量能获得背景干净、分辨率高的双向电泳图谱,为帕金森病的生物标志物和药物靶点的研究打下了基础。     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

DMSO, an Organic Cleanup Solvent for TCA/Acetone-Precipitated Proteins, Improves 2-DE Protein Analysis of Rice Roots

Protein extraction is a critical step in a two-dimensional electrophoresis (2-DE)-based proteomic analysis. Dimethyl sulfoxide (DMSO), a small organic molecule, is widely used as a solvent in biological sciences. In this study, we modified the cleanup step of the commonly used trichloroacetic acid (TCA)/acetone method of protein extraction by using 20?% DMSO/acetone as a solvent to wash TCA?Cacetone-precipitated pellets. The improved protocol (TCA/acetone?CDMSO) was compared with the TCA/acetone and phenol extraction method based on the protein yield, the number of spots, and resolution on 2-DE maps. TCA/acetone?CDMSO washing increased protein yield, and improved the resolution in 2-DE with less background, streaking, and smearing. This method also produced the highest number of protein spots. To our knowledge, the present study is the first to use DMSO as a cleanup solvent for TCA/acetone-precipitated proteins to enhance their quality prior to 2-DE.     

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.     

Proteome profile of human urine with two-dimensional liquid phase fractionation

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.     

Solubilization,two-dimensional separation and detection of the cardiac myofilament protein troponin T

Proteomics strongly relies on the separation of complex protein mixtures, with two-dimensional electrophoresis (2-DE) being a commonly used technique. However efficient separation requires adequate solubilization of the original sample which will determine whether all proteins are accurately represented. Cardiac muscle has presented a particular challenge to solubilization. Here we have optimized the solubilization, separation and detection of the myofilament protein troponin T (TnT). Human left ventricular tissue from a rejected donor transplant heart was homogenized under 19 different conditions and subjected to 2-DE and Western blot analysis for TnT. The optimal conditions for isoelectric focusing of intact TnT requires homogenization in 6 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, with the addition of NaCl (2.5 M final concentration). TnT degradation products present in this severely damaged heart however, were differentially extracted from both each other and the intact molecule under the various conditions used. Despite adequate focusing of TnT it was found that a nonglutaraldehyde silver staining protocol, that is compatible with subsequent mass spectrometry, has greatly reduced sensitivity for TnT compared to Coomassie blue. Thus, care is required to avoid misrepresentation of troponin T in proteomic analysis in cardiac tissue.     

柑橘叶片和果皮总蛋白质提取及双向电泳体系的建立

以春甜橘(Citrus reticulata Blanco‘Chuntianju’)果皮和叶片为材料,分别用Tris-HCl、尿素/硫脲(Thi/Urea)、三氯乙酸/丙酮(TCA)和酚(Phe)等4种方法提取柑橘总蛋白质,从蛋白质产量、单向SDS-PAGE和双向电泳等方面进行比较。结果表明,4种方法的分离效果存在较大差异,不论是以柑橘叶片还是果皮为材料,均以TCA法最好,且双向电泳图谱分辨率较好,蛋白点清晰、均匀、基本没有条纹,且蛋白点多。这说明TCA法不仅能很好地去除柑橘果皮、叶片中存在的大量干扰物质,而且还能得到稳定的蛋白点。     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis

We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.     

Quantitative protein profiling using two-dimensional gel electrophoresis,isotope-coded affinity tag labeling,and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.     

Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis

Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.     

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.     

3种不同油桐种仁蛋白质提取方法比较研究

一种有效的蛋白质提取方法是蛋白质双向电泳成功分离的关键。油桐种仁可以用来制取工业用桐油,但其中富含大量的能干扰蛋白质提取的物质,并能影响双向电泳图谱的分辨率。本文中对油桐种仁蛋白质采用丙酮提取（方法A）、苯酚抽提（方法B）以及丙酮—苯酚结合提取（方法C）,并通过蛋白质双向电泳技术对这3种方法的提取效果进行比较研究。结果显示：采用方法C所提取的蛋白质样品,其蛋白浓度高达到8.1 μg·μL^-1;并且该方法对油桐种仁中的高分子量、低分子量蛋白均有较强的提取能力;此外,其蛋白样品经过双向电泳所得到的蛋白质点和图谱分辨率也较其余两种方法好。     

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.     

An improved sample preparation method for analyzing mycobacterial proteins in two-dimensional gels

Two-dimensional gel electrophoresis (2-DE) is currently a widely used analytical method for resolving complex mixtures of proteins. Sample preparation has a marked influence on 2-DE pattern. To reduce impurities and to increase the low-abundance proteins, protein precipitation is often used for the preparation of samples before 2-DE. In this study, we revealed that addition of SDS prior to TCA precipitation of mycobacterial cell extract proteins increases the resolution of the 2-D gel pattern.     

Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics

Comparing different proteomes by classical two-dimensional electrophoresis is challenging and often complicated by substantial gel-to-gel variation. Separating two or more protein samples labelled with different fluorescent dyes in one single gel, as in two-dimensional difference gel electrophoresis, reduces this variability considerably. Recent technological innovations, specifically the introduction of a pooled internal standard, even further improve the quantification accuracy and statistical confidence of this method. In addition, decreasing the sample complexity by one of several protein or organelle fractionation procedures increases the number of spots investigated by this protein differential display methodology.     

A method for the two-dimensional electrophoresis of leaf proteins

Proteins extracted from green leaves of tobacco were subjected to analysis by two-dimensional electrophoresis. It was found that electrophoretic separations were unsatisfactory when leaf extracts were analyzed directly without prior extraction of pigments, phenols, and lipids by acetone treatment. The plant pigments and several phenolic compounds present in leaves presumably interacted with the proteins and created charge heterogeneity, streaking, and other artifacts. It was found that these problems could be overcome by treatment of leaf extracts with acetone followed by solubilization and electrophoresis of the acetone-treated proteins. Leaf extracts were prepared by grinding deribbed leaf disks in a buffer containing 5 mm potassium carbonate, 9.5 m urea, 0.5 dithiothreitol, 2% Nonidet P-40 detergent, 500 μg/ml l-lysine, and 2% Ampholines. The extracts, after centrifugation to remove cell debris and insoluble material, were treated several times with ice-cold acetone. The acetone-precipitated proteins were treated with nucleases, reprecipitated with cold acetone, and then resuspended in the above grinding buffer. The presence of l-lysine and Ampholines were required for good electrophoretic separations. The resuspended proteins were subjected to two-dimensional electrophoresis and proteins detected by staining and or fluorography. At least 300 distinct proteins could be recognized in radioactive samples. The method gives reproducible patterns even after repeated freezing and thawing of the samples.