New Technology Spurs on Proteomics

Graphic: Leza BerardoneOne recent morning at the Applied Biosystems proteomics research center in Framingham, Mass., scientist Jason Marchese patiently used a pipettor to place tiny samples onto a 2-inch-by-2-inch plate. He was surrounded by technology as simple as 2-D gel electrophoresis apparatus and as cutting-edge as a high-throughput system that uses automated robotics for multidimensional liquid chromatography separation of proteins and an automated workstation that uses the latest in mass spectrometry advances to identify and analyze proteins. The only scientist working in a large room filled with machines for proteomics research, Marchese talked about how he can load protein sample onto machines in the afternoon and return the next day to a database full of newly identified proteins. But in spite of major advances like this, inadequate technology still holds back the field of proteomics. Applied Biosystems is just one of a number of biotechnology companies working hard to provide novel tools to identify more quickly and precisely the thousands upon thousands of proteins produced by the human body. Now that the human genome is mapped, researchers in academia, government, and industry have turned their attention to mapping proteins. And biotechnology companies are responding by developing the needed tools for the job. Already a multimillion-dollar business, proteomics is expected to grow quickly into a multibillion-dollar business, according to market estimates. "It was important to map the genome, but clearly, proteins are where the action is," says Leigh Anderson, president of the proteomics subsidiary of Large Scale Biology Corp. in Vacaville, Calif. An early player in the proteomics industry, Anderson points to the promise--and value--that proteins hold for new diagnostic tests, therapeutic treatments, and medicines. But discovering and analyzing proteins is a daunting task, and there is still a long way to go before wide-scale protein identification is complete, or even possible. Proteins are far more numerous, fragile, and changeable than genes, and they can be difficult to find and measure. As Anderson puts it, the question now is, "What technology do we need?" It's not an easy question to answer, because the field is still young and the job is so big. Several recent technology advances have greatly improved the picture for proteomics research. Processes that once took months may now take just a few days or less. Biotechnology companies focusing on proteomics now hope to further reduce the process times down to a few minutes while also improving quality. Capturing Proteins Just a decade ago, the only way to identify a protein was to isolate it on a 2-D gel and then sequence it; a painstaking process that took days and sometimes weeks and could involve a lot of material. Two-dimensional gel electrophoresis is still a workhorse tool in proteomics, and it has become increasingly useful (see also, "2D Glasses,"). But 2-D gels can be labor-intensive, and they do not always provide reliable quantitative information. Additionally, they simply cannot identify certain classes of proteins, including membrane, highly acidic or basic, and low-abundance proteins. Courtesy of Ruedi AebersoldUsing ICAT reagents, researchers can easily compare the expression level of proteins from two samples, such as normal and diseased cells. Now, a new, complementary technology called ICAT™ (isotope-coded affinity tags) Reagent helps address many of the problems with 2-D gels. ICAT provides a new method for quantification and identification of proteins from complex samples using mass spectrometry analysis. The technology has received widespread praise throughout the industry for being the first tool that allows both pre-fractionation and quantification of relative expression levels of proteins, including low-abundance and membrane proteins, in a high-throughput model. It also is capable of identifying those proteins that 2-D gels miss, such as membrane proteins. "It lets us do things that weren't possible with 2-D gels," explains Dave Hicks, marketing manager for the Applied Biosystems proteomics research center. Developed by Ruedi Aebersold at the University of Washington,1 ICAT is currently licensed by Applied Biosystems. ICAT works by using a label that contains the stable isotope deuterium to label all the proteins (e.g., in cell extracts) via their cysteines, mixing them together, and digesting the samples with a protease. Peptides that contain the label are bound and then further separated. These labeled peptides are then loaded into a mass spectrometer and the ratio of the various peptide pairs is measured. As an additional benefit, ICAT could theoretically make any separation technology quantitative, including high-performance liquid chromatography and capillary electrophoresis. Since the proteins are pre-mixed, the relative ratio stays the same no matter how much protein is added during fractionation. A Role for Protein Biochips? Another new development that promises major advances in proteomics is the protein biochip. A number of companies are developing chips in the hopes that they will become a key tool for measuring large numbers of proteins in biological samples. "Separation technology is the key technology today for proteomics research, especially protein arrays," comments William E. Rich, CEO of Ciphergen Biosystems in Palo Alto, Calif. Protein biochips draw on the technology developed for genomics, but they have proven challenging to develop because proteins are far more difficult to analyze compared to DNA. Also called protein or antibody arrays, protein biochips hold the potential to measure protein-protein interactions, protein-small molecule interactions, and enzyme-substrate reactions. They may also allow differential profiling, such as distinguishing the proteins of a healthy cell from those of a diseased cell. The chips hold value for identifying potentially relevant biomarkers and for pharmaceutical discovery-based research. Among the companies working in this area, Ciphergen has developed a protein biochip system, the ProteinChip® System, that incorporates mass spectrometry (in particular, surface enhanced laser desorption/ionization [SELDI]), and biochip technology in a single, integrated platform. The chips allow researchers to capture, separate, and quantitatively analyze proteins directly on the chip. The arrays (protein molecular weights) are read directly by the SELDI process without radioactive or fluorescent labels or genetically engineered tags that may interfere with the protein. Large Scale Biology and Biosite Diagnostics Inc. of San Diego recently announced a collaboration to develop protein biochips. Large Scale Biology plans to provide 2,000 to 5,000 protein targets from its Human Protein Index™ as well as expressed proteins produced with its Geneware®‚ technology. Biosite will then use its high-throughput Omniclonal™ phage display technology to generate high-affinity antibodies to the targets, enabling the creation of antibody arrays in a variety of formats, including biochips. Based on this, the companies intend to market a broad antibody and target package. "In a couple of years we should be able to offer high-throughput, low-cost arrays," Anderson says. He expects that the resulting collection of antibodies will span many of the commercially important human proteins. Another leader in this area is Zyomyx of Hayward, Calif., which specializes in miniaturized protein biochip architectures containing high-density arrays. The company has recently collaborated with MDS Proteomics in Toronto (See also, "On the Fast Track in Functional Proteomics,") on research to identify protein pathways. In related technology, Bio-Rad Laboratories of Hercules, Calif., recently unveiled a platform for multiplexed, fluorescent immunoassays. Called the Bio-Plex™ protein array system, it is intended to allow researchers to extract more data from small samples. The platform builds on an integrated system of Bio-Rad application software, calibration and validation protocols, along with optimized assay panels. It is intended as a tool to speed the drug discovery process. Continuing Advancements in Mass Spectrometry Improved 2-D gels, ICAT, and protein biochips are important subunits advancing protein research, certainly, but they depend on mass spectrometry (MS) technology. Now highly accurate, and increasingly fast and automated, MS is widely viewed as the cornerstone proteomics tool for characterizing proteins. It determines the identification of large numbers of proteins and peptides, and it holds the promise of even more rapid identification and characterization in the future as the technology continues to advance. The focus today is on developing a new class of mass spectrometers that provide higher throughput and automation and more powerful and precise protein analysis, according to Hicks at Applied Biosystems. In one effort to achieve higher throughput, Applied Biosystems is developing a new MS/MS system based on the matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) technology.2 MALDI-TOF,3 first released in 1990, was the first system to enable rapid protein identification by using a technique called peptide mass fingerprinting. The company's newest system, the Voyager™ TOF/TOF, which is still in development, is the first tandem-TOF platform. The system uses a high-throughput MALDI source and two TOF mass analyzers with a timed ion selector and collision cell for both MS and MS/MS analysis of biomolecules. Kratos Analytical Inc. of Chestnut Ridge, N.Y., has recently released an MS system based on MALDI-TOF technology as well. The AXIMA CFR promises a combination of high mass accuracy, resolution, and sensitivity with high-throughput sample handling. Using a 384-spot microtiter sample, the AXIMA-CFR generates seamless postsynaptic density of proteins with an ion gate that allows reliable selection of parent signals for fragmentation from complex mixtures, a camera for viewing samples using nonuniform matrix preparations and unconventional samples, and a novel laser beam-focusing mechanism to maximize the yield of ions extracted from the source to the flight tube. Advances in MS/MS technology continue because MS alone is often not enough for identifying all proteins in a sample. The electrospray ionization tandem MS (ESI-MS/MS) method typically used for sequencing one or more protein cleavage products has led the way to more precise protein identification. An approach involving direct MS/MS of intact proteins for simultaneous protein identification and detection of post-translational modification appears promising for analyzing small molecules. Applied Biosystems, along with its joint venture partner MDS SCIEX of Concord, Ontario, is currently advancing the Quadrupole TOF technology for this purpose. One application of the technology is the characterization of post-translational modifications in proteins. More Technology Needed ASAP Despite the many new developments moving proteomics research forward at an increasingly rapid pace, a number of barriers still hold the field back. Technologies are needed still to better integrate the steps required for protein work and alleviate bottlenecks that occur between sample processing, separation, identification, and characterization. The process of loading proteins onto an MS plate, generating spectra, and searching protein databases takes time. A number of companies--including Genomic Solutions of Ann Arbor, Mich.; Oxford GlycoSciences of Oxford, England; Proteome Systems of North Ryde, Australia; and Large Scale Biology--have speeded up the process by automation. Advances in high throughput must be balanced, however, with high-quality data, explains Dave Walker, Bio-Rad's protein discovery business unit manager. Computer technology also must keep up with protein discovery. "Advances in proteomics technology must be accompanied by high-speed computing to allow fast data analysis," Hicks notes. A number of companies are focusing on the challenge of developing information technology especially for proteomics, including Compaq, which is collaborating with Celera, and IBM, which is collaborating with MDS Proteomics. Once the technology barriers to proteomics are overcome, however, our understanding of how proteins drive the human body may grow exponentially. Or as Ciphergen's Rich comments, "Scientists have known for a long time that proteomics is a necessary reality to understanding biology. If we focus on proteins now, proteomics has potentially revolutionary applications." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist. References 1. S.P. Gygi et al. "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nature Biotechnology, 17:994-9, 1999. 2. D. Wilkinson, "Pursuing proteomes," The Scientist, 14[12]:28, June 12, 2000. 3. B. Sinclair, "MALDI-TOF goes mainstream," The Scientist, 13[12]:18, June 7, 1999.

New Technology Spurs on Proteomics

Interested in reading more?

Become a Member of