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Seeing (and picking) spots Although chromatographic methods are gaining momentum, many researchers still prefer to separate proteins with 2-D gels.
Katie Cottingham
F
or years, 2-D gel electrophoresis has been a mainstay in the field of protein biochemistry. With the technique, researchers separate proteins by isoelectric point (pI ) in the first dimension, then by molecular weight in the second dimension. In addition to providing pI and molecular weight information, 2-D gel analyses can reveal whether a protein is modified or whether a sample contains different isoforms of a protein. Instead of studying one or a few proteins at a time, researchers are now analyzing whole proteomes consisting of thousands of proteins. In 2001, John Yates and colleagues at the Syngenta Agricultural Discovery Institute and the Scripps Research Institute proposed an alternative to gel-based methods for identifying many proteins simultaneously. In the multidimensional protein identification technology (MudPIT), also known as the shotgun proteomics technique (Nat. Biotechnol. 2001, 19, 242–247), proteins are digested into peptides, which are then separated by LC before MS analysis. According to company representatives, preferences for either 2-D gel or LC workflows are generally divided geographically. Whereas U.S. researchers are turning to LC-based methods in large numbers, European researchers continue to embrace 2-D gel technology. Robert Marchmont of GE Healthcare says that shortly after the MudPIT method was published, some experts had even predicted that researchers would stop performing separations with 2-D gels. Marchmont says, however, that 2-D gels are still being heavily used and are a workhorse for protein profiling and expression proteomics. “Although there are limitations with the [2-D gel] technology, [gels] are still extremely powerful tools in proteomics in terms of their power of resolution, throughput, and ability to separate posttranslational modifications,” says Marchmont. 2-D gels can provide data that cannot yet be obtained with gel-free methods, say experts. “The major point is that if you need to link biological relevance and posttranslational modifications, then the quantitation must take place on the isolated and intact protein,” explains Detlev Suckau at Bruker Daltonics (Germany). “A shotgun approach at the proteome level © 2005 AMERICAN CHEMICAL SOCIETY
cannot provide this information.” Marina Pekelis at Bio-Rad says that 2-D gels “give a good visual presentation of several interrelated proteins, which could be used as a biomarker.” According to Doris Terry at Purdue University, researchers can obtain much higher sequence coverage with 2-D gels/MS than with LC/MS. But some researchers, such as Chao-Xing Yuan at the University of Pennsylvania, say they will only maintain the gel equipment they currently have and do not plan to invest more money in the technology. According to Yuan, LC/MS is faster and less labor-intensive than gel work, and more proteins can be identified in one shot with gel-free methods. Because of the dichotomy among proteomics researchers, company representatives find it difficult to assess the market for 2-D gel accessories, such as imagers and spot excisers. Some representatives report that the market is steady, whereas others say it is increasing, albeit slowly. Tables 1 and 2, which are meant to be representative rather than comprehensive, list several commercially available 2-D gel imaging systems and all-in-one systems that include imagers and spot excisers.
Imaging spots After researchers run a 2-D gel, they typically visualize the protein spots by staining the gel with a colorimetric dye, such as silver stain or Coomassie Blue stain, or a fluorescent O C T O B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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dye, such as SYPRO Ruby, Deep Purple, or a Cy dye. Some researchers say that they simply use an office computer scanner to obtain an image of silver- or Coomassie-stained gels. But to visualize fluorescently labeled proteins on 2-D gels, scientists use more advanced imaging systems; most of these systems can also provide pictures of gels stained with colorimetric dyes or those that are radioactively labeled. Chemiluminescent reactions performed on blots of the gels can be detected by some systems. Yuan says that the choice of imager depends on how much money a researcher has to spend and whether he or she wants a versatile instrument. Imagers vary widely in price, from a few thousand dollars for a simple digital camera and illuminator setup to ~$100,000 for a scanner with many lasers that can detect a wide range of dyes. Imagers combined with spot 398 A
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excisers in all-in-one systems can be even more expensive. Most imagers use either CCD cameras or laser scanning technology. Laser scanners are generally more expensive than CCD cameras, but experts say that they offer better image quality. A recent innovation in the 2-D imager area is scanning CCD systems. Both GE Healthcare and PerkinElmer Life and Analytical Sciences offer models with this technology. Scanning CCD systems offer the high resolution of laser scanners but at a lower price. “Unlike the typical CCD systems [in which] you have a magnification on the camera, our system takes a number of sequential frames across the [gel]. It merges those images together to [form] one large image of high resolution,” explains Elaine Scrivener at PerkinElmer Life and Analytical Sciences.
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Although some instruments provide very high resolution, most proteomics researchers don’t need it. Often, the imagers were also designed for use with microarrays and other applications that require higher resolution. “The optimal resolution [for 2-D gels] is between 100 and 200 µm,” says Pekelis. “When people [set our instrument] to 100 µm, they intentionally oversample it to make sure they are not missing any details.” She adds that higher resolution settings are not typically used for 2-D gels because those settings generate huge files that don’t really provide much extra information. Once a suitable image is obtained, researchers can perform several software analyses and choose spots for further study. Protein amounts are often determined and compared within the same gel or between multiple gels. Some software packages perform triangulation, which allows researchers to lay one gel image over another to match spots. Most companies that sell imaging systems also sell analysis software that can be used with their own instruments or with imagers sold by competitors. Several companies, such as Nonlinear Dynamics and Decodon (Germany), specialize in analysis software and do not sell imagers or other accessories.
cal pathway,” says Suckau. He says that with the Proteineer system, however, data are transferred from one part to the other so that at the end, users see a gel image with spots that are linked to their MS identifications. Robots minimize researcher intervention. According to Gordon Nye at LEAP Technologies, the 2D iD and 2D iDx systems also track data and can interface with laboratory information management systems. Depending on the number of spots a researcher wants to cut out of a gel, a spot exciser can take as long as 1 h to complete its job. Thus, many instruments include measures to prevent gel dehydration and contamination. Spot pickers are often enclosed to shield the gel from airborne particles and to keep the gel in a humid environment. Some spot pickers also periodically spray the gel with water or buffer. The Genomic Solutions and LEAP Technologies products both include HEPA filters to further prevent contamination. In addition, Genomic Solutions offers further humidification and cooling options. To reduce or eliminate spot-to-spot contamination, many instruments wash the picking tip after cutting each gel piece.
Cut it out
Future innovations
The next step in the workflow is to excise the interesting spots. Most spot excisers, or spot pickers, are sold with an imaging system. This system can be used as either a primary imager or a secondary one to capture a picture of the gel just before it is cut and to make sure the spots are still in the same locations. Typically, spot excisers are not sold with analysis software, but they are compatible with most packages on the market. Frank Smith of Genomic Solutions says that an advantage of directly picking spots from an all-in-one system is that the gel is handled less, and this prevents shrinkage and warping. He adds that these changes in the gel’s geometry can result in inaccurate and unreliable spot picking. On the basis of customer feedback about throughput issues, however, GE Healthcare representatives decided to sell their Ettan Spot Picker without an imager. Jasmine Gruia-Gray of GE Healthcare points out that separate imagers and spot excisers can prevent bottlenecks and allow for higher throughput. “While the gels are being imaged [on a separate imager], other gels can be picked,” she explains. Also, some experts say that the imagers that are sold with spot cutters are not of the best quality. Terry says that in some cases, faint spots that are observed with an imager are not visible on an imager–spot cutter system, for example. Some all-in-one systems include tracking mechanisms to ensure that information about each spot is carried with it through the imaging, spot cutting, and MS steps. For example, the Bruker Proteineer SP spot exciser and imager is part of a larger Proteineer solution that includes a digestion platform, a mass spectrometer, and a database system. All data are tracked through all the steps by a transponder or bar code. “Typically, if people buy [components] from different sources, each part does the job it was designed to do, but there is no information exchange downstream in the analyti-
Although many parameters on imagers and spot excisers are already optimized beyond users’ needs, experts say that two key aspects of these instruments will improve in the future: throughput and visualization of multiple dyes and stains. Currently, most spot pickers and imagers can handle as many gels as can fit on their illumination or cutting surfaces; this means that with available instruments researchers can analyze multiple small gels at the same time or a single large gel. An exception is the Investigator ProPic II, which has two trays that double its capacity. Therefore, researchers can handle two large gels or a combination of a large gel and many small gels simultaneously. Terry predicts that other companies will emulate this feature to entice customers who are interested in highthroughput studies. The speed of imaging and cutting is another factor that can increase throughput. According to Kumar Bala at GE Healthcare, customers are often interested in the speed of the systems, which is important if one is running 20 or 30 gels for an experiment, for example. To address this need, faster imagers and spot cutters are being introduced onto the market. Bala says that more researchers are using fluorescencebased platforms. Companies will expand the wavelength ranges that imagers can detect to account for new dyes that are being developed, he says. 2-D gel electrophoresis is still the preferred protein separation technology for many researchers, especially those in Europe. Gels have several advantages that LC methods lack; this leads experts to conclude that the techniques are complementary. Certainly, researchers will be seeing spots for some time to come. Katie Cottingham is an associate editor of Analytical Chemistry. O C T O B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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