Immobiline-based two-dimensional gel electrophoresis: an optimised protocol for resolution of human colonic mucosal proteins

An optimised protocol for the production of two-dimensional protein patterns of human colonic mucosal cells using immobilised pH gradients in the first dimension is presented. We tested a wide variety of solubilising agents and electrophoretic parameters, separately and in combination. Protein solubilisation was found to be best using a lysis solution containing 9 M urea, 2% Triton X-100, 2% 2-mercaptoethanol, 0.8% Pharmalyte pH 3-10 and 8 mM phenylmethylsulfonyl fluoride (PMSF). Horizontal streaking of basic proteins in the first dimension was virtually eliminated by a combination of washing Immobiline strips in 100 mM asorbic acid, pH 4.5, for 24 h before use and isoelectric focusing samples at 40 degrees C. The presence of 2% glycerol in the first dimension resulted in tighter resolution throughout the entire pH range. The use of these conditions may prove to have broad applicability to the generation of optimally resolved Immobiline-based two-dimensional protein patterns of many other tissues.     

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.     

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.     

Autoimmune pancreatic disease: preparation of pancreatic juice for proteome analysis

The identification of pancreatic proteins is generally hampered by the high content and activity of proteases produced by this organ. The aim of this work was the development of a protocol for the analysis of pancreatic juice by two-dimensional (2-D) gel electrophoresis allowing consistent and reproducible protein analysis encompassed by high-resolution protein 2-D maps and subtle protein spot recognition without substantial losses due to proteases. Immobilized pH gradient (IPG) strips were used for the first dimension, the second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the key step was the sample preparation technique. Improvements were achieved by using several protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanine (TLCK)-HCI, Complete) to prevent degradation of the proteins. The application of different pH-ranges was a valuable step for getting an overview of the expressed protein pattern. These investigations resulted in well-resolved 2-D maps with a high reproducibility.     

Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology. These efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies. Proteomics can no longer be equated with a single two-dimensional electrophoresis gel. Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity. In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow-range immobilised pH gradients) to increase the resolution of proteins contained on two-dimensional gels. We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome. Subproteomics, utilising up to 40 two-dimensional gels per sample will become a powerful tool for near-to-total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research.     

Horizontal two-dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension.

A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of CSF samples. This high-resolution method is a tool worthy of consideration for the research of CSF proteins and disease-specific changes in different neurological disorders.     

Degradation of individual intracellular proteins analyzed by two-dimensional gel electrophoresis and computerized video densitometry

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse-chase-labeling of cells using radioactive amino acids [35S]methionine, two-dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two-dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell-type-specific proteins were significantly enhanced (p less than 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.     

A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes

Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis. This was demonstrated with different samples. In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE. Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma. With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.     

Two-dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients.

Two-dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin-layer gradient horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte-based techniques, immobilised pH gradients prevent the loss of proteins of pI greater than 8; accordingly, basic components, including basic proline-rich proteins, can now be resolved. A two-dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow-range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5-5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI approximately 3.75-4.75 and Mr approximately 32,000; although low levels occurred in some normal individuals, there was less heterogeneity (pI approximately 3.75-4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins.     

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.     

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.     

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.     

Identification of changes in <Emphasis Type="Italic">Triticum durum</Emphasis> L. leaf proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum ‘Ofanto’). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4–7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed.     

A two-dimensional protein map of Caenorhabditis elegans

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.     

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.     

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.     

Computerized, comprehensive databases of cellular and secreted proteins from normal human embryonic lung MRC-5 fibroblasts: identification of transformation and/or proliferation sensitive proteins

Databases of protein information from human embryonal lung fibroblasts (MRC-5) have been established using computer analyzed two-dimensional gel electrophoresis. One thousand four hundred and eighty-two cellular proteins (1060 with isoelectric focusing and 422 with nonequilibrium pH gradient electrophoresis, in the first dimension) ranging in molecular mass between 8 and 234 kDa were separated and numbered. Information entered in the database (in most cases for major proteins) includes: protein name, HeLa protein catalog number, mouse protein catalog number, proteins matched in transformed human epithelial amnion cells (AMA) and peripheral blood mononuclear cells (PBMC), transformation and/or proliferation sensitive proteins, synthesis in quiescent cells, cell cycle regulated proteins, mitochondrial and heat shock proteins, cytoskeletal proteins and proteins whose synthesis is affected by interferons. Additional information entered for a few transformation-sensitive proteins that have been selected for future studies includes levels of synthesis and amounts in fetal human tissues. A total of four hundred and seventy-six [35S]methionine labeled polypeptides (258 isoelectric focusing; 218, nonequilibrium pH gradient electrophoresis) secreted by MRC-5 fibroblasts were separated and recorded (J. E. Celis et al., Leukemia 1987, 1, 707-717). Information entered in this database includes molecular weight and transformation sensitive proteins. These databases, as well as those of epithelial and lymphoid cell proteins (J. E. Celis et al., Leukemia 1988, 9, 561-601), represent the initial stages of a systematic effort to establish comprehensive databases of human protein information. In the long run, these databases are expected to offer a useful framework in which to focus the human genome sequencing effort.     

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.     

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.      

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。