Genomics & Proteomics

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Product Release

Capturing Proteins Using Antibody Arrays

Antibody microarrays are enabling researchers to perform high-throughput proteomic analyses

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Schematic view of the procedure to screen protein-protein interactions using antibodyArray. (Source: Hypromatrix)
Experts disagree on the exact number of meaningful genes in the human genome, but proteins clearly far outnumber genes, perhaps by a factor of ten or twenty, especially after considering post-translational modifications. When all the possibilities are tallied, distinct proteins number between half a million and one million.

As the proteomic equivalents of gene microarrays, antibody microarrays enable parallel, multiplexed, miniaturized, high-throughput proteomic analysis that includes individual protein detection, protein expression profiling, and interactions between proteins and drugs, ligands, antibodies, or other proteins.

Antibody microarrays use immobilized antibodies affixed to glass, membranes, microplate wells, mass spectrometer plates, silicon, plastic, or other surfaces to capture less abundant proteins of interest from complex biological fluids and tissues. Chip substrates depend on the desired detection and antibody immobilization methods. In the early days of peptide microarrays, developers used silicon chips because flat, high-quality silicon was less expensive than glass with similar properties. Hypromatrix Inc., Worcester, Mass., has tried several different substrates but settled on nitrocellulose membranes and glass substrates coated with nitrocellulose membranes for antibody immobilization. According to company president John Hou, nitrocellulose coupled with proprietary chemistry keeps antibodies in their active state and makes spots easier to detect.

Complementing the Binding Capacity
With only about 500 antibodies commercially available, covering the entire human proteome with antibody microarrays won't happen any time soon. Still, companies continue to chip away at the proteome with innovative antibody-generating technologies. Epitomics Inc., South San Francisco, has developed a suite of antibody-generating methods. TargetMAb generates rabbit monoclonal antibodies (MAbs) for proteins of all classes directly from DNA plasmids. MultiMab multiplexes immunization of many antigens to create antibodies several at a time. Through its PathoMAb technology, Epitomics immunizes rabbits directly with disease tissues or cells, generating thousands of MAbs against both cell surface molecules and intracellular targets. Using a subtractive method to compare antibodies generated in this manner from those in normal tissues, scientists identify disease-specific antibodies rapidly.

Some companies have found a way to complement the binding capacity of antibodies, or to eliminate antibodies from their protein recognition chips altogether.

The ProteinChip Biology system, from Ciphergen Biosystems Inc., Fremont, Calif., consists of instrumentation, ProteinChip Arrays, and software for data collection and analysis, including comparison of the presence and abundance of proteins in crude samples. Like other protein analysis chips, ProteinChip arrays can probe individual proteins, DNA-protein interactions, and protein-protein associations. Ciphergen also has a ProteinChip interface for its QStar instruments, allowing data collection in single MS and MS-MS mode directly from the ProteinChip arrays.

By incorporating built-in chromatography chemistries, ProteinChip does not rely solely on antibody-protein or other high-affinity interactions. For example, Ciphergen offers arrays containing weak cation exchange, strong anion exchange, metal affinity capture, reverse phase, hydrophobic interaction, normal-phase silicate, and inert gold surfaces.

Aspira Biosystems Inc., South San Francisco, Calif., eliminates the "antibody limitation" by creating all the molecular recognition surfaces it needs through a type of molecular imprinting technology known as ProteinPrint. Aspira first creates a peptide corresponding to a signature sequence in the target protein. Next, polymerizable monomers self-assemble around the peptide and are cross-linked in place. The peptide is then removed, leaving behind an artificial molecular recognition pocket that is complementary to the peptide in both shape and chemical functionality. When the polymer is exposed to a sample of denatured protein, target proteins bind to the recognition pockets. Washing, staining, and detection are carried out as with traditional antibody detection systems.

ProteinPrint offers several advantages over traditional antibodies. Scientists can create recognition sites without knowing the full target sequence, without purchasing or creating antibodies. Typically, Aspira targets unique COOH-terminal seven-peptide sequences for denatured proteins, or an internal sequence for active proteins. On the minus side, the targeted region must somehow be accessible to the recognition site, whereas natural antibodies take care of that detail. Whereas molecular imprinting is faster and less expensive than raising and purifying antibodies, it does take time.

SomaLogic, Boulder, Colo., uses photoaptamers—modified, single-stranded DNA or RNA which, like antibodies, bind proteins with the affinity and specificity normally associated with antibodies. Photoaptamers differ from conventional aptamers in that thymidine is substituted with brominated deoxyuridine, which gives the molecules the additional capability of cross-linking to target proteins under the influence of ultraviolet light, hence, the "photo" in "photoaptamer." Like antibodies, photoaptamers can be fabricated into arrays and used for protein capture.
Applications: wherever you find proteins
Medical diagnostics are an obvious commercial opportunity for antibody microarrays, but to be useful in this context, chips are limited to a few key analytes and must incorporate built-in sample preparation and readout. For example, the Triage Cardiac microarray from Biosite Diagnostics Inc., San Diego, which is used in hospitals and other emergency medical settings, tests for just three cardiac markers. Biosite also is working on an antibody-based stroke diagnostic microarray that will test five stroke-related protein markers.

A more esoteric, albeit further-off medical application, termed "personal proteomics," is more in line with the sophistication that developers envision for antibody microarrays. Like personal genomics or personal medicine, personal proteomics seeks to sub-define an illness according to specific molecular characteristics—in this case, protein expression. For example personal genomics seeks to differentiate diagnostically similar tumors according to the genes expressed by those tissues. The idea is that genomically distinct tumors may be associated with radically different prognoses and require different therapies. Personal proteomics takes this idea a step further: identical-looking tumors exhibiting similar genomics are differentiated on the basis of their protein expression profiles.

The other significant application of antibody arrays is research proteomics, which stresses research-based protein function analysis, determination of enzyme activity, antibody cross-reactivity studies, selection of proteins from phage or ribosome display libraries, and epitope mapping.

Hypromatrix's AntibodyArray chip is used to study protein-protein interactions, post-translational modifications, and protein expression patterns. "Our chips are available for less than $1,000, take 6 hours to process, and give many fewer false positives because they don't create interactions, they just detect them," says Hou.

Hypromatrix offers three antibody arrays. Its 400-antibody signal transduction array, says Hou, "covers a lot of ground and can be used as a starting point for protein expression analysis." Hypromatrix also makes an apoptosis array containing 150 antibodies, and a cell-cycle array with 60 antibodies. Custom arrays using any combination of the 400 well-characterized antibodies are also available.

According to Dev Baines, of Prometic Life Sciences, Montreal, Quebec, Canada, today's antibody microarrays are too specific to be used to mine all proteins of interest in a sample. That is why Prometic focuses on group-specific chemical affinity ligands, rather than antibodies, for protein capture. High-abundance proteins such as albumin are first removed chromatographically. Then low-abundance proteins of interest are captured by Prometic's synthetic chemical ligands, which are less specific than antibodies but have the potential for picking out many more diverse proteins than any collection of antibodies. "Instead of focusing on active site or docking mechanisms, our affinity technology targets the 500 or so unique folds that occur in almost every protein. Essentially what we're doing is to scale down what we've done successfully with chromatography columns to array-sized devices, which allows parallel processing. Proteins collected in this fashion are then subjected to 2D analysis."

(above) Scanning electron micrograph of lower part of microarray device (ultrasonic welding of a clear upper plastic part creates a capillary space that contains the sample fluid) illustrating the reaction chamber where plasma contacts labeled antibody reagents. (Source: Biosite)

(above) The Zyomyx Human Cytokine Biochip, with 30 biologically relevant cytokines, is the first in a series of protein profiling biochips designed by Zyomyx to facilitate highly sensitive and comprehensive, multiplex protein expression profiling using minimal sample amounts.
Although clinical proteomics is the ultimate goal of many antibody microarray efforts, not everyone believes chips are the answer to devilishly complex proteomic puzzles. "Clinical proteomics is strictly comparative," says Lisa Bradbury, senior scientist at Ciphergen Biosystems Inc., Fremont, Calif., who contrasts chip-based protein profiling with more conventional 2D proteomics. "If you use an antibody array to profile late-stage versus early-stage colon cancer, you may miss important proteins because you can't cover everything. Proteomic methods are unbiased because they are not limited by selected antibodies. The end point is to define distinct populations through differential protein expression. Those differences don't necessarily need to tie in with disease mechanisms, but they must at least be signature differences."

In drug development, protein chips will benefit companies looking for subsets of "responders" for clinical trials. After commercialization of proteomic-based discoveries, antibody chips will help determine which patients will benefit from those drug therapies. In every case, treatment options are limited by what is available. "This revolution isn't going to happen overnight," says Larry Cohen, CEO of Zyomyx Inc., Hayward, Calif., "but in the long term, it's quite possible."

Zyomyx' claim to fame is its activity-retaining protein immobilization technologies, which combine nanotechnology and biochemistry. In February 2003, the company launched its Protein Profiling Biochip system and its first protein biochip product, the Zyomyx Human Cytokine Biochip containing 30 cytokines."

All arrays are not created equal
Genomics has progressed much more rapidly than proteomics because complementary gene-gene interactions are more predictable and easier to bring about than protein-protein interactions.

Protein function, including affinity for other proteins, depends on the molecule's primary, secondary, and tertiary structure; for genes, primary structure is usually enough to retain activity. Although gene hybridization interactions are strong and specific, antibody-protein interactions vary greatly and suffer from unpredictable cross-reactivity. Antibodies are difficult to make and are available for only a small number of proteins, whereas automated oligonucleotide synthesizers can create gene probes by the dozens. Finally, genes are easily amplified using polymerase chain reaction (PCR) or other techniques, but no such method exists for proteins.

Reproducible, reliable protein immobilization was worked out during microarrays' "peptide days" through processes that are now quite familiar: attach a spacer molecule to the substrate and add the antibody of interest via a covalent sulfhydril, amine, or hydroxyl group linkage. Antibody arrays still require robotic manufacture if high reproducibility is desired. Typical spot creation methods include contact printing, piezoelectric spotting, and photolithography.

Even though they employ standard immobilization and spacer chemistry, antibody arrays are much more difficult to fabricate and operate than gene chips, says Bruce Kimmel, director of protein therapeutics at Diversa Corp., San Diego. "Regardless of the platform, you need to be able to do a sandwich assay, which requires a capture agent and detection reagents. Calibrating such a system is extraordinarily difficult when you have hundreds of different capture events occurring on the same chip. Binding reagents need to work in the same buffers and have the same washing parameters."

Unlike DNA affixed to microarrays, currently there is no antibody collection that represents the entire proteome. So which antibodies go into arrays? "Choosing them is part science, part art," says Hou of Hypromatrix. "At the very least, you want proteins that give you the best opportunity to capture what you're looking for. One approach to that is to use arrays of well-studied proteins related to whatever your experiment is trying to show. And of course, you can't use an antibody that doesn't exist or which is not available at the right purity."

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(Source: Ciphergen Biosystems)
Genes and proteins: comparing apples and oranges
Comparing antibody arrays to DNA arrays is an "apples and oranges" proposition. Gene chips are able to measure transcriptional mRNA profiles in multiplexed, parallel fashion because DNA is homogeneous and gene probes are easily synthesized or purchased in several useful formats. "You could say that no great technological breakthroughs were required for gene arrays to progress to where they are today," says Gunars Valkirs, vice president of discovery at Biosite Diagnostics Inc. "The same is not true for antibody arrays. Antibodies exist for only a small fraction of the proteome. Even if 10,000 antibodies were available, the cost of producing a 10,000- protein array would be prohibitive."

Density is another huge digression point between DNA and antibody chips, says Valkirs. "With current technology, antibody arrays can't be multiplexed effectively beyond a certain point, say 100 assays per chip, because of serious signal-to-noise issues. It's very easy to optimize one assay on a chip, or even 10. For 100 assays, however, you need to add labeled antibodies for each one of those tests, which means at least 100 times the noise. That wouldn't be so bad except you may still only be looking for a couple of proteins that require high sensitivity, in which case the signal-to-noise [ratio] becomes unacceptable."

Valkirs also noted that maintaining interassay and interchip precision and specificity is difficult with antibody arrays. "Unless something remarkable happens on the detection side, specifically label-free detection that does not require a second labeled antibody, antibody arrays will be limited in the number of simultaneous assays they can provide."

Inch wide, mile deep
Because proteins outnumber genes by such a large margin, and with relatively few readily available antibodies, proteomics by necessity covers a much narrower slice of its domain than does genomics. However, it also cuts a lot deeper, says Larry Cohen of Zyomyx. "You really only need to make two measurements to know what's going on with a gene: its sequence and when it generates its messenger RNA. Those measurements are not trivial, but that's basically all you need. It's not that simple with proteins. Because proteins are responsible for how cells function, you'd like to measure their structures, interactions, and expression levels, preferably in highly parallel fashion."

Although antibody microarrays only investigate a tiny fraction of the proteome, the information they give is still valuable. "DNA arrays were on the market long before we knew the entire genome sequence," says Cohen, "but they were still exciting and worthwhile tools. Although protein arrays only investigate a subset of the proteome, they remain highly valuable for a variety of applications."

The question, "Why use protein arrays?" is related to the deeper question, "Why proteomics?" The point of medical genomics is to discover the immutable biochemical factors governing disease. Ultimately, however, scientists come around again to proteins because those are the agents responsible for what goes on in the cell. Not coincidentally, proteins are also drug discovery's principal target. "You simply can't get protein concentrations, or even confirm a protein's existence, from DNA," says Cohen. "Even mRNA only really infers what's going on by giving some idea of the potential for synthesizing a protein. The only way to be sure of proteomic status is to measure proteins directly."

Angelo DePalma, PhD
DePalma is a freelance writer based in Newton, N.J.

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