Two-dimensional Gel Electrophoresis. Rabab M. Aly Department of Clinical Pathology Mansoura University, Egypt

Transcription

1 Two-dimensional Gel Electrophoresis Rabab M. Aly Department of Clinical Pathology Mansoura University, Egypt

2 1 Introduction Proteomics, or the high-throughput identification and analysis of proteins, is an emerging technology facilitated by advancements in mass spectrometry, genome sequencing/annotation and computer-based peptide search algorithms. Protein separation for proteome investigations relies upon time-honored techniques such as liquid chromatography (FPLC, HPLC and caplc) and two-dimensional (2-D) gel electrophoresis (GE). Improvements in 2-D electrophoresis methodology over the last ten years have improved gel to gel reproducibility and ease-of use. Classical 2-DE has also direct applications, such as phenotyping of genetic variants and post-translational modification (PTM) characterization, in particular phosphorylations, glycosylations, deamidations and much more. Two-dimensional electrophoresis is one of the most commonly used techniques in proteomics. In 2-D PAGE, proteins are separated according to isoelectric point (pi) by isoelectric focusing (IEF) in the first dimension and according to size by sodiumdodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (Klose, 1975; O Farrell, 1975; Gorg et al., 2000). It has a sole capacity for the resolution of large number of proteins in a or 106 dynamic ranges in cells, permitting the consecutive analysis of many gene products (Inagaki and Katsuta, 2004). Electrophoresis can be one dimensional (one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. 2 One-dimensional Gel Electrophoresis 1D-GE, is an analytical separation technique that is used for all protein chemistry, is beneficial for proteomic analysis. ID-GE provides information about the molecular size, amount, and purity of a protein sample. ID-GE is the standard first step in immunoblotting and immunodetection. In 1D-GE, proteins are separated according to their molecular weights. First, the protein is solubilized with a buffer system containing a thiol reductant and SDS. Then the gel is subjected to an electric field and protein-sds complexes migrate through polyacrylamide gel at different rates. Smaller proteins migrate further through the gel than larger proteins so that the mixtures of proteins are resolved into bands in order of molecular weight. 3 Two-dimensional Gel Electrophoresis In 2D GE the first separation includes IEF, allowing separation of intact proteins. Today this is performed on an immobilized ph gradient (IPG) strip containing ampholytes to create ph gradients of wide or narrow ranges. After separation into zones by IEF, a treatment with a thiol reductant and SDS, the second dimension separates by mass using ordinary SDS-PAGE. 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research, where resolution of large number of proteins on a single gel is sometimes necessary. The strip is joined with the SDS slab gel. Separating protein mixtures into reproducible fractions is needed for providing the highest resolution of proteins and it is the key of success of 2-DE procedure. Sample preparation methods include extraction with simple solubilization solutions, complex mixtures of chaotropic agents, detergents or reducing agents. Elec-

3 trophoresis is then performed on polyacrylamide gel, separating the protein- SDS complexes according to size only. The initial separation for 2-D PAGE by IEF was originally done in capillary gels with ph gradients generated by carrier ampholytes (Hoving et al., 2000). The daunting task of learning to handle flimsy capillary gels plus the poor reproducibility of carrier ampholyte IEF led to the wide acceptance of IPGs for IEF. The ph gradients of IPGs are generated by means of buffering compounds that are covalently bound into porous, polyacrylamide gels (Wildgruber et al., 2000). The ph gradients, fixed as they are in IPG gels, remain stable over extended run times at very high voltages, a requisite for highresolution separations. IPGs are cast on plastic backing sheets and are cut into mechanically stable strips that are easily manipulated. Whereas the classical 2-DE has definite limitations with respect to resolution, reproducibility and protein-loading capacity, 2DE using IPGs has proved to be amazingly flexible in terms of the requirements of proteome analysis. With a few notable exceptions (Ünlü et al., 1997; Righetti et al., 2004), 2-D PAGE is now done almost exclusively with IPGs as the IEF media. IEF SDS-PAGE First dimension, tube gel or strip gel Second dimension, slab gel Figure 1: Overview of 2D gel electrophoresis. In the first dimension (left), one or more samples are resolved by isoelectric focusing (IEF) in separate tube or strip gels. IEF is usually performed using precast immobilized ph-gradient (IPG) strips on a specialized horizontal electrophoresis platform. For the second dimension (right), a gel containing the pi-resolved sample is laid across to top of a slab gel so that the sample can then be further resolved by SDS-PAGE (source: Hames, B.D. and Rickwood, D. Eds. (1990) Gel Electrophoresis of Proteins: a Practical Approach, 2nd ed. Oxford University Press, New York). 2-DE has been a core technology of proteomics that can separate complex protein mixtures in cells and tissues prior to mass spectrometry (MS) analysis (Klose, 1975, Dunn & Görg, 2004). 2-DE-based proteomics not only enables identification of proteins expressed but also provides quantitative data of protein expression and post translational modification (Rabilloud, 2002), thereby affording a global analysis of protein behavior and a survey of novel diagnostic markers and drug targets of various diseases. Detection of low abundant, hydrophobic, high molecular weight, and basic proteins has been a challenging issue for increasing the recovery of proteome by 2-DE. However, recent progress in 2-DE technologies is gradually overcoming at least part of these problems. Improvements in the detection of hydrophobic and basic proteins are reported elsewhere (Görg, et al., 1997; Rabilloud, 1998; Tastet et al., 2000; Hoving, 2002; Dunn and Görg, 2004).

4 4 Detecting Low-Abundance Proteins Because the cellular expression levels of many important proteins, such as, signaling molecules and regulatory proteins are low, detection of low abundant proteins is of particular importance. A simple way for the detection of these proteins would be to load more protein samples to 2- DE gels or to increase the sensitivity of protein detection. Broad dynamic ranges of protein expression in cells are challenges to detect low abundant proteins by proteomics with 2-DE (Corthals et al., 2000) and without 2-DE. The dynamic range of proteins expressed a cell is estimated to be or 106, while that of standard 2-DE gels for protein detection is less than (Rabilloud, 2002). Low-abundance proteins are of great interest in proteomic research and can be studied with 2-D PAGE. However, almost by definition, the concentrations of low-abundance proteins in a sample are near or below the lower detection limits of 2-D PAGE and its associated protein stains. It is necessary to increase the relative amounts of low-abundance proteins in the sample in order to be able to visualize them in the gels. Merely increasing the protein load to the 2-D gel is often insufficient, because highabundance proteins will dominate the gels and can hide low-abundance proteins. Moreover, at high protein loads, resolution is lost and, therefore, so is the ability to distinguish closely spaced protein spots. Considerable effort is being devoted to the development of pre-fractionation methods as a means for enriching the content of low-abundance proteins in samples for 2-D PAGE. The basic idea behind prefractionation is to segregate sample proteins into distinguishable fractions containing limited numbers of proteins. Sequential extraction, described above, is an example of one type of approach to prefractionation. Chromatographic methods have also been exploited. Ion exchange (Butt et al., 2001) and hydroxyapatite (Fountoulakis et al., 1999) matrices have been used to fractionate complex protein patterns prior to 2-D PAGE.The objective of separating proteins using 2D-PAGE is twofold: (a) identifying new proteins and (b) measuring their relative abundance between comparative samples. Beside that 2D-PAGE resolves large numbers of proteins, the Visualization of these proteins is accomplished with staining techniques which enables the relative abundances of the proteins to be quantified. After staining, the protein spots are aligned and scanned with different categories of imageacquisition devices that are used with 2-D PAGE to measure their individual intensities. It has been challenging to ensure direct spot-to-spot comparison between two separate gels. This was accomplished by the development of 2-D differential in-gel electrophoresis (DIGE) which overcame this limitation by separating many distinct protein mixtures in a single 2D-PAGE gel (Ünlü et al., 1997). In a typical 2DDIGE, up to three different protein samples can be labeled with fluorescent dyes prior to twodimensional electrophoresis. Then, the three samples are mixed and put in the same gel. After the gel electrophoresis, the gel is scanned with the excitation wavelength of each dye. So that each sample can be seen separately. The actual running of 2-D PAGE is rather straightforward and easily learned (Fountoulakis et al., 1999) However, sample preparation for 2-D PAGE is another matter. For the most part, successful 2-D PAGE depends on efficient extraction and solubilization of proteins. Unlike the situation for DNA, there is no universal sample-preparation method suitable for all proteins. Each source of protein presents its own sample preparation challenges. Proper sample treatment should begin from the moment the material is collected. Care must be exercised to prevent proteolysis following cellular death (Inagaki et al., 2004). Once proteins have been extracted from the source material, they must be prepared for 2-D PAGE. The major goal of sample preparation is to solubilize as many proteins as possible and to maintain their solu-

5 bility throughout the 2-D PAGE process. Firstly, proteins are denatured to their constituent polypeptide chains so that polypeptide sequences can be matched to their corresponding gene sequences. Secondary sample-preparation concerns are the removal of non-proteinaceous material that may interfere with 2-D PAGE and the prevention of artifactual modifications of polypeptides. Proteins are extracted from source material by well established cell-disruption methods then solubilized and denatured by means of chaotropes, detergents, and reducing agents. Protein extraction can be done directly into a solubilization solution or extracted proteins can be diluted into a solubilization solution. Solubilization solution is also incorporated into the matrix of the IPG strip to maintain protein solubility during IEF. 5 Visualization of proteins Proteins separated by gel electrophoresis can be visualized by a number of methods using different types of stains. Various stains interact differently with the proteins and some of the stains used are not even specific for proteins. The degree of sensitivity is also different. Visualization of proteins in gels is accomplished with staining techniques (Rabilloud, 1999; Rabilloud, 2000; Pennington, 2001).Despite the availability of a wide variety of specific stains, the majority of 2-D PAGE gels are stained with Coomassie Brilliant Blue (CBB), some type of silver stain and some molecular Probes. For proteomics work, protein stains must be compatible with MS and that has limited the choice of silver stain to those that do not include gluteraldehyde treatment or oxidation steps (Yan et al., 2000). Coomassie Blue and molecular probe stains are both compatible with MS. When formulated as a colloidal sol (Neuhoff et al., 1988), colloidal CBB is very easy to use and can be made environmentally benign (Nivinskas et al., 1996). Colloidal CBB is essentially an endpoint stain, meaning that gels can be left in it overnight for convenience. A post-stain water wash is important with colloidal CBB. The wash removes excess colloidal dye particles from the gel surface and also drives the dye molecule into the proteins in the gel. Thus, the wash increases the signal-to-noise ratio of the stained gel. Colloidal CBB is the least sensitive of stains. Its detection limit is about 10 ng of protein per spot. Colloidal CBB stains a wide range of proteins and can respond linearly over two orders of magnitude in protein amount (depending on the proteins). Imageacquisition instruments range from simple cameras and light boxes to sophisticated laser-based fluorescence detectors (Patton, 1995; Miller et al., 2001). For subsequent digital image analysis, gel images must be captured electronically. The three categories of image-acquisition devices used with 2-D PAGE are document scanners, charge coupled- device (CCD) cameras, and laser-based detectors. 6 Advantages of 2D-PAGE Resolution, detection, quantitation, and reproducibility of proteins by electrophoresis have been enhanced through several advances. The 2-D SDS-PAGE and 2DDIG have high resolving power and enable the detection of large number of proteins on a single gel plate. By using 2D-PAGE, reproducibility has been a big concern when profiling protein mixtures but this can be resolved by the use of 2D-DIGE. The introduction of IPGs has enhanced the resolution by enabling the analyst to tailor the ph gradient for maximum resolution using ultrazoom gels with a narrow ph gradient range. 2-DE applies important advantage in a modern proteomics setup is that it offers a very flexible and valuable platform to initially

6 screen the proteins of interest, because 2D gels combine high resolution, low cost and image display of the results. In 2D-DIGE, the protein samples are labeled with fluorescent dyes and then separated by 2D- PAGE. Different protein samples are labeled with different fluorescent dyes, mixed together and separated by the identical gels. The gels are scanned by laser scanners and the image of 2D-PAGE for multiple samples is obtained from single gels and so it can enhances reproducibility and quantitation. The use of computers allows acquisition, data analysis, spot detection, normalization, reporting and exporting of data. 2D-DIGE is an important tool, especially for clinical laboratories where 2D-PAGE can detect the disease aberrations. It can be applied to the studies on lung cancer and pancreatic cancer. The size of protein spots changes in parallel with expression level of proteins, so that 2D-PAGE can achieve quantitative comparison between multiple samples and allows rapid identification of protein changes between two samples on the same gel plate without influences of gel-to-gel variations. 7 Sample Preparation 7.1 From Cells or Tissues The sample treatment is the key to obtain reasonable results. The protein composition of the cell lysate must be reflected in the pattern of the 2-D gel without any losses or modifications. Too much salt, like washing cells with PBS, and amphoteric buffers in cell cultures, like HEPES, have to be avoided. The chemicals used have to be of the highest purity. A typical denaturing buffer (lysis buffer) is 9 M urea, or 7 M urea plus 2 M thiourea, 2-4% non-ionic. or zwitterinonic detergent, 1% dithiothreitol (DTT) and 0.5% carrier ampholytes. The high urea/thiourea concentration is needed to get proteins into a single conformation by cancelling the secondary and tertiary structures, to get hydrophobic proteins into solution, and to avoid protein-protein interactions. Thiourea improves the solubility of membrane proteins. To establish the ph gradient, IPGs, such as with Immobilines, the Bromophenol is very useful as a control dye. Nucleic acids, lipids, and salts must be removed; for example, salts can be removed by dialysis or precipitation, lipids with an excess of detergent (> 2%), and nucleic acids by sonication, or specific extraction. PMSF (phenylmethylsulphonyl-fluoride) is frequently used as an inhibitor of proteolysis; it must be added to the sample prior to the reducing agent. Anti-protease cocktails containing other protease inhibitors are less toxic and more effective, but some of these inhibitors might lead to charge modifications of some proteins. Protein precipitation can be very effective for diluted samples or plant samples: the content of the cell lysate is precipitated with 10% TCA (trichloroacetic acid) in acetone; the pellet is washed with acetone, dried under vacuum, and resuspended with lysis buffer. Moreover, the proteases are inhibited. Exceptionally, the tissue is boiled for 5 minutes in 1-2% SDS before they are diluted with lysis buffer, for example for plants or organisms with tough cells. Optimized procedures for different sample types do exist; however a general procedure is not available (Baudin & Bruneel, 2004; Bruneel et al., 2005; Gorg et al., 2009). 7.2 From Biological Fluids Both blood plasma and serum can be used; the choice of the anticoagulant to obtain plasma samples is not yet standardized, but EDTA is often preferred because it does not interfere with IEF and acts as metalloprotease inhibitor as well. Then the plasma or the serum isolated after centrifugation is stored at -

7 80 C when possible. The urines must be sampled from either a specimen or the urines of 24 hours, centrifuged to eliminate the mineral and organic pellet, and stored at -80 C. The cerebrospinal fluid is better analyzed without storage; alternatively, it can be centrifuged and stored at -80 C. For other biological fluids, not any protocol is yet standardized (Lahm& Langen, 2000). 8 First-dimension Isoelectric Focusing (IEF) It includes different methods including horizontal polyacrlamide system. 8.1 Using Dry Strips IEF is performed in 0.5 mm-thin IPG-gel-strips cast on plastic backing. The film-supported gels are easy to handle; IPGs are very reproducible, in particular because the fixed gradients are not modified by the sample composition; moreover, detergents and reducing agents can be added without ph gradient disturbing. Several strips can run in parallel, up to twelve using actual materials. 8.2 Using Tubes In the original method for high resolution 2-D electrophoresis, the IEF step was carried out with carrier ampholytes generating ph gradients in gel rods, as called tube gels. Electric field is applied first to establish the ph gradient; then, the sample is loaded onto the acidic end of the gradient, the electric field is applied again to separate the proteins during the gradient drift to the cathode, and the run is stopped after a defined time period. 8.3 Using Horizontal System Horizontal polyacrylamide (PA) systems have a number of advantages over the vertical ones when ultrathin gels polymerized on support films are used, and particularly for IEF. Handling is simple, in particular for staining, washing, dehydration of the gel; ready-made gels are available with or without IPG; buffer strips are used instead of large buffer volumes; cooling is easy to perform efficiently. The same denaturing buffer as above can be used for sample loading, usually by a strip rehydrated with the sample then laid down on the acidic part of the gel. Agarose gels for IEF are now available because the agaropectin residues have been removed; however, the electro-endosmosis flow is not completely eliminated. Separations in agarose gels, usually containing % agarose, are more rapid than PA gels. Macromolecules larger than 500 kda can be separated since agarose pores are larger than those of PA gels. Moreover, its components are not toxic and cannot interfere with the separation. Nevertheless, it is difficult to prepare stable agarose gels with high urea concentrations because urea disrupts the configuration of the helicoidal structure of the polyoside chains. As well as with PA and agarose gels, IEF must be carried out at a constant temperature, usually 10 C. Exceptionally, the temperature can be stated at 37 C, for example for the study of cryoprotein, such as IgM, increasing their solubility, or at below 0 C for the analysis of ligand bindings or enzyme-substrate complexes. It is recommended to use marker proteins of known ph for controlling the ph gradient. The isoelectric point of the proteins in sample can be measured on the ph calibration curve. Flatbed systems can use both concepts for establishing ph gradients, i.e. carrier ampholytes and (IPGs).

8 When an electric field is applied, the negatively charged carrier ampholytes migrate towards the anode, the positively charged ones to the cathode. They align themselves in between according to their pi and will determine the ph of their environment. To maintain a gradient as stable as possible, strips of filter paper soaked in the electrode solution are applied between the gel and the electrodes (an acidic solution at the anode, and a basic solution at the cathode); nevertheless, these electrode solutions are not necessary for short gels. Carrier ampholytes are also very useful for preparative separations and titration curve analysis. Problems with carrier ampholytes can arise when long focusing times are necessary. First, as with tubes the cathodal drift can take away part of the proteins out of the gel. Moreover, a gel can burn through at the conductivity gaps, in particular created when highly viscous additives are used. Because of some limitations of the carrier ampholytes system, an alternative method was developed: IPGs. They are built with acrylamide derivatives with buffering groups, as called Immobilines, by copolymerization of the acrylamide monomers in a PA gel. To be able to buffer at a precise ph value, at least two Immobilines are necessary, an acid and a base. A ph gradient is obtained by the continuous change in the ratio of Immobilines. In practice, IPGs are prepared by linear mixing of two different polymerization solutions with a gradient maker, as for pore gradients. Since the gradient is fixed in the gel, it stays unchanged all along the separation time even with viscous additives such as urea and non-ionic detergents. IPG can be exactly calculated in advance and adapted to the separation problem reaching very high resolution with up to 0.01 ph units per cm. The gradient is not influenced by proteins and salts in the solution. The use of IPGs is restricted to PA gels only. New IEF systems try to expand the ph range in both directions by using very acidic or basic narrow ph gradients; they are based on additional types of Immobilines. IPGs can also be used with a perpendicular urea gradient to detect various mutations in proteins, and for preparative separations. But, flatbed IEF, even being the best classical IEF system, is really not well adapted for fist dimension of 2-DE; dry strips are most often preferred (Westermeier, 2001; Baudin, 2010). 9 Second-Dimension SDS-PAGE Before the second dimension, the IPG strips are equilibrated in the specific buffer adapted to SDS-PAGE separation, in particular with reducing agent such as DTT or DTE, and by iodoacetamide to block the thiol groups by alkylation; finally, the strip is equilibrated in Tris buffer, plus urea, glycerol, and SDS. SDS-PAGE is then performed on either horizontal or vertical systems. 9.1 Using Flatbed System Horizontal flatbed systems can be used with similar results. The temperature has to be regulated, for example with a cooling plate. Film-supported SDS PA gels for the second dimension are much easier to use. Another advantage of flatbed systems is the facilitated image analysis. Nevertheless, the steps in silver staining require more time, and the film can show fluorescent background at certain wavelengths. 9.2 High-Resolution 2-DE For the complex protein mixtures, high-resolution and high purity of spots can solely be achieved by adequate special resolution using large gel sizes (up to 1 m). Less complex protein mixtures are usually studied in medium sizes to miniformate gels. The latter are useful for optimization of sample preparation. There are ready-made gels available for large and small formats (Weiss & Gorg, 2009).

9 10 2-DE procedure Briefly, the proteins is resuspended in 150 µl of a 2-DE sample buffer containing 9 M urea, 65 mm dithiothreitol (DTT), 2% Pharmalyte, and 1% bromophenol blue and then centrifuged at C 4 and 23000g for 30 min to remove debris. The supernatant is then mixed with a rehydration buffer consisting of 8 M urea, 4% CHAPS, 0.5% IPG buffer 3-10 NL, 19 mm DTT, and 5.5 mm Orange G to a final volume of 350 µl. The first dimension is performed by in-gel rehydration for 12 h in 30 V on 18 cm ph 4-7 linear or ph 3-10 nonlinear IPG strips. The proteins are then focused at Vh at a maximum voltage of 8000 V (3). The second dimension (SDS-PAGE) is performed by transferring the pi focused proteins (IPG strips) to homogeneous or gradient home-cast gels on gel bonds. The electrophoresis is performed at V, 10 C, and ma, overnight. Molecular probe staining is done according to manufacturer s instructions. Briefly, gels to be stained with Sypro Ruby is directly placed in a fixing solution containing 10% methanol and 7% acetic acid for at least 20 minutes after 2-DE. Gels are then washed 3x10 minutes under agitation with Milli-Q water before approximately 400 ml of probe stain are added and incubated in room temperature over night. Silver staining of gels are done according to Shevchenko, with some few modifications (Shevchenko et al., 1996). Proteins are fixed by incubating the gel in 50% methanol and 5% acetic acid for at least 20 minutes directly after 2-DE and then incubated with 50 % Methanol for 5 minutes, followed by Milli-Q water for 10 minutes. In the sensitizing step the gel is incubated with 0.02 % sodium thiosulphate for 1 minute, followed by 2x1 minutes washing with Milli-Q water. The gel is then immersed in 0.1 % silver nitrate solution for 20 minutes before excess of silver is washed away by 2x1 minute in Milli-Q water. Next, the gel is developed in 0.04 % formaldehyde in 2 % sodium bicarbonate solution for 2x1 minute. The exact developing time is optimized depending of the protein amount in the gel. Finally, the reaction is stopped by incubation 1x5 min in 0.5 % glycine and the gel is washed with Milli-Q water for 2x20 min. The images of the protein patterns are analyzed by a CCD (Charge-Coupled Device) camera digitizing at 1340*1040 pixel resolution in a UV scanning illumination mode for Sypro Ruby stained gels or at 1024*1024 pixel resolution in white light mode for silver stained gels using a Flour-S-Multi Imager in combination with a computerized imaging 12- bit system. The unit of the UV light source is expressed in counts while the unit of the white light source is expressed as optical density (OD). Gel images are evaluated by spot detection, spot intensities and geometric properties. 11 Applications of 2-DE Proteomics, which is the study of the entire protein complement expressed by a genome in a cell or a tissue, holds a key position in the new biology. It emerged from the long work on comprehensive protein visualization on 2-D gels, in particular using mass spectrometry (MS) and revitalized by the development of peptide sequence databases. These tools allow differential expression studies with many applications as well as in fundamental biology as in medicine and pharmacology Fundamental Biology Many examples could be given showing the enormous amount of work which has been realized in cell biology. The complete proteome of Escherichia coli is now available; those of Helicobacter pylori, Sal-

10 monella sp., Bacillus subtilis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Haemophilus influenzae,, and of many more other bacteria are in progress. In the biology of plants, Arabidopsis thaliana (a model for plant physiology study) proteome will be soon complete, as well as that of chloroplasts. In animal biology, main models are now more or less sequenced at the genomic level, and more or less completed at the proteome level, for example for Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens sapiens (Celis et al., 1998). HUPO (Human Proteome Organization) has the ambition to determine the entire proteome of all the human tissues, such as blood plasma or serum, and cell lines (Jung et al., 2000; Bruneel et al., 2003; Pernet et al., 2006) DE and Drug Discovery Proteomics represents a powerful approach of providing valuable information on target drug design, creating a new paradigm that will accelerate downstream drug development. As one of the important developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease. A number of biotechnologies, including proteomics and a number of cellular methodologies, have been developed. Proteomics development faces different challenges, including both the traditional biology and the emerging bioinformatics. New technologies include twodimensional gel electrophoresis, and activity-based assay are increasing the utility of proteomics in the drug-discovery process. Screened proteins as tumor markers for many cancer as in bladder, gastric breast, lung and pancreatic cancer, can be done by proteomic analysis (2-DE) of cancerous tissue. Particularly in cancer, it is useful to distinguish between diagnostic, prognostic, and predictive markers (Srinivas et al., 2001; Lehmann et al., 2007). Moreover, diagnostic markers are used to aid histopathological classification that is often a key for choosing between therapy modalities, including surgery, chemotherapy, radiotherapy and their combinations. Unfortunately, there are only a few markers which can predict treatment outcome. At least two approaches are available for cancer proteomics, one is the search of plasma markers; another is the examination of the tumor, for example using laser capture micro-dissection. Various protocols for solubilisation have been applied with or without enzymatic digestion, in particular with needle aspiration, surface scrapping or mincing of tumor tissue in buffer. Many examples could be given on the 2-DE analysis of tumors: in colorectal carcinomas, lung cancer, ovarian cancer, prostate cancer and leukemia (Banks et al., 1999). 12 Conclusion 2-DE can separate thousands of proteins with important features: it has extremely high resolving power, it can tolerate crude protein mixtures, and with relatively high sample loads; moreover, proteins separated in 2-D gels can be further analyzed. Most often, first dimension is IEF in an immobilized ph gradient, and second dimension is SDS-PAGE. Confirmation of protein identity can be performed by 2-D gel. The technique of 2-DE has been considerably improved during the past decades and new improvements regularly emerge from both industrial manufacturers and academic laboratories.

11 References Banks, R.E., Dunn, M.J., Forbes, M. A., et al. (1999). The potential use of laser captures microdissection to selectively obtain distinct populations of cells for proteomic analysis. Electrophoresis, 20, Baudin, B. (2010). Protéomique, spectrométrie de masse et analyses multiples, 46, Cahier de formation Bioforma Ed, Paris. Baudin, B. & Bruneel, A. (2004). Introduction to proteomics: Goals, technical aspects and applications to fundamental biology, drug discovery and clinical chemistry. Recent Research and Development in Biophysics and Biochemistry, 3, Bruneel A, Labas V, Mailloux A. et al. (2005). Proteomics of human umbilical vein endothelial cells applied to etoposideinduced apoptosis. Proteomics, 2005, 5: Bruneel, A., Labas, V., Mailloux, A., et al. (2003). Proteomic study of human umbilical vein endothelial cells in culture. Proteomics, 3, Butt, A., Davison, M.D, Smith, G.J, Young, J.A, Gaskell, S.G, Oliver, S.G and Beynon, R.J. (2001). Chromatographic separation as a prelude two-dimensional electrophoresis in proteomics analysis. Proteomics, 1, Celis, J.E., Ostergaard, M., Jensen, N. et al. (1998). Human and Mouse databases: novel resources in the protein universe. FEBS Letters, 430: Corthals, G.L, Wasinger, V.C, Hochstrasser, D.F and Sanchez, J. C. (2000). The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis, 21, Dunn, M.J & Görg, A. (2004). In Proteomics: from Protein Sequence to Function (eds. S.R. Pennington and M.J. Dunn), BIOS Scientific Publishers, Oxford, pp Fountoulakis, M., Taka.cs M.F., Berndt, P., Langen, H., Taka, B. (1999). Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis, 20, Görg, A., Drews, O., Lück, C. et al. (2009). 2-DE with IPGs. Electrophoresis, 30: Görg, A., Obermaier, C., Boguth, G., Csordas, A., Diaz, J.J and Madjar, J.J. (1997). Very alkaline immobilized ph gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins, Electrophoresis 18, Görg, A, Obermaier, C., Boguth, G., et al. (2000). The current state of two-dimensional electrophoresis with immobilized ph gradients. Electrophoresis, 21, Hoving, S., Gerrits, B., Voshol, H., Muller, D., Roberts, R.C and van Oostrum J. (2002). Preparative two-dimensional gel electrophoresis at alkaline ph using narrow range immobilized ph gradients. Proteomics, 2, Hoving, S., Voshol, H., and Ostrum, J. (2000). Towards high performance two-dimensional gel electrophoresis using ultrazoom gels. Electrophoresis, 21, Inagaki, N., Katsuta, K. (2004). Large Gel Two-Dimensional Electrophoresis: Improving Recovery of Cellular Proteome., 1, Jung, E., Hoogland, C., Chiappe, D., et al. (2000). The establishment of a human liver nuclei two-dimensional electrophoresis reference map. Electrophoresis, 21, Klose, J. (1975). Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik, 26, Lahm, H.W. & Langen, H. (2000). Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis, 21:

12 Lehmann, S., Dupuy, A., Peoc h, K., et al. (2007). Présent et futur de la protéomique clinique. Annales de Biologie Clinique, 65, Miller, M.D, Acey, R.A, Lee, L.Y, Edwards, A.J. (2001). Digital imaging considerations for gel electrophoresis analysis systems. Electrophoresis, 22: Neuhoff, V., Arnold, N., Taube, D., Ehrhard,t W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R Electrophoresis, 9, Nivinskas, H., Cole, K.D. (1996). Environmentally benign staining procedure for electrophoresis gels using Coomassie Brilliant Blue. Bio-Technique, 20, O Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Che, 250, Patton, W.F. (1995). Biologist's perspective on analytical imaging systems as applied to protein gel electrophoresis. J Chromatogr A., 698, Pennington, S.R. Dunn, M.J (Eds.), Proteomics: From Protein Sequence to Function, BIOS, Oxford, UK, 2001, Pernet, P., Bruneel, A., Baudin, B., et al. (2006). A module for two-dimensional gel electrophoresis database creation on personal Web sites. Proteomics and Bioinformatics, 4, Rabilloud, T. (1998). Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis, 19, Rabilloud, T. (1999). Proteome research: two-dimensional gel electrophoresis and identification methods. Principles and practice, Springer, Berlin, Germany. Rabilloud, T. (2000). Detecting proteins separated by 2-D gel electrophoresis Anal. Chem, 72, Rabilloud, T. (2002). Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics, 2, Righetti, P.G, Castagna, A., Antonucci, F., Piubelli, C., Cecconi, D., Antonioli, P., Astner, H., et al. (2004). Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches. J. Chromatogr. A., 1051, Shevchenko, A., Wilm, M., Vorm, O., Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem., 1, Srinivas, P.R, Srivastava, S., Hanash, S. et al. (2001). Proteomics in early detection of cancer. Clinical Chemistry, 47, Tastet, C., Charmont, S., Chevallet, M., Luche, S., and Rabilloud, T. (2000). Structure-efficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis. Proteomics, 3, Ünlü, M., Morgan, M.E, and Minden, J.S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis, 18, Weiss, W. & Görg, A. (2009). High-resolution two-dimensional electrophoresis. Methods in Molecular Biology, 564, Westermeier, R. (2001). Electrophoresis in practice (third edition), Wiley-VCH, ISBN, , Germany. Wildgruber, R., Harder, A., Obermaier, C., Boguth, G., Weiss, W., Fey, S.J., Larsen, P.M., and Gorg, A. (2000). Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized ph gradients. Electrophoresis, 21, Yan JX, Wait R, Berkelman, T., Harry, R.A. (2000). Westbrook JA, Wheeler CH, Dunn MJ. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis, 21,