Drug Discovery and Biotechnology Trends – Biochips 2 – Protein Chips: Putting Proteomes to Use with Microarrays

Different cells contain different proteins. Even the same cell’s protein content changes over time. Accordingly, any organism’s complete collection of proteins cannot be easily determined. Fortunately, evolution in protein microarrays helps scientists identify and quantify proteins faster than ever.

by Mike May and Gary Heebner

ADVERTISERS Affymetrix DNA microarrays, based on the principles of semiconductor technology 408-731-5000 www.affymetrix.com Ciphergen Biosystems, Inc. protein microarray systems and related products that discover, characterize, and assay proteins from native biological samples 510-505-2100 www.ciphergen.com Genetix [UK] laboratory automation products and services for microarrays and sequencing +44 1425 624 600 www.genetix.com Genetix [USA] 877-436-3849 Genomic Solutions DNA microarrays, fabrication instruments 734-975-4800 www.genomicsolutions.com Leica [Germany] instruments and systems for imaging analysis, digital cameras +49 6441 29-0 www.leica-microsystems.com Leica [USA] 847-405-0123 MWG [Germany] products and services for genomic research including microarrays, oligonucleotides, sequencing, and laboratory automation +49 8092 8289-0 www.the-mwg.com MWG [USA] 336-812-9995 Phalanx Biotech Group DNA microarrays for gene expression 650-988-6898 www.phalanxbiotech.com Takara Bio, Inc. kits and reagents for molecular biology research +81 77 543 9254 www.takara-bio.co.jp/english

IN THIS ISSUE: • Proteome complexity • Capture molecules • Connecting peptides to chips • Automated microarray makers • Ready to use microarrays • Measuring interactions

The companies in this article were selected at random. Their inclusion 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 second of four supplements this year on biochips. The first appeared in the 27 February issue of Science. The next will appear in the 9 July issue.

No one knows precisely how many proteins make up any organism’s proteome. In humans, the proteome may outnumber the genome by 10 to 30 times in sheer numbers of species. In fact, the complexity is likely very much higher when the plethora of post-translational modifications is considered. Nonetheless, microarrays help scientists identify proteins from different cells and under various conditions, which can be used in both basic research and biomedicine. Tony Pawson, director of research at the University of Toronto’s Samuel Lunenfeld Research Institute, says, "We are entering an exciting phase, but I would say that we have just gotten started."

In fact, protein microarrays themselves are just becoming widely available and more broadly applied. These devices—like proteomics itself—remain new and largely under development. "The difficulty with proteins," says Pawson, "is that they are more complex than DNA, and when you put them on chips they tend to denature." Consequently, much of the work in protein microarrays involves learning to make proteins that remain functional on a chip.

As protein microarrays grow increasingly stable and reliable, these devices will bring high throughput technology to proteomics. Pawson says, "To understand the language of protein interactions, catalytic specificities of proteins, and the identification of small molecules that regulate protein activity, we need chip based assays. This is essential if we want to look in a relatively fast way at the levels and functions of multiple proteins in a cell." As DNA chips boosted genomics, protein microarrays will transform proteomics.

Anti- and Other Bodies
When it comes to capturing proteins, antibodies come to mind as the hook. Although antibodies can recognize proteins with great sensitivity and selectivity, only a minority of the several thousand available antibodies work well with the multiplex assay format used on protein chips. Joachim Feldwisch, project manager of arrays at Affibody AB, says, “The major issue in protein microchip development is the lack of high quality content in the capture molecules to be immobilized onto the chips. At present there are just a few hundred very good antibodies available for chip applications.” Cambridge Antibody Technology and Dyax, among other companies, create large libraries of antibodies that can be applied to protein arrays. Nonetheless, creating new antibodies takes a fair amount of time and labor.

Instead of using antibodies themselves as the hook, Affibody AB created an antibody mimic called an Affibody molecule. Feldwisch says, “We have chosen one of the five antibody binding domains of Staphyloccocus aureus protein A as the scaffold for making Affibody molecules.” Three helical bundles make up this scaffold, which includes just 58 amino acids. At present, the scaffold can be mutated in more than a dozen spots to create libraries of more than three billion different Affibody molecules. Birger Jansson, director of research at Affibody, adds, “We can easily express Affibody molecules in E. coli and produce them inexpensively.”

Affibody makes these capture agents available to customers who want to make protein microarrays. Feldwisch says, “Our current strategy is to be an affinity ligand supplier. We deliver the content that another company could use to make chips or use in drug discovery.” A customer can send a protein or even a primary sequence of amino acids, and Jansson and his colleagues will prepare Affibody molecules in return.

SomaLogic also uses a unique capture molecule called a photoaptamer. An aptamer is just a single strand of nucleic acids, which can bind with target molecules. At SomaLogic, scientists replace some of the thymine nucleotides with 5-bromo-2-deoxyuridine (BrdU). Then, ultraviolet light causes the aptamer to bond covalently with its target protein. These photoaptamers generate high specificity because the bond develops between a BrdU at a specific location in the capture molecule and a specific amino acid in the target protein.

Getting Attached
No matter what kind of molecule serves as a protein microarray’s hook, it must be attached to the microarray surface. “This isn’t a new problem,” says Vincent Smith, group leader at NextGen Sciences, “because the issue of attaching antibodies or capture molecules to surfaces has been a fundamental part of immunoassay technology for many years.” For investigators who want to make their own arrays, Eppendorf, MicroSurfaces, Miragene, TeleChem International, and other companies offer treated glass slides that allow attachment of proteins. For instance, PerkinElmer’s HydroGel coated slide creates a three-dimensional surface that was designed to keep proteins active during a variety of assays, including protein-protein interaction studies, protein profiling, and sandwich assays.

Creating uniformity in the attachment of capture molecules is a crucial goal in making protein microarrays, according to Smith. “But getting uniform attachment with different molecules is difficult,” he says. To enhance uniformity, scientists at NextGen Sciences use a BioTag, which is a sequence of 15 amino acids that connects a protein to a microarray. “This peptide is quite small,” says Smith, “and it can be attached to any protein sequence by PCR, standard cloning techniques, and recombinant protein expression.” With a BioTag attached to a protein, a biotin molecule gets attached to the tag. That makes the protein-tag combination ready for spotting on a streptavidin-coated slide. “Once you bind this tag to the surface, it is hard to remove,” says Smith, “because the biotin-streptavidin bond is so strong.”

By attaching a BioTag to a specific point on a protein, say, the C-terminus, this system controls the orientation of attachment. In other words, a capture protein’s binding site can be oriented precisely on a microarray. In addition, Smith points out that attachment via the BioTag should keep the protein itself away from the array surface and in solution. This gives the protein a good chance of retaining full activity. Currently, NextGen Sciences’ protein microarrays remain in development. Nonetheless, Smith says, “We are creating chips for antibody and protein arrays that will be useful in studying breast cancer.”

Automating the Arraying
Getting a sample to a microarray often involves liquid handling steps. To complete these steps, scientists use products that range from simple hand-held devices, such as a pipette, to automated work stations and robotic systems that transfer small sample volumes or even spot proteins on glass slides. For automated work stations, scientists can turn to a variety of companies, including Applied Biosystems, Qiagen, and Tecan.

To make a complete protein microarray, a researcher would require a substantial collection of equipment. Most important, one product must work well with the next to create uniform and effective chips. To make things easier, some chip-building scientists prefer an integrated system—all from a single vendor—that was designed to make protein microarrays. Companies that make complete systems for in-house chip fabrication include Genetix, PerkinElmer, and TeleChem International.

At TeleChem, for example, scientists developed a micro microarrayer called the SpotBot, which prints samples from 384-well plates into microarrays. Todd Martinsky, founder and executive vice president at TeleChem International, says, “The Stealth Micro Spotting Device, equipped on most microarrayers, is the most widely used microarray technology in the world.” He adds, “We are like the Intel of the microarray robot world, with more than 2,500 installations around the world.” This device can put down hundreds of spots using only 0.25 microliters of sample. The SpotBot’s record is 50,400 spots on one chip. Nonetheless, Martinsky says, “You are realistically looking at a few thousand spots, because of time factors.”

For people who want to make more microarrays and in less time, TeleChem created its NanoPrint. Martinsky calls this a high throughput microarrayer, which can print onto 210 slides in one batch. As Martinsky explains, “The NanoPrint uses linear servo motors that run on magnets so there are no moving parts to break, cause inaccuracies, or generate heat. This makes the system more reliable and accurate than traditional systems, and at the same time makes it easier to keep your proteins cold while they are being printed into microarrays.”

Martinsky expects a productive future for protein chips. “We’ve been hearing a lot about the difficulty of making protein microarrays,” he says, “but DNA was hard too. It was really hard.” He adds, “Protein microarrays are difficult, but all of the technical hurdles are being overcome.” In fact, he points out that TeleChem and an autoimmune diagnostic company are working on diagnostic chips that should hit the market next year and premade antibody microarrays due out by summer.

Other companies also expect high demand for systems that make complete protein microarrays. PerkinElmer’s ProteinArray Workstation, for instance, makes microarrays from small volumes of proteins. In addition, this system can make microarrays on standard glass slides or chips treated with PerkinElmer’s HydroGel.

Ready-To-Use Arrays
Instead of going to the trouble of making protein microarrays, lots of scientists would rather purchase them, all ready to go. A variety of companies—including BD Biosciences Clontech, RayBiotech, and Zyomyx— already make chips that can hunt down cytokines, identify the proteins in a sample, and even determine if a protein has been activated by phosphorylation.

For instance, Peter Wagner, chief technology officer at Zyomyx, says, “We ship fully developed, implemented systems consisting of antibody-loaded chips together with all necessary reagents and instrumentation to customers. That is our theme.” This product platform is called the Zyomyx Protein Profiling Biochip System. Wagner says that it’s extremely easy to use and provides the highest quality in multiplexed expression profiling analysis. He says, “You just take an antibody-loaded chip from a bag, put it in the machine, and add your samples in microtiter plate formats.” The chip processing work station can run 12 chips at a time in a fully automated fashion. “A researcher can collect 14,000 data points in just a few hours,” Wagner says.

Last year, Zyomyx released a cytokine-chemokine chip that provides 180 microassays of 30 different proteins simultaneously in full multiplexed mode. “That chip applies to many therapeutic areas,” Wagner says. “In any inflammatory process or complex disease, those proteins are key targets.” Zyomyx also released a mouse chip, and Wagner promises a sequence of upcoming launches of additional microarrays.

From BD Biosciences Clontech, investigators can purchase the Ab Microarray. It contains 500 monoclonal antibodies on a glass surface. This chip can screen for proteins that correlate with a physiological or pathological process.

Instead of catching proteins with antibodies, Ciphergen Biosystems is known for using chromatographic surfaces to capture unknown proteins from crude samples. William E. Rich, president of Ciphergen Biosystems, says, “I think the future of biochips is very bright, because by miniaturizing the separation processes of biochips, you reduce the cost of chips and reagents and increase speed. All of the benefits come into play.” To get those benefits, Ciphergen did start by using antibodies as the capture molecules. “But the capacity of the chips was too low,” Rich says. “The nonspecific binding was a problem, too.” So, scientists at Ciphergen captured proteins on chromatographic surfaces and then detected them with surface enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF-MS).

Recently, Ciphergen also announced a new product line that uses antibodies, proteins, and other biological molecules as capture agents. “These chips can look at signal transduction pathways, different expression levels, and protein-protein interactions and have the full benefits of SELDI-TOF-MS detection,” Rich says.

Aiming at Interactions
Some companies—including Biacore and Jerini—make microarrays that are designed to specifically study interactions between proteins or peptides. “In drug discovery and development and in identifying potential drug targets,” says Stefan Löfås, vice president and chief scientific officer at Biacore, “we need to know the signaling pathways, and these are the protein-protein interactions.” He adds that such knowledge can tell scientists how drugs might interact with cellular functions and, perhaps, even expose potentially toxic side effects.

Biacore’s scientists developed a chemical coating for microarrays that gives customers a variety of ways to attach proteins. Löfås says, “The surfaces of our chips keep proteins active and functional for looking at protein-protein interactions or interactions with other molecules.” Then, Biacore’s instruments use an optical technology called surface plasmon resonance (SPR) to measure changes in the refractive index near the surface, which correlates directly with changes in mass. This technology uses no labeling, yet it can measure binding or detachment events in real time. “This provides kinetic information,” Löfås says. “You get more detailed information about the interactions.”

Löfås finds unique benefits from SPR. First, he likes the lack of labeling. “With SPR,” he says, “you can actually get detailed information without changing the structure of the captured molecule.” Second, he points out the value of real-time measurements. “All traditional methods look for endpoint measurements,” he says, “but with SPR we can get information about binding before, after, and in between.”

Interactions also take center stage in protein and peptide microarrays from Jerini. It’s no wonder given that Mike Schutkowski, director of Jerini peptide technologies, says, “The majority of all the events in life are triggered by protein-protein interactions.” Jerini’s PepSpots, for example, maps protein interactions. In addition, Jerini makes chips that examine enzymatic activities of proteases and kinases, and these are called PepStar arrays and PhosphoSite-Detector arrays, respectively. Schutkowski points out similarities between the benefits of studying protease activity and phosphorylation. He says, “Proteases and kinases belong to the largest gene families, and they are involved in dysfunction. Too much activity causes defined illnesses, and if you inhibit these enzymes you can expect an effect, too.” To follow all of this activity, Jerini’s PepStar microarray, for instance, includes 2,300 different human sequences in triplicates on a single slide. “This provides a tool to search for the natural substrates of target kinases in just an hour or two,” Schutkowski says.

As mentioned at the start, protein microarrays remain quite young. As a result, scientists still face many challenges in this technology—from improving the efficiency and specificity of capture molecules to higher throughput ways to analyze the results. All of that work, however, could lead basic researchers and pharmaceutical scientists to new and useful knowledge, especially as they learn more about signaling pathways that are controlled by proteins.

Mike May (mikemay@mindspring.com) is a freelance writer and editor based in Madison, Indiana, U.S.A. Gary Heebner (gheebner@cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

WEBLINKS ADVERTISERS Affymetrix DNA microarrays, based on the principles of semiconductor technology 408-731-5000 www.affymetrix.com Ciphergen Biosystems, Inc. protein microarray systems and related products that discover, characterize and assay proteins from native biological samples 510-505-2100 www.ciphergen.com Genetix [UK] laboratory automation products and services for microarrays and sequencing +44 1425 624 600 www.genetix.com Genetix [USA] 877-436-3849 Genomic Solutions DNA microarrays, fabrication instruments 734-975-4800 www.genomicsolutions.com Leica [Germany] instruments and systems for imaging analysis, digital cameras +49 6441 29-0 www.leica-microsystems.com Leica [USA] 847-405-0123 MWG [Germany] products and services for genomic research including microarrays, oligonucleotides, sequencing, and laboratory automation +49 8092 8289-0 www.the-mwg.com MWG [USA] 336-812-9995 Phalanx Biotech Group DNA microarrays for gene expression 650-988-6898 www.phalanxbiotech.com Takara Bio, Inc. kits and reagents for molecular biology research +81 77 543 9254 www.takara-bio.co.jp/english FEATURED COMPANIES Affibody protein microarrays based on antibody mimics www.affibody.com Applied Biosystems automated work stations www.appliedbiosystems.co BD Biosciences Clontech protein microarrays www.clontech.com Biacore International AB systems to study molecular binding www.biacore.com Cambridge Antibody Technology libraries of antibodies www.cambridgeantibody.com Ciphergen Biosystems, Inc. instruments and arrays for proteomics research www.ciphergen.com Dyax Corporation libraries of antibodies www.dyax.com Eppendorf AG treated glass slides for microarrays www.eppendorf.com Genetix instruments and supplies for array fabrication www.genetix.com Jerini AG peptides and protein arrays www.jerini.com MicroSurfaces, Inc. treated glass slides for microarrays www.proteinslides.com Miragene treated glass slides for microarrays www.miragene.com NextGen Sciences, Ltd. discovery acceleration company www.nextgensciences.com PerkinElmer Life and Analytical Sciences instruments and supplies for array fabrication las.perkinelmer.com Qiagen GmbH automated work stations www.qiagen.com RayBiotech, Inc. cytokine microarrays www.raybiotech.com SomaLogic, Inc. protein capture photoaptamers www.somalogic.com Tecan automated work stations www.tecan.com TeleChem International, Inc. instruments and supplies for array fabrication www.arrayit.com University of Toronto's Samuel Lunenfeld Research Institute nonprofit research institute www.mshri.on.ca Zyomyx, Inc. protein microarrays www.zyomyx.com

Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. The companies and organizations in this article were selected at random. Their inclusion 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 article was published as a special advertising section in the 14 May 2004 issue of Science