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Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. This is the final special supplement this year on Advances in Proteomics. The first three appeared in the 25 March, 29 April, and 27 May issues of Science. By studying the ways in which proteins react with each other, researchers hope to achieve two goals: learning more about the ways in which cells function, and developing new drugs to treat disease. But the nature of the entities makes exploration of their interactions a difficult task. "Proteins are vibrating, extremely complex molecules in a constant state of modification, both initiating and driving interactions with many molecules," says Allan Simpson, vice president for research and analysis at GE Healthcare. "It requires a large range of technologies to handle and study them." Nevertheless, several life scientists have taken up the challenge. "The study of protein-protein interactions has probably seen an exponential increase," says Anke Cassing, associate director of corporate strategy at Qiagen. "A lot of researchers are focused on protein-protein interactions," adds Frank Montagne, chief scientific officer for Elchrom Scientific. "These are key mechanisms for the discovery of new signaling pathways." To facilitate the study of those mechanisms, increasing numbers of vendors have introduced new tools and technologies. "We've introduced new protein biochip arrays to study protein interactions," says William Rich, CEO of Ciphergen Biosystems. "We use SELDI-TOF-MS to detect protein interactions directly from the arrays, which provide advantages over elution-based mass spectrometry methods." Ken Miller, product manager of Thermo Electron, points to two other areas of advance. "The sensitivity of analysis capabilities of mass spectrometry has increased," he says. "And a lot of tools have emerged for very selective and effective affinity isolation of proteins and protein complexes." Lothar Germeroth, CEO of German firm IBA, outlines other recent improvements in tools. "Methods for stable isotope protein labeling for quantitative proteomics can further the efficiency of mass spectrometry," he explains, "since the technology enables the identification of dynamic changes in the composition of protein complexes in different cellular states." Improvements in tools for studying protein-protein interactions cover a wide spectrum. "There are approaches now that not only discover or confirm the interaction but allow you to drill deeper and characterize where the interaction contact surfaces reside on the binding partners," says Robert Vigna, technical marketing manager for Pierce. Two Emerging Technologies As its name implies, tag chemistry pinpoints specific proteins with appropriate chemical tags. "Proteins can be expressed with tags on one or both ends," GE Healthcare's Simpson explains. "Purification is now relatively easy, as the technology is available to capture the tag." IBA's Germeroth extends that point. "Tag-based methods allow you to apply fully processed and modified protein baits to identify the corresponding prey in a native environment," he says. "The advent of tag-based methods such as tandem-affinity purification [TAP] has improved the speed and accuracy of determination of protein-protein interactions." IBA, which advertises itself as "The TAG Company," markets the One-STrEP protein-protein interaction kit. "Our system is based on the Strep-tag technology," Germeroth says. "Strep-tag's small size, physicochemical balance, and mild elution conditions allow for a mild and rapid purification of protein complexes in only one step without compromising any protein in these complexes. The technology is ideally suited for automated determination of protein-protein interactions which the pharma industry strongly demands." QIAGEN has expanded its comprehensive portfolio of Ni-NTA products into the area of bead based assays. In LiquiChip assays, the remarkable selectivity of the Ni-NTA chemistry for His-tagged proteins is used to couple proteins to capture beads, thus enabling multiplex interaction assays. Ciphergen, has just introduced what it calls Equalizer Beads. "It is basically a bioseparation procedure that uses combinatorial methods to build a chemical library onto beads with a diversity of 1 million to 10 million different affinities," Rich says. "When you put these beads into a very small sample of serum, each bead finds the protein for which it has the most affinity. When you wash away all the excess beads with high abundant proteins, you have concentrated up all the low abundant proteins attached to the beads." Initially, Ciphergen is offering the technology as a service. "But eventually, as its development is improved, we will sell it as a product," Rich says. Chromatographic Separations GE Healthcare's ÄKTAxpress is a dedicated chromatography system for multidimensional purification of tagged proteins. "We have integrated knowledge into the system so that researchers needing to purify proteins do not need to be skilled chromatographers. It's a protein factory on a lab bench," Simpson says. For chromatographic professionals, the company offers alternative systems, including the ÄKTA Explorer.
Ciphergen, meanwhile, has developed products that eliminate the need for traditional chromatographic separations. The company's ProteinChip Arrays provide a variety of surface chemistries that allow researchers to optimize protein capture and analysis. "For us, the arrays have always been a miniaturized form of chromatography that scientists can use to rapidly predict optimum chromatographic conditions to scale up protein purification," Rich says. "And if you use it with our Equalizer technology, it works even better." The other traditional approach, two-dimensional (2-D) gel electrophoresis was developed specifically to separate protein mixtures. "Where the aim is to catalog the entire protein repertoire of a cell or tissue as comprehensively as possible, 2-D gel electrophoresis and liquid chromatography can be seen as complementary separation methods," Qiagen's Cassing says. "Two-D gel has quality comparable to chromatography," Elchrom's Montagne agrees. "But it's cheaper." Anatoli Tassis, manager of business development and applications development for oligos at Elchrom, points to another advantage of 2-D gels. "You use both the molecular weight and the bioelectric potential to reach your goal," she says. "That gives you better characterization."Bio-Rad Laboratories, GE Healthcare, and Invitrogen have electrophoresis systems with the units, power supplies, and accessories required to perform protein separations. Elchrom recently introduced Blot-EX, a new nonacrylamide, high-resolution electrophoresis precast hydrogel specifically designed to increase protein transfer efficiency during Western blotting of proteins. "This is specifically for low expressed proteins," Montagne says. "We aimed it at the pharmaceutical industry searching for new drugs." At a more general level, Qiagen's Qproteome fractionation tools provide efficient isolation of targeted subsets of proteins. "The kits are easy to use and don't require any special equipment," Cassing says. "And our PhosphoProtein Purification Kit is the only product on the market for complete separation of phosphorylated proteins from unphosphorylated proteins." Another vendor, Syngene, has introduced DYMENSION, a software program that can analyze a typical 2-D gel image in seconds. Three Means of Characterization Proteins usually function in families or pathways and interact with related proteins. Researchers often begin their studies by trying to understand how a protein with unknown function relates to other known proteins. The two-hybrid method allows researchers to identify related proteins. The method, available in bacterial, yeast, and mammalian models from such companies as Promega and Stratagene, can identify protein-protein interactions, protein cascades, and mutations that affect protein-protein binding. "Nothing really compares with the original two-hybrid for screening thousands of potential interactions," says Andrew Farmer, director of cell and molecular biology at BD Biosciences Clontech. "But it can produce a lot of false positives." To overcome that problem, Clontech has developed the BD Matchmaker Two-Hybrid System 3. "The tighter you can make the selection, the less the chance that false positives will come up," says Grigory Tchaga, the company's R&D director for protein and PCR Technologies. "In addition, scientists can use Clontech's pBridge Vector to create a three-hybrid system to screen for interactions that involve three proteins. And the BD Matchmaker One-Hybrid System reveals protein interactions with DNA sequences." Another way to compensate for yeast two-hybrid's false positives involves pairing the method with SPR, otherwise known as protein interaction analysis, which allows researchers to gather real-time functional data about binding events. "If you use yeast two-hybrid, you need to confirm your data with a more reliable method," says Stephan Löfas, chief scientific officer of Swedish firm Biacore. "Here is where we come in. Our technology permits you to go into the details to confirm whether or not you have seen a real interaction. Also, you don't need to do the type of labeling that would disturb the interactions." Protein Interaction Analysis and MS Mass spectrometry also works well in collaboration with other separation methods. "2-D gel is a protein purification mechanism that reduces a complex sample to a number of discrete spots," Thermo Electron's Miller explains. "Mass spectrometry allows you to identify what those spots are." To perform the identification, scientists combine electrospray mass spectrometry with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) techniques. In addition, Miller continues, "Mass spectrometry paired with liquid chromatography seems to identify many more proteins than 2-D gels." Pierce has paired mass spectrometry with another technology – the use of cross-linking reagents. "After in-gel tryptic digestion of the cross-linked complex, MS analysis of the resulting cross-linked peptides is facilitated," Vigna explains. "Bioinformatic analysis of these peptide masses can give information regarding the contact interface of the binding partners." Several well-known companies, including Applied Biosystems and Bruker Daltonics, manufacture mass spectrometers. Thermo Electron has adapted its Finnigan LTQ family of mass spectrometers by adding additional detectors and a new orbitrap. "These instruments provide a much higher quality of data, accuracy, and resolution," Miller says. "They also create the ability to do top-down proteomics – determining exact protein isoforms. And we have added a MALDI source to the LTQ, giving a very rapid way to introduce samples at the front end. That permits thousands of experiments a month instead of the hundreds or dozens using conventional mass spectrometry." Challenges remain for scientists who study protein-protein interactions. But the emergence and application of new tools and technologies has given researchers the opportunity to meet them more efficiently and more rapidly.
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Science. ISSN 0036-8075 (print), 1095-9203 (online)
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