2-D Glasses

Suppliers of 2-D Electrophoresis Equipment Courtesy of Bio-RadBio-Rad's Mini-PROTEAN 3 system Graphic: Leza BerardoneThe inevitable inventory of genes that will be produced by the Human Genome Project heralds the start of a new era: the Age of Proteomics. Although DNA is the blueprint for life, it is the set of proteins that are actually transcribed and translated that determine the function of a particular cell. Proteomics is the study of the complete protein complement of the cell, tissue, or organism. Central to this burgeoning field is two-dimensional (2-D) gel electrophoresis.1,2 Several thousand different proteins are expressed at any one time in a particular sample tissue or organism; 2-D gel electrophoresis makes it possible to resolve these proteins. Scientists may compare samples from normal and diseased tissues, or from tissue samples harvested over the course of development, to identify those proteins whose expression levels change. With the 2-D method it is even possible to distinguish functionally distinct proteins encoded by the same gene, such as mRNA splice variants and proteins bearing post-translational modifications (e.g., methylation, glycosylation, and phosphorylation). Two-dimensional gel technology was first described more than 25 years ago by Patrick O'Farrell.3 However, recent advances have made the technique more popular, by improving what was often a harrowing and time-consuming experience. Fragile polyacrylamide tube gels containing drift-prone carrier ampholytes are now replaced by immobilized pH gradient (IPG) strips that allow for simultaneous "in-gel" rehydration and sample application, offer mechanical stability due to plastic backing, provide an increased range of pH values, and most importantly, improve reproducibility between gels and among different laboratories.4 New stains and staining techniques now permit proteins present in low abundance to be detected in one step. Matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry (MS) allows the analysis and identification of very small amounts of protein isolated from the gel. These advances have combined to make 2-D electrophoresis a more attractive option for the analysis of complex protein mixtures. Perhaps the greatest demand for better 2-D methods and higher-throughput equipment arises from researchers involved in drug development. Pharmaceutical companies can radiolabel drug compound candidates and use 2-D electrophoresis to identify protein targets. These protein targets may also become disease markers, leading to early detection and diagnosis of a given disease. In any event, the race to make sense of the genome has forced 2-D gel technology forward. Of premier importance to the success of the 2-D method is sample preparation. Some researchers choose to prefractionate the protein components from specific organelles to reduce the number of proteins in the sample, thereby improving results. Prior to electrophoresis, the protein mixture must be solubilized and denatured without affecting the protein's isoelectric point (see below). Proteins are therefore solubilized in solutions that usually include urea and one or more non-ionic or zwitterionic (compounds that contain charged moieties, yet whose net charge is zero) detergents. Anionic detergents, such as sodium dodecyl sulfate (SDS), interfere with isoelectric focusing and must either be removed by acetone precipitation or diluted into an excess of non-ionic or zwitterionic detergent such as NP-40. Reducing agents such as dithiothreitol (DTT), and carrier ampholytes are also commonly added.5 Finally, contaminants (such as salts, nucleic acids, polysaccharides, and lipids) must be removed and the sample must be protected against liberated proteases. The first dimension of the 2-D technique resolves proteins by isoelectric focusing (IEF). All proteins have a net charge (the sum of the charges of the amino acid side chains). The pH at which the net charge of the protein equals zero is the isoelectric point (pI). IEF separates (focuses) proteins on the basis of their charge or pI by electrophoresis across a polyacrylamide gel containing a pH gradient. Even proteins that differ from each other by only one amino acid residue can be separated in this manner. Precast, dehydrated, immobilized pH gradient gel strips are widely used for the IEF step and are available in a variety of pH ranges that can be overlapped for even higher resolution.4 Because the voltage necessary for IEF is generally high (up to 3,500 V) the gels are typically run horizontally on a flatbed system complete with a cooling plate. After completion of IEF, the proteins are resolved in the second dimension via SDS-polyacrylamide gel electrophoresis (PAGE). SDS not only denatures proteins; it also applies a uniform negative charge to the surface of the proteins, allowing separation based on relative molecular weight. The IPG strips must first be equilibrated with buffer, SDS, urea, reductant, and glycerol. The equilibrated strips are then affixed to the top of a vertical SDS-polyacrylamide gel and electrophoresed. Finally, the separated proteins are visualized. Proteins labeled with isotopes are detected by autoradiography; other methods of detection commonly used are silver staining, Coomassie Brilliant blue staining, and fluorography. The resultant two-dimensional array of spots, each of which corresponds to a single protein species, is then analyzed with specialized computer software. Getting New Glasses Suppliers of 2-D gel electrophoresis equipment often create integrated systems for proteomic analyses. For instance, Biometra of Göttingen, Germany, manufactures the IEF-Module for low sample throughput, the Mini-IEF for high sample throughput, and the Mini-Focus for flat-bed IEF. Biometra also offers the Minigel and Minigel-Twin, Multigel and Multigel-Long, and Maxigel Slabgel systems for the second dimension. BioDocAnalyze, a light-sensitive CCD video system for detection and quantification of proteins, is also available. This modular video system is available either as a core "economy" set, consisting of camera, digitization card, and image analysis software, or as a complete package that adds a computer, monitor, printer, dark hood, and transilluminator. Amersham Pharmacia Biotech (APBiotech) of Piscataway, N.J., offers a number of products for high-throughput 2-D gel analysis. APBiotech's IPGphor™ Isoelectric Focusing System combines two processes, the rehydration of Immobiline™ DryPlate or DryStrips with the sample solution, and IEF, into a single step for the first-dimension separation. The Multiphor™ II Flatbed System can be used for either IEF or SDS-PAGE. The Hoefer™ DALT System allows casting of up to 23 large format gels and runs up to 10 vertical SDS-PAGE gels simultaneously. The new large format Ettan™ DALT II system is compatible with 24-cm IPG strips for high resolution and high throughput, running up to 12 vertical SDS-PAGE gels at one time. APBiotech also stocks the ImageScanner™ and Typhoon™ 8600 for protein spot selection, ImageMaster™ 2-D Elite gel matching software, the Ettan spot picker, and Ettan MALDI-ToF for protein identification. ProSpot, a high-throughput spot handling platform, stacks up to 12 second-dimension gels, performs spot picking, digesting, and sample dispensing onto MALDI target slides; this product and Lab WorkFlow tracking software, which will integrate the Proteomic suite, are scheduled to be launched later this year. Finally, APBiotech offers the new 2-D difference in gel electrophoresis (DIGE) technology. According to Jasmine Gray, product manager for Proteomics, DIGE consists of "labeling up to three [different] protein samples with CyDyes™ and running them on a single gel at the same time," thereby eliminating complex gel-to-gel comparisons. The technology simplifies detection of proteins that are differentially or uniquely expressed. Courtesy of Bio-RadA selection of 2-D electrophoresis products from Bio-Rad Bio-Rad Laboratories of Hercules, Calif., and Micromass of Manchester, U.K., have developed the ProteomeWorks™ System. Tom Slyker, Proteome program manager at Bio-Rad, sees the system as "an exciting opportunity to establish global maps of proteomes, using 2-D in an integrated system." In addition to first- and second-dimension cells, ProteomeWorks has a spot cutter that automatically excises protein spots selected and driven by PDQuest™ software. Following gel excision, proteins are spotted to a M@LDI™ target plate in the MassPREP digest station for automated destaining and enzymatic digestion in preparation for obtaining peptide mass fingerprints with the M@LDI 2-D-Gel-MS analyzer. The WorksBase™ software for Proteomics integrates data from PDQuest, the ProteomeWorks spot cutter, the MassPREP Station, and Micromass' MassLynx™ and ProteinLynx™ systems. Genomic Solutions of Ann Arbor, Mich., also offers a fully integrated system, the Investigator™ Proteomic Solution. The system includes a 2-D electrophoresis system (using Duracryl™ high-tensile strength acrylamide gels), gel processor, 2-D analysis software, Flexys® robot for automated spot excision, ProGest™ protein digestion station, ProMS™ MALDI preparation station, and MALDI-ToF mass spectrometer. Genomic Solutions also offers contract laboratory services. Courtesy of FermentasFermentas' 2-D electrophoresis systemOther companies specialize on specific elements of proteome analysis. C.B.S. Scientific of Del Mar, Calif., provides tube gel electrophoresis systems for the first-dimension, IEF tube gel adapters, vertical mini-gel systems, and adjustable slab gel systems for the second-dimension. Fermentas of Hanover, Md., sells mini, maxi, and modular electrophoresis systems for both the first- and second-dimension. Invitrogen Corp. of Carlsbad, Calif., offers Novex® precast mini-gels and pre-made buffers for both IEF and 2-D separations. The 2-D mini-gels are available in both traditional Tris-glycine and NuPAGE® formulations with preformed wells to accept either gel slices or tube gels. The Novex ZOOM Gel™ line is designed to accept 7-cm IPG strips from any manufacturer. Invitrogen also offers SERVALYT PRECOTES® and SERVALYT PreNets®, horizontal IEF gels bonded to inert plastic sheets or netting to simplify handling. SERVALYT carrier ampholytes are available for those who are casting their own IEF gels. Greg Bambeck, vice president of research and development for Zaxis of Hudson, Ohio, states that Zaxis' precast 2-D gels are "the purest, most uniform, highest-resolution gels in the world." Zaxis gels are purported to have 140 percent better resolution than other precast gels, allowing one to visualize more spots. Zaxis gels are compatible with IEF tube gels and IEF-IPG strips, and their cassettes are adapted for a variety of other companies' apparatuses. The Z-Fast System, a high speed electrophoresis mini-gel running system for the second dimension, runs four times faster than other mini-gel systems. Zaxis' colloidal Coomassie "Burst" stain is about an order of magnitude more sensitive than standard Coomassie Blue stain (in the range of silver stain). It is possible to use 2-D technology even if setting up a complete 2-D gel electrophoresis system is not feasible. Many contract laboratories, such as Kendrick Labs of Madison, Wis., offer 2-D services for academia, government institutions, and industry. Kendrick Labs provides a sample preparation kit and a mailer in which to send samples. They offer a basic 2-D service, custom pH gradients, slab gels for high- or low-molecular weight proteins, manual- or computer-generated gel comparisons, Coomassie blue staining, silver staining, SYPRO Ruby staining, autoradiography, fluorography, and laser densitometry. Soft Contacts Following 2-D electrophoresis is the data analysis step. Typically two gels, representing for instance wild-type versus mutant cells, are compared to identify those proteins whose expression levels vary significantly. Matching gel spots by eye is laborious and subject to error, but new computer software with spot detection and matching algorithms has revolutionized this process. Compugen, based in Princeton, N.J., and Tel Aviv, offers Z3 image analysis software that automates spot detection and alignment of gel images, computes differential expression, and includes tools for data mining and querying. This software can extract comprehensive and accurate information from repeated experiments, making accurate, large-scale analyses possible. Courtesy of Decodon GmbhComparative gel analysis using Delta 2D from Decodon GmbH Melanie 3, available from Geneva Bioinformatics of Geneva offers analysis for identification, quantification, and matching of gels, in addition to comprehensive annotation, advanced statistical, classification, querying, warping, and reporting functions. Delta 2D, from Decodon GmbH of Greifswald, Germany, employs image processing algorithms to align spots on different gels, is able to compare highly dissimilar gels, offers global and exact warp to accelerate matching, and produces well-balanced dual-channel images for direct visual comparison. Redwood Shores, Calif.-based Scimagix's 2-D Gel Analysis & Mining Application features automatic spot detection and unwarping, automatic registration of all spots into Oracle®, and the ability to search all experiments for "protein signatures." APBiotech's Imagemaster 2-D™ Elite software and 2-D database software provide automatic spot detection, background correction, quantification of spot density, and spot matching of an unlimited number of gels. The software is also capable of search and analysis across experiments for quantitative pattern relationships, and of combining densitometry studies with mass spectrometry data. Bio-Rad's PDQuest software compares gel images and identifies protein expression differences between samples and selects spot cuts in order from least to highest quantity of protein, minimizing carryover contamination from spot to spot. Overlapped spots are resolved and targeted for cutting so that protein mixtures are reduced. One can also pool the same low-abundance spot from multiple gels into a single target plate well. The Phoretix 2-D gel analysis software version 5, from Nonlinear Dynamics Ltd. of Newcastle upon Tyne, U.K., has many useful features such as auto-matic matching, with the option to manually intervene, and gel averaging from a group of matched gels. "Spot filtering" assists in locating spots demonstrating changes in protein expression. The user can click on any spot in an image and acquire information--in the form of a comparisons table, montage, histogram, or animation--about how that protein has changed through a gel series. Other features include full calibration and normalization facilities and background subtraction to accurately quantify spot material; molecular weight and pI determination by two methods; and a Web page builder. Using the Phoretix 2-D Gel Database, a click on any spot will search for all proteins that undergo a similar expression pattern. Nonlinear Dynamics also offers TotalLab, a general image analysis product, and is set to release Progenesis, a fully automated, comprehensive 2-D analysis package. Because it is the most powerful method to analyze large collections of proteins, two-dimensional gel analysis remains the core proteomic technology. Yet advances in electrophoresis equipment and analysis software are making the technique more and more accessible to scientists previously intimidated by lengthy procedures and disappointing outcomes. These advances have combined to produce a sort of renaissance in protein analysis. Now, using 2-D glasses, the secrets of the cell can be brought into focus. Hillary E. Sussman (hes01@health.state.ny.us) is a freelance writer in Albany, N.Y. References 1.L. Defrancesco, "One step beyond," The Scientist, 13[1]:16, Jan. 4, 1999. 2.S. Borman, "Proteomics: Taking over where genomics leaves off," Chemical and Engineering News, 78(31):31-7, July 31, 2000. 3. P.H. O'Farrell, "High resolution two-dimensional electrophoresis of proteins," Journal of Biological Chemistry, 250:4007-21, 1975. 4. A. Gorg et al., "The current state of two-dimensional electrophoresis with immobilized pH gradients," Electrophoresis, 21:1037-53, April, 2000. 5. T. Berkelman, T. Stenstedt, "2-D electrophoresis: Using immobilized pH gradients," Amersham Pharmacia Biotech, 1998.

2-D Glasses

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