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1.
《Electrophoresis》2017,38(3-4):408-416
Serum levels of Prostate‐Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pI s and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.  相似文献   

2.
Image analysis of two‐dimensional gels is a crucial step in a proteomic workflow and has a direct impact on obtained qualitative and quantitative data. Since the analysis is a complex process and creates large data amounts, the use of a respective software is inevitable. There are only a few papers published addressing the issue of analysis‐based variance; therefore, our aim was to highlight the discrepancy of received results when different commercially available image‐tools are used for gel analysis especially in terms of comparability of the obtained outcome when the same digital image set is used. A set of six gels (three replicates per group) of real‐life samples was created and examined with two different versions of PD‐Quest (Bio‐Rad) (version 6.1 and its update version 8.0) and with an external image‐tool Delta 2D (Decodon) (version 3.6). Replicate groups were analyzed and compared with each other with regard to volume ratios of a group of significantly changed spots. The study points out significant variations among results depending on the software package used, underlining the importance of a careful investigation of post‐experimental processes to receive comparable and reliable results.  相似文献   

3.
《Electrophoresis》2018,39(12):1488-1496
Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin‐binding proteins. The method used a fluorescent carboxyfluorescein‐diacetate‐labeled cisplatin analogue (CFDA‐cisplatin), two‐dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA‐cisplatin‐containing protein adducts in the acidic pH range (pH 4–7). Based on this analytical method we extended the identification of intracellular cisplatin‐protein adducts to the alkaline pH range (pH 6–10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA‐cisplatin‐binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D‐3‐phosphoglycerate 1‐dehydrogenase.  相似文献   

4.
Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics.  相似文献   

5.
Maltol, ethyl maltol, vanillin, and ethyl vanillin are important food additives as flavor enhancers. To quantify the four additives in milk powder, a novel 2D liquid chromatographic (2DLC) method was developed in this article. In such a 2DLC system, the target fractions eluted from the first dimensional column (C4) are stored onto the trapping column (C8) for subsequent analysis; after that, they were switched into the second dimensional column (C18) by a two‐position six‐port switching valve. A one‐step sample preparation method was used prior to 2DLC chromatographic analysis, which was easy and convenient. After optimization of all experimental parameters, the new method was validated in terms of linearity, LODs, and LOQs, intra‐ and interday precision, and accuracy. A conventional single‐dimensional liquid chromatographic method was also proposed in this work for comparison. In order to evaluate the applicability of the new 2DLC method, five brands of commercial milk powder samples (n = 8) were analyzed. Vanillin and ethyl vanillin were detected in two samples, respectively. It is showed that the 2DLC method is effective in quality control programs of milk powder products.  相似文献   

6.
Claeys D  Geering K  Meyer BJ 《Electrophoresis》2005,26(6):1189-1199
Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.  相似文献   

7.
Kim J  Kim SH  Lee SU  Ha GH  Kang DG  Ha NY  Ahn JS  Cho HY  Kang SJ  Lee YJ  Hong SC  Ha WS  Bae JM  Lee CW  Kim JW 《Electrophoresis》2002,23(24):4142-4156
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.  相似文献   

8.
Purification of high‐purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two‐dimensional preparation method was successfully developed for the preparation of high‐purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first‐dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high‐purity compounds in the second‐dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high‐purity compounds from complex samples.  相似文献   

9.
Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.  相似文献   

10.
In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large‐scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide‐level fractionation, the first dimension of separation was performed with high‐pH reversed‐phase chromatography (pH‐RP) and strong cation exchange chromatography (SCX). Regarding protein‐level fractionation, C8 protein reversed‐phase (C8‐RP Prot) and high‐recovery protein reversed‐phase (hr‐RP Prot) were used instead. The second dimension consisted of a reversed‐phase nano‐HPLC on‐Chip coupled to an electrospray ionization quadrupole time‐of‐flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide‐level pH‐RP and the hr‐RP Prot protein‐level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide‐level SCX, with 509 proteins identified, was the worst method. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

11.
KN Valente  LH Choe  AM Lenhoff  KH Lee 《Electrophoresis》2012,33(13):1947-1957
Optimized 2DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2DE, which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility-enhancing agents and the relatively low-throughput nature of 2DE limit the systematic study of sample preparation. In this work, design of experiment (DOE) approaches are used to optimize protein recovery by altering the levels of four solubility-enhancing agents (urea, DTT, CHAPS, and SDS) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS-PAGE and 2DE recovery. DOE methodologies are presented as relatively high-throughput procedures for optimizing 2DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT), Chinese hamster ovary (CHO) cell lysate (8 M urea, ≥2% CHAPS, ≥32.5 mM DTT), and Escherichia coli cell lysate (8 M urea, 4% CHAPS, 65 mM DTT), with optimized conditions increasing 2DE protein recovery at least 50% compared to suboptimal conditions.  相似文献   

12.
As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5 mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80 ± 8% extraction yield obtained without SDS to 48 ± 2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4 ± 0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 μg mL−1 depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kg protein mass)−1 resulted for spiking concentrations of 10 mg As L−1. The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.  相似文献   

13.
We present the study on the sample transfer characteristics of two different microfluidic interfaces for 2D‐CE . These interfaces were manufactured using two different microfabrication technologies: one was obtained via the classical photolithography—wet etching—anodic‐bonding process; and the other was obtained via the selective laser‐induced etching process. The comparison of the two interfaces, and an intact capillary as a reference, was made via the CE separation of amino acids (arginine and lysine) under different bulk flow conditions, with and without applying bias potential to the secondary channels. The influence on peak shapes, migration times, and repeatabiliy were evaluated.  相似文献   

14.
Li ZB  Flint PW  Boluyt MO 《Electrophoresis》2005,26(18):3572-3585
Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.  相似文献   

15.
Reed PW  Densmore A  Bloch RJ 《Electrophoresis》2012,33(8):1263-1270
We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.  相似文献   

16.
Hu Y  Wang G  Chen GY  Fu X  Yao SQ 《Electrophoresis》2003,24(9):1458-1470
The defense mechanism by which cells combat metal stress remains poorly understood. By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level. By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals. This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis. From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes. According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups. By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively. One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details. Significant changes in SOD1 expression were observed throughout all 15 DIGE gels.  相似文献   

17.
The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps.  相似文献   

18.
Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High‐quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE‐42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.  相似文献   

19.
Researchers frequently use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D‐PAGE method, which ‘inverts’ the dimension of protein separation of the conventional 2D‐PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first‐dimension separation was conducted on a gel‐free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second‐dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D‐PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D‐PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D‐PAGE. Overall, the i2D‐PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.  相似文献   

20.
A competent extraction method of plant proteins for 2-D gel electrophoresis   总被引:1,自引:0,他引:1  
The efficient extraction of high‐quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea‐/thiourea‐ and NP‐40‐containing solution. Protein profiles were examined on pH 3–11 non‐linear IEF strips and SDS‐PAGE and compared with extracts using the established method of acetone‐10% TCA/0.07% 2‐mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1–8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2‐mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250–150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25–15 kDa. The MALDI‐TOF‐MS of differential spots from acetone containing 10% TCA/0.07% 2‐mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.  相似文献   

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