Karen De Jongh
Cell Therapeutics, Inc.
Two-dimensional gel electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. The first dimension, isoelectric focusing, employs either gels containing carrier ampholytes or immobilized pH gradients; the second dimension is typically SDS gel electrophoresis. The utility of this technique lies in its enormous resolving power; of the 5,000-10,000 proteins present in a cell, approximately 2,000 can be resolved in a single electrophoretic run. Not only does this provide a method for detecting small changes in the levels or properties of proteins in response to changes in cellular conditions, but it also allows the isolation of proteins in quantities sufficient for structural analysis by a number of techniques including amino acid analysis, amino-terminal sequencing, peptide mass fingerprinting, and tandem mass spectrometry. A critical point in this overall process is optimization of electrophoresis, as well as the subsequent analytical procedures. This workshop on two-dimensional gel electrophoresis was organized to address some of the issues involved in successful application of this technique. The four presentations delivered during this workshop are summarized below.
Andrew J. Link, University of Washington, "Using Two-Dimensional Gel Electrophoresis to Display and Characterize Prokaryotic Proteins".
The use of two-dimensional gels for purification and characterization of proteins from E. coli and for identifying deviations between observed protein properties and predictions from the genome was described. Based on the open reading frames that are present in databases, the theoretical appearance of a two-dimensional gel can be constructed. Molecular weight, isoelectric point, and abundance of mature gene products can be precisely measured experimentally using two-dimensional gels. The mature amino-termini can be determined by sequencing, and information on a protein's subcellular location can be obtained by cell fractionation before electrophoresis. Differences between the predicted and observed two-dimensional gel can indicate errors in DNA sequencing, post-translational modifications, processing such as signal peptide removal, or proteolysis within cells or during sample preparation.
Two methods were described for preparing total cell extracts. The first involves heating for 4 minutes to 95[[ring]]C in 2% SDS, and the second uses sonication with a micro-tip. Both methods require DNase and RNase treatment to reduce viscosity. As a benchmark for calculating protein concentration when making total cell extracts, E. coli grown to stationary phase in rich media with a cell density of 3 x 109 cells/ml will yield approximately 800 ug protein from 1 ml of culture. Samples should be loaded onto the first dimension gel in 9 M urea, 4% NP-40, 2% ampholyte, and 1% DTT, with the final concentration of SDS less than 0.3%. Osmotic shock has also been used to enrich for proteins from the periplasmic space 30-fold. Sequencing the amino-termini of these periplasmic proteins after two-dimensional gel electrophoresis revealed that 85% were novel compared to proteins sequenced from a total cell extract; upstream fractionation can allow identification of otherwise undetected proteins, in addition to providing information about subcellular location.
The first dimension isoelectric focusing gels were prepared with various ampholyte blends. For the first dimension, 10 to 250 ug of protein are loaded onto a 1.2 x 150 mm isoelectric focusing gel and focused for 14 hours at 800 V. The second dimension gels were 1.5 x 160 mm standard SDS-PAGE separating gels without stacking gels. After electrophoresis, proteins were electroblotted to PVDF and detected by staining with colloidal gold for analytical gels (10-40 ug protein loaded) or by Coomassie blue for preparative gels (200-250 ug protein loaded). All E. coli proteins transferred at least partially, but electrotransfer of high molecular weight proteins was incomplete.
Protein spots from preparative gels can be subjected to amino-terminal sequencing or in situ digestion. Less than 10% of the spots analyzed by sequencing contained multiple proteins. Most (95%) E. coli proteins were not blocked: 388 proteins from a single two-dimensional gel were identified by amino-terminal sequencing, and 299 contained unique amino-termini. Of these, there were matches to 166 open reading frames in the databases, and 77 sequences represented novel proteins. Tandem mass spectrometry after in situ digestion of spots from two-dimensional gels was also used for protein identification by correlating observed peptide masses and masses predicted from databases using a program called SEQUEST. Samples stored on PVDF membranes for up to five years at room temperature can be analyzed in this manner. This is the preferred analytical method for blocked proteins and a method with the potential to identify modified amino acids and protein mixtures.
Scott D. Patterson, Amgen, Inc., "From Analytical 2-D Gels to Identification of Spots from Preparative 2-D Gels".
Practical aspects of sample preparation and enrichment, parameters that are important for running reproducible gels, and identification of peptides by immunoblotting, sequencing, limited 1-D mapping, and peptide-mass mapping were addressed with an emphasis on analysis of eukaryotic proteins.
Several tips for improving the reproducibility of two-dimensional gels were presented. The importance of using high quality reagents and preparing large volumes of solutions and buffers from the same batches of reagents was stressed. Because artifactual changes such as charge modifications may result from TCA precipitation, lyophilization or acetone precipitation was recommended for concentration of samples when necessary. The preferred method for removing DNA and RNA from samples employs DNase and RNase, because shearing is somewhat less effective. For carrier ampholyte isoelectric focusing, low salt concentrations are necessary, and the final SDS concentration of samples should be 0.3-0.5%. The importance of using a blend of ampholytes that will provide good buffering across the entire range of desired pH was outlined. To improve reproducibility batches of tube gels should be cast using the same lot of ampholyte for the entire project, and a low current should be used so that proteins do not precipitate at the point of sample loading. The upper buffer may be pre-warmed to 30[[ring]]C to prevent sample precipitation, and acetone precipitation of the sample followed by re-solubilization in sample buffer may improve the quality of the isoelectric focusing, presumably due to removal of some adverse component that is soluble in acetone. Because carrier ampholytes are not stable above pH 7.5, non-equilibrium methods (known as NEPHGE, non-equilibrium pH gradient electro-phoresis) must be employed for basic proteins. With isoelectric focusing in immobilized pH gradients, samples should be solubilized in urea/NP-40, and SDS should be avoided altogether. For samples containing high levels of salt, the voltage may be decreased resulting in desalting during an extended sample loading time. To increase sample loads, immobilized pH gradient gels may be re-hydrated directly in the sample rather than in buffer as has typically been done.
For maximal reproducibility of the second dimension gel, it was recommended that highly purified acrylamide stock solutions be used and that Tris buffers be made from weighed acid and base rather than titration. To avoid convection currents when preparing the second dimension gels, both gel plates and solutions should be at the same temperature, and gels should be run at the temperature at which they were poured. In addition there should be sufficient SDS present in the running buffer that it is not required in the second dimension gel. Isoelectric focusing gels should be equilibrated briefly in the second dimension gel's running buffer (1-2 min for 1.2 mm tube gels, longer for immobilized pH gradient gels).
Pre-fractionation can be used to assist protein identification. Due to limitations in sample load on the first dimension gel, a whole cell lysate may not yield sufficient quantities of a protein, and pre-fractionation allows increased loading of particular proteins while keeping the total protein load constant. For example, Triton X-114 extraction provides samples enriched in proteins that partition into aqueous and detergent phases at elevated temperatures. Simple enrichments such as this also yield information about subcellular locations of particular proteins, as well as translocation in response to specific treatments. Other methods to increase the quantities and resolution of specific proteins involve:
* The use of larger carrier ampholyte isoelectric focusing gels. On 3 mm gels, 0.75-1.0 mg of protein may be loaded. Samples should be solubilized in octyl-glucoside instead of NP-40, and the isoelectric focusing gel should also be cast with octyl-glucoside instead of NP-40, which enables efficient equilibration of gels before electrophoresis in the second dimension;
* The use of modified narrow-range immobilized pH gradients. On broad-range gels, 3-5 mg of protein can be loaded, but narrow-range gels work well with sample loads of about 15 mg; and
* Rehydrating immobilized pH gradient gels with sample. With preparative gels where milligram quantities of protein are loaded, resolution may be poorer than with analytical gels where sample loads are in the microgram range. However, if the protein of interest is well resolved, this decrease in resolution may not be problematic.
Methods for protein identification following two-dimensional gel electrophoresis include (1) in-gel digestion or blotting to PVDF, enzymatic digestion, HPLC, and subsequent amino-terminal sequence analysis, (2) immunoblotting, and (3) MALDI-MS and peptide mass searching following either in-gel digestion in the presence of Tween-20 or digestion from PVDF membranes in the presence of reduced Triton X-100. Care should be taken when interpreting peptide mass mapping data, because the peptide masses obtained from a particular digest do not always match the highest ranked protein identified from the database search. This may be due to post-translational modifications of proteins, proteolysis, insufficient resolution of proteins in the gel resulting in generation of peptides from a mixture of proteins, or an incorrect match. To avoid incorrect identification by peptide mass mapping, a number of additional strategies can be used. These include digestion of a portion of the sample with a second protease or on-probe deuteration. Alternatively, the sample can be chemically modified by methyl esterification or iodination, for example, and the chemically modified peptide masses can be compared with predicted masses. The most certain method for protein identification is to obtain part of its sequence either by Edman degradation or MS/MS, providing a peptide sequence tag.
Andrew Gooley, Macquarie University, "From Proteins to Proteomes: Rapid Protein Identification from 2-D Gels".
The concept of proteome research was introduced. In contrast to the genome, the proteome is an entity that may be altered depending on cell type, tissue, developmental change, or physiological conditions. In addition, for many genes more than one protein product exists due to alternative splicing, post-translational modification, or cellular processing. Two-dimensional gel electrophoresis provides a powerful technique for monitoring these processes, and at Macquarie University amino acid analysis is being used as the first step in a hierarchical process to identify proteins separated by this technique. The amino acid analysis procedure has been automated so that 300 samples may be analyzed per week in a single station. Protein identification based on the results of amino acid analysis is carried out using ExPASy, which may be accessed through the URL address http://expasy.hcuge.ch/. This software generates ranks and scores that provide a level of confidence in the accuracy of the proteins identified.
The ExPASy search generates a list of proteins ranked according to score. Confident identifications are made when a score of less than 30 is obtained for the first ranked protein and the score for the second ranked protein divided by the score for the first ranked protein is greater than 2. Additional information from techniques such as classical Edman degradation or MS/MS is needed when scores are above 30. Protein identifications can be confirmed by sequence tags, the sequence of the first three residues. Sequencing cycles are usually optimized to allow assignment of as many cycles as possible, but this is unnecessary when only three residues are required, so cycle times may be significantly shortened for sequence tagging. For example, at Macquarie University, a Beckman sequencer is being used for this purpose with cycle times of 20 minutes, and after the sequence tag is generated, the same sample is used for amino acid analysis.
As an example of the use of this technique, 30 proteins from a blot of an E. coli two-dimensional gel were amino acid analyzed. Of these, 21 were identified using ExPASy with the highest rank, 6 were identified but were not ranked number 1, and 3 were not in the database. To improve the accuracy of identification, mass and pI windows were also included in the identification software. The identifications were confirmed by sequence tags.
Gerald Latter, Cold Spring Harbor Laboratory, "Construction and Analysis of Two-Dimensional Gel Databases".
This presentation addressed practical aspects of computer analysis of two-dimensional gels. Coomassie-stained gels can be used for quantitative analysis with a film scanner, but silver-stained gels are not generally useful for quantitation. For radiolabeled proteins, autoradiography and fluorography can be used for generating images to obtain quantitative information as long as calibration standards are used, but phosphorimager analysis is a better method due to its high dynamic range and linearity. For accurate quantitation and comparisons, multiple gels of a high quality should be analyzed. For analysis of gel images on film, results from several exposures may be merged into a single image to avoid problems due to the inherently low dynamic range.
Various software packages with different features are available for analysis of two-dimensional gels. Typically the software first goes through a detection process. Background subtraction should be done to remove any streaking and to flatten the threshold. Software models for spot detection and quantitation segment the edges of the spot or use a Gaussian model that separates overlapping spots. Spots between gels can be matched initially by selecting landmarks and then allowing the software to match additional spots. The software should allow normalization either to the total amount of protein loaded or to a single spot or set of spots. Mismatches can be detected by various methods including vector analysis, and the software typically has an editing capability to allow correction of mismatches. Comparison of levels of spots between two gels may use a scatter plot or a histogram of spot ratios, so that anything that deviates from a Gaussian curve is suspected of being altered. For comparison of many gels at once, a technique of cluster analysis is used that detects changes in sets of spots in three to six gels.
A number of two-dimensional gel databases exist, including quantitative databases for S. cerevisiae and REF52, and annotative databases for E. coli and human keratinocytes. There are five two-dimensional gel databases available on the World Wide Web, and an annual database issue of Electrophoresis provides an additional resource to the major databases available. When building or using a database, experiments may be linked by different methods depending on the software employed. Ideally the software should be capable of detecting and storing spot sets and should allow annotation of spots. A number of two-dimensional gel software packages are available commercially, and selection of a particular package depends on the type of analysis required. Typical analyses in a core facility may simply involve comparison of a single protein in two samples, or identification of a few spots, which may not necessarily require complicated software. For detection of minor changes in many spots, statistical analysis and sample repetition is typically done, and so sophisticated software would be needed. Consideration of the methods employed for computer analysis of two-dimensional gels should take into consideration the nature of the experimental problem and the goal of the project.
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