Vol. 5, No. 5, pp 2628, 30, 3233.
The matching game
Protein-based biochips are making strides in proteomics and diagnostics.
For several years, as knowledge of the human genetic complement expanded, promises were made with regard to our understanding of how cells function normally and how disease progresses when they do not.
To a large extent, this promise was based on genomic analysis using DNA or gene chips, tools that distinguish the slightest changes in gene sequence or RNA expression patterns. But as researchers learned more about the biochemistry of metabolism and disease, they began to realize that this promise would go largely unrealized if they relied on gene chips alone.
Every cell in a population of individuals contains largely the same genes, and although the expression of these genes may change with time or conditions, the main participant in cellular metabolism is not nucleic acid but protein. It only makes sense, therefore, to look to proteins to convey messages about human health and disease. To that end, a new industry has followed on the heels of DNA microarrays, and we are entering the era of protein chips and antibody arrays.
Unfortunately, although these chips are useful for global protein studies, they are not very specific. For that reason, companies such as Prolinx (Bothell, WA, www.prolinxinc.com) have developed affinity systems such that a particular moiety on the chip binds directly to a partner attached to the protein of interest. In the Prolinx chip, the chemical pair is phenylboronic acid and salicylhydroxamic acid; other pairs include biotin and streptavidin, and nickel and hexahistidine.
For even more specific binding, however, nothing can compete with the use of antibodies or antibody mimics. Within the range of antibodies, monoclonals offer the tightest binding; polyclonals offer diversity of binding, which may become critical if you still want your bound protein to function; and single-chain or phage display molecules offer greater stability. Following in the single-chain vein, antibody mimics are small proteins that have been engineered to interact with their partner protein. These include the trinectins of Phylos (Lexington, MA, www.phylos.com) and the affibodies of Affibody (Stockholm, www.affibody.com).Detection
Regardless of which attachment method is used, detecting interactions between proteins and ligands or other proteins is the name of the game. To that end, various technologies have been established to identify points of interaction, some of which are derivativeof DNA chip technologies. Companies such as Ciphergen and LumiCyte (Fremont, CA, www.lumicyte.com) rely on surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS), whereby laser light desorbs and ionizes proteins bound to a chip (Figure 1). The ionized molecules then enter a mass spectrometer, where their composition and mass are detected (www.seldi.org). With SELDI-MS, the bound proteins do not have to be labeled.
Another technique that does not require protein labeling is surface plasmon resonance (SPR), as exemplified by the system developed by Biacore. In chip-based SPR, target proteins are bound to the chip surface, and a baseline refractive index is established. A mixture of potential ligands or proteins is then passed over the chip, and interactions between the solution-phase molecules and the chip-bound proteins are measured as a change in the refractive index.
Alternatively,many laboratories rely on the use of fluorescent or radioisotope tags. The tagcan be attached directly to the proteins in the solution being tested or can be a component of a secondary antibody such as those used in Western analysis.
To a large extent, though, how the proteins are detected depends on what instruments are available to a researcher and, even more so, what application the researcher has in mind.
In February, Emanuel Petricoin, a researcher at the U.S. FDA (Bethesda, MD), and his colleagues presented their experiences in using serum protein patterns to identify individuals with ovarian cancer (1). Petricoins group incubated Ciphergens C16 hydrophobic interaction protein chips with serum from women with and without ovarian cancer and elucidated patterns of proteins that were specific to the cancer. These patterns then served as a template against which blind serum samples were tested. The pattern matching correctly classified 63 of 66 noncancerous or benign samples and all 50 cancerous samples.
After proper validation, serum proteomic pattern analysis might ultimately be applied in medical screening clinics as a supplement to diagnostic workup and assessment, Petricoin wrote. A negative value, if the sensitivity remains at 100% on further trials, could be used for reassurance, whereas a positive value might be sufficient to warrant further investigation.
While Petricoins group only looked at protein patterns, other groups have been vocal about the need to identify the actual proteins associated with the disease. For some of these people, protein chips present an ideal method.
Take prostate cancer, where there is only the prostate-specific antigen (PSA), which is secreted in sera, says Moncef Jendoubi, founder of Milagen, Inc. (Richmond, CA, www.milagen.com). The PSA is secreted as a result of the enlargement of the prostate as we age and is not indicative of cancer. We applied almost 15,000 antibodies, each recognizing a separate protein, and identified, in individuals suffering from prostate cancer, 164 proteins that are not PSA.
While Milagen is making strides with its antibody program, other companies are entering the diagnostic protein chip market. In early 2001, Large Scale Biology Corp. (LSBC, Germantown, MD, www.lsbc.com) announced a collaborative agreement with Biosite Diagnostics (San Diego, CA, www.biosite.com). By combining LSBCs Human Protein Index with Biosites high-throughput Omniclonal phage display technology, the companies hope to generate antibody arrays against various human diseases.
Similarly, this past January, NextGen Sciences (Alconbury, U.K., www.nextgensciences.com) initiated a collaborative project with Cytomyx (Cambridge, U.K., www.cytomyx.com) and Carlos Caldas of the University of Cambridge (U.K.) to generate a range of protein chips targeted at breast cancer.
My research group has been studying breast cancer at the gene and mRNA levels using human biopsy cell lines as well as primary tumors with linked, appropriately anonymized clinical information, says Caldas. This initiative allows us to access new technology in protein expression analysis and to combine this with the genomics and transcriptomics information we are already gathering.
Prognosis and pharmacoproteomics
You can imagine having an antibody chip for predictive medicine, says Jendoubi. Once you have identified a number of proteins secreted in sera or urine, you can segregate the proteins by which are linked to early disease, the onset of metastasis, who does and does not tolerate treatment, toxic effects, and who is prone to resistance or relapse.
Fundamentally, you establish a pharmacoproteomic profile of an individual. Like pharmacogenomics, which allows researchers and clinicians to predict the response of an individual to drug treatment on the basis of his or her genetic profile, the evolving field of pharmacoproteomics allows drug developers and clinicians to further subdivide the treated population.
Like others, we believe that the availability of protein biochips designed to evaluate toxicity and efficacy may dramatically change the research process, says Gunars Valkirs, vice president and chief technical officer of Biosite Diagnostics.
In the construction of protein interaction maps, protein chips offer advantages over the more traditional methods. In yeast two-hybrid screening, one protein is fused to the DNA-binding domain of a transcription factor (the bait), and the proteins in the mixture to be analyzed are fused to the transcriptional activation domain of another protein (the prey). When the bait and prey interact in vivo, they activate the transcription of a reporter gene.
One of the main advantages of a protein chip is that whereas the researcher has little control over the conditions under which the fusion proteins interact in the yeast cell, there is ample opportunity to modify the reaction conditions on a chip. This effectively eliminates many of the false positive interactions that are inherent in the yeast two-hybrid system. Furthermore, although the expression of a natural transcription factor can trigger a reaction in the yeast system, this same protein is just another spot on the protein chip, again limiting the risk of false positives.
Protein arrays also facilitate the identification or characterization of the enzymatic activity of proteins. This is potentially useful to the pharmaceutical industry, in which companies are searching for small molecules that bind to specific proteins in the proteome or interrupt the proteins activity. In 2000, Gavin MacBeath and Stuart Schreiber of Harvard University (Cambridge, MA) described their experiences in developing an array of peptides and determining which one was a substrate for a particular kinase (3). They incubated the slides in the presence of a kinase and radiolabeled ATP. As anticipated, each of the substrates was radiolabeled by its respective kinase. Similarly, Snyders group probed the yeast array describedin the preceding paragraphwith various phospholipids and identified 150 lipid-binding proteins (Figure 2).
Randall C. Willis is a senior associate editor of Modern Drug Discovery. Send your comments or questions regarding this article to email@example.com or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.