Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4-8 and 7-10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5-10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4-8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7-10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4-8 or 7-10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.     

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.     

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.

The number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,     

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis.

A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.     

Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (G?rg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.     

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).     

Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

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.     

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Detection of the hypusine-containing protein (HP = eIF-5A) in crude yeast extracts by two-dimensional western blots.

The hypusine-containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF-5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies, the soluble protein fraction of the yeast was resolved on two-dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4-7; second dimension: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

A human myocardial two-dimensional electrophoresis database: protein characterisation by microsequencing and immunoblotting.

This communication briefly describes how a human heart two-dimensional electrophoresis (2-DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2-DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (M(r)), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4-7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2-DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.     

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.     

A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell

We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis. These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions. The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection. The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots. The Protean IEF cell had the next highest number of spots detected per gel. In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel. Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed. A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed.     

Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]     

Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients

In this study, metabolically radiolabeled Escherichia coli cell extracts were used to systematically evaluate protein recoveries at each step of two-dimensional (2-D) electrophoresis and using different sample application methods. Sample application using sample cups resulted in better protein recovery compared with sample loading by rehydration when the Multiphor system was used. At least 50% or more of an E. coli extract was lost when high protein amounts (500 microg) were loaded by rehydration using this system, which employs separate holders for rehydration and isoelectric focusing (IEF). In contrast, when the IPGphor system was used, rehydration sample loading consistently yielded the highest overall protein recoveries. These improved protein recoveries were due to integration of rehydration and electrophoretic separation in a single unit. Even at high protein loads (500 microg), less than 15-20% of the proteins were lost when proteins were loaded by rehydration using sample buffer containing 2% carrier ampholytes in the ceramic immobilized pH gradient (IPG) strip holders used for both rehydration and IEF. Regardless of the loading conditions used, carrier ampholytes in the sample buffer increased protein recoveries. Use of thiourea did not significantly affect protein recoveries but did improve protein resolution in 2-D gels as expected. In summary, these results show the best protein recoveries are obtained for all protein loads when samples are applied to IPG strips during rehydration using a single device for both rehydration and IEF. In contrast, the poorest recoveries are obtained when rehydration and IEF are performed in separate devices, and losses increase dramatically with increasing protein loads using this approach.     

Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12.     

Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.     

Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (相似文献