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.     

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.     

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.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

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,     

High resolution two-dimensional mapping of tissue-specific polypeptides in the desert locust, Schistocerca gregaria.

High resolution two-dimensional gel electrophoresis (2-DE) using immobilized pH gradients was used to map the tissue-specific polypeptides of the desert locust, Schistocerca gregaria. Highly specific comprehensive 2-DE reference maps ("master gels") were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4-7 range in the first dimension and within the 14-94 kDa molecular mass range in the second dimension.     

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.     

Dialysis-assisted two-dimensional gel electrophoresis

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.     

Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis

The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

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。     

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).     

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.     

Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3–10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times.      

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.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

pI-based phosphopeptide enrichment combined with nanoESI-MS

IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.