by Laura Lane
Protein is a hot topic in more circles than just the life sciences. From its brain-building power to its muscle-boosting might, protein easily draws the attention of anyone interested in maximizing their potential. No matter what crowd you belong to, this diverse and complex group of molecules inspires investigation into how each affects health and well-being. Accomplishing that is much easier said than done. Fortunately, protein microarrays
have arrived. These high-throughput devices provide the type of technology required to fully understand all the multi-faceted and wide-ranging spectrum of properties.
With the relatively recent appreciation of the varied set of proteins that arise from a single gene, protein microarrays allow researchers to make the most of every minute at the bench. With hundreds to thousands of proteins – either enzymes, antibodies, peptides
or other types – on a single platform, microarrays enable the characterization of multiple proteins within one assay. Many protocols mirror the universal enzyme-linked immunoabsorbent assay (ELISA). Some use ELISA variations or other new approaches that harness the binding specificity of antibodies.
All strategies encompass efforts to minimize background noise while optimizing the signal of binding events. The challenge involves capturing proteins at concentrations as low as nanograms per milliliter among proteins measured in grams per milliliter. For serum, “protein abundance can change by as much as 10,000-fold on stimulation,” according to a paper published in the journal Nature Reviews Drug Discovery1. Companies have attempted to resolve this problem with a number of innovations that optimize both binding specificity and sensitivity.
The fragile nature of proteins also requires the preservation of their precise native structures while handling and planting them on to solid substrates. Thus, developing the array involves selecting a platform that can “retain their functionality and stability after spotting since proteins are sensitive to the physical and chemical properties of the support,” according to a paper published in a recent issue of the journal The International Journal of Biochemistry and Cell Biology”2.
You may be able to avoid the problem by using peptide arrays. With peptides of up to 12 amino acid residues, these arrays allow you to study the exact epitopes involved in binding. “We’re not putting entire proteins down,” says Chris Hebel, director of business development at LC Sciences, a custom service company that both produces and processes the peptide arrays. “We’re just looking at that primary structure binding.”
However, with less than a year under their belt, peptide arrays have yet to find a specific application. “We envision some creative scientist out there will figure it out.” Hebel says.
For that reason, protein and antibody arrays dominate the market, which has developed various means to surmount the challenge of maintaining structural integrity. In general, you’ll find arrays with either two-dimensional or three-dimensional foundations. Two-dimensional supports are known to keep the cost of arrays low and provide a low fluorescent background. However, fixing proteins involves covalent binding that can possibly alter the structure and binding activity. On the other hand, these proteins usually attach to the support in an orientation that exposes the active site required to bind to the target proteins. With three-dimensional supports, proteins tend to attach in random orientations. But these proteins retain their natural binding activity in the gel-like substances that circumvent binding by simply entrapping the proteins, which remain intact with the nurturing, aqueous environment.
Nitrocellulose membranes
have become especially popular for affixing proteins. Used for decades in blotting protocols, nitrocellulose has proven its prowess for keeping proteins “very happy,” says Alex Vodenlich, president and chief executive officer of GenTel Biosciences. But in its traditional form, nitrocellulose is too thick and porous for the high degree of sensitivity required for arrays.
“So we shrank it from 15 microns to a nanometer-thin film that still binds proteins but removes the problem of high background interference,” Vodenlich says. “It has great characteristics for immunoassays.”
GenTel offers the nitrocellulose film on several formats. The company’s base unit is a one- by three-inch glass slide. To increase efficiency, you can purchase a device that holds four individual slides, for an array of 64, all of which can be simultaneously processed. GenTel also offers a 96-well format with a three- by five-inch plate. Once printed, the nitrocellulose then assembled into the 96-well plastic frame, which can then be scanned like an ELISA plate.
“We call this ‘ELISA on steroids’: Instead of using one antibody per well, per sample, you can measure multiple analytes for each well and sample,” he says.
Both ThermoFisher Scientific and Invitrogen
have signed on to use GenTel’s nitrocellulose. Thermo uses the film to make a 16-well ExcelArray slide, which is printed with a variety of antibodies to simultaneously quantify up to 12 different proteins. Choose from arrays ready for studying proteins involved in inflammation, angiogenesis and chemotaxis.
“ExcelArray combines the ease of working with pre-printed slide arrays with the excellent quantitation and sensitivity of ELISAs,” says Mitch Gaver, director of cell pathways marketing at Thermo Fisher Scientific. “This system will not only show if a biomarker is present, but also if it increases or decreases in specific diseases or conditions.”
Invitrogen uses the film on one- by three-inch slides for its ProtoArray line of human protein arrays. The company’s latest array features 8,000 full-length proteins.
“The development is driven by the voice of our customers,” says Jennifer Cannon, business area manger of protein analysis at Invitrogen. “Some customers say they want more of the proteome [all within one array]. That was certainly the case when we had the 1,800-, 3,000- and 5,000-protein arrays. But, now with the 8,000-protein array, we don’t have researchers saying they want more.”
Perhaps equally important to obtaining the global view of a high density array is focusing on a smaller group of relevant proteins. For example, researchers studying a specific pathway may encounter difficulties with a high density array. “You may have cross-reactivity of the small molecules and get conflicting data,” Cannon says. “More is not better in this case.”
Many researchers, however, remain at the discovery phase for which high density arrays provide the hypothesis-developing information. “We’re now at the stage of looking big,” says Dorit Zharhary, director of research and development at Sigma-Aldrich
Israel. “Scientists are trying to find which proteins are relevant for diagnosis and prognosis that may be used as disease biomarkers.”.”
Zharhary points to the popularity of the Sigma-Aldrich’s larger arrays over the smaller ones. “There is a demand for the larger arrays,” she says. “This made us realize that people aren’t so much looking for a needle in the haystack as they are looking for something they’re not already thinking of.”
Aiding this search are reverse phase arrays in which whole cell lysates are printed as an array, which can then be probed for the presence of expressed proteins. “Rather than each spot containing a discrete protein, lysed cells are printed on the surface instead,” says Duncan Hall, commercial director at Arrayjet Limited, which offers an entire line of inkjet microarrayers suitable for printing cell lysates. “This application of protein microarray is increasing in popularity.”
Eventually, efforts to plumb the depths of the proteome will lead to the resolution of specific patterns of proteins that correlate with specific diseases and conditions. Unlike the single-analyte diagnostic tests that provided information on the presence and abundance of individual proteins, protein microarrays place those proteins in the context of networks of “interacting, modifying, antagonistic and synergistic proteins…[that] are much more likely to be descriptive of biological processes”1.
References:
1 Kingsmore, SF. “Multiplexed Protein Measurements: Technologies and Applications of Protein and Antibody Arrays,” Nature Reviews Drug Discovery, 5(4):310-320, April 2006.
2Kopf E and Zharhary D, “Antibody Arrays – An Emerging Tool in Cancer Proteomics,” The International Journal of Biochemistry and Cell Biology, 39:1305-1317, 2007.
