Protein microarray technology, the process of immobilizing multiple proteins on to a single solid support and using miniaturized assays to better understand protein interactions, has seen steady growth in recent years. Facilitating the study of protein synergy with antibodies, other proteins, drugs, DNA, lipids, or peptides, and with considerable utility in identifying post-translational modifications (PTMs), protein microarrays are driving proteomics research. With their simple, high-content format, these powerful tools represent a cost-effective solution to rapidly generate vast quantities of robust and reproducible data. They also offer exceptional green credentials in comparison with lower throughput techniques such as ELISA.
More informative than mass spec
“Mass spectrometry (MS) is the most powerful technique on the planet for identifying minute amounts of known protein, but for making novel discovery, Snapshot Proteomics™ protein microarrays are far more informative,” says Christian Loch, chief executive and science officer at AVMBioMed. Describing how this technology is based on an assay originally developed by Marc Kirschner at Harvard, Loch explains that microarrays containing approximately 23,000 individual human proteins—a far larger number of proteins than has ever been identified in any MS-based experiment—are used to capture PTMs from complex mixtures like cell extract or serum.
“Many consumers assume that proteomics and mass spectrometry are synonymous,” he says. “However MS readouts are, strictly speaking, genomic, since they mostly ignore the effects of splice variation and PTM, instead using peptides to inform which of approximately 20,000 human genes were expressed in the sample. Splice variation creates 100,000 unique transcripts from these 20,000 genes, while PTM creates 20 million unique proteins from the transcripts. Snapshot Proteomics informs these key processes, allowing researchers to ascertain exactly which proteins were made. This information is captured by examining activities and interactions of expressed genes across 23,000 substrates. Snapshot Proteomics therefore expands the data pool 1000-fold, providing a detailed intracellular image that is based on substrate-specific alterations.”
Data is extracted from Snapshot Proteomics arrays in a method analogous to a Western blot, with improved sensitivity afforded by enzymatic amplification. “More traditional methods of studying PTM, such as immunoprecipitation with PTM-specific antibodies followed by tandem MS (IP-MS/MS), involve extensive manipulation and labor under poor reproducibility,” says Loch. “While top-down approaches preserve PTM by avoiding trypsinization, they inform considerably fewer (~1200) proteins.”
A technology offering many benefits
“A major advantage of protein microarrays is that they are highly sensitive to targets at a wide range of concentrations,” notes Valerie Jones, Ph.D., director of sales and marketing at RayBiotech. “This contrasts with in vivo studies and mass spectrometry, where abundant proteins may mask the analyses of less abundant targets.” Loch adds that because the most informative proteins (e.g. enzymes and transcription factors) are often low abundance, this represents a significant advantage. “A further benefit of protein microarrays is that they require less sample than antibody microarrays,” says Jones.
Also keen to stress the many advantages of protein microarrays is Daniel J. Schwartz, director of business development at Grace Bio-Labs. “When it comes to simplicity, protein microarrays have clear benefits over antibody sandwich microarrays,” he notes. “These are typically straightforward and elegant assays, requiring little optimization or detection reagent investment, and they are also extremely robust, providing reproducible data under a variety of conditions and using a range of sample types. This facilitates very high-density arrays, yielding large quantities of data for minimal investment. Furthermore, a single protein microarray plate can provide the same depth and accuracy of data as hundreds of standard 96-well ELISA plates, saving huge quantities of time and plastic waste.”
One-Minute Read
- Protein microarrays are highly sensitive to targets at a wide range of concentrations.
- Protein microarrays require less sample than antibody microarrays.
- When it comes to simplicity, protein microarrays have clear benefits over antibody sandwich microarrays.
- Customization makes protein microarrays well-suited to serve a wide variety of research interests.
- Protein microarrays are increasingly being used for allergy screening and vaccine development.
Easily customizable
Providing ready-made protein microarrays related to allergies, cancer, and autoimmune diseases, RayBiotech also offers custom-made arrays based on their high-quality protein library. “We specialize in producing recombinant proteins in E. coli and HEK293 cells,” says Jones, “and we’re continually increasing our protein library to accommodate the diverse needs of our customers. This is especially pertinent to protein microarray technology since many researchers prefer to use customized arrays based on their project’s objectives.”
Jones points out that this applicability to customization makes protein microarrays well-suited to serve a wide variety of research interests. “Protein microarrays have successfully been used as high-throughput kinase assays to characterize drug specificity,” she says. “Additionally, the application of protein microarrays for autoantibody profiling is often exploited to help characterize autoimmune diseases and certain cancers.” Further utilities include serological assays measuring immune responses against a large number of antigens in allergy screening; cancer biomarker discovery or validation by probing high densities of known disease state tissue lysates using specific antibodies or cell types; and epitope mapping for antibody development. “With the emergence of large molecule therapeutics and with heightened concerns about experimental reproducibility, we’re seeing increased work to profile antibody selectivity across the 23,000 immobilized prey proteins,” Loch adds.
Image: Protein microarrays have many different applications. Image courtesy of RayBiotech.
Optimized protein microarray substrates
“Our protein microarray surface chemistry is based on the well-known protein-binding properties of nitrocellulose,” says Schwartz. “Included within our portfolio are non-porous (PATH®) and porous (AVID, NOVA, and SuperNOVA) protein substrates, all of which provide preservation of native protein conformation, high signal-to-noise ratio, long-term array stability and compatibility with virtually all detection modalities. In fact Grace Bio-Labs PATH slides are used as the substrate for the highest density commercial microarray on the market, the HuProt Array from CDI Laboratories. To increase workflow throughputs and reproducibility, we also offer array well chamber devices suitable for use with automated liquid handling or manual applications.”
Elaborating that Grace Bio-Labs provides custom arraying services with piezo-electric non-contact arraying or solid pin instrumentation, Schwartz reports that microarrays produced using the company’s substrates can be analyzed using commercially available instrumentation. “Confocal scanners such as those offered by Molecular Devices, Innopsys, Agilent, and Tecan are compatible with our substrates for researchers detecting with fluorescence,” he adds. “Grace Bio-Labs substrates can also be imaged with near-IR wavelength detection modalities on scanners like those offered by LI-COR, or even using standard CCD acquisition devices such as cellphones for colorimetric applications.”
Schwartz also notes that it is possible, using different wavelength fluorescent reporters, to detect multiple reactants to each immobilized protein on the arrays. “For example, with immunogenicity testing, several antibody isotypes from a single serum sample can be simultaneously measured for reactivity against the same analyte using isotype-specific detection antibodies labeled with different fluorophores. This further increases the already considerable amount of data that can be gained from a single experiment.”
An increasingly popular technology
General consensus indicates a growing demand for protein microarrays, not only among the research community but in point of care and clinical research as well. “We’ve seen a steady rise in peer-reviewed publications since 2001,” says Schwartz, “with two specific areas of increased activity being allergy screening and vaccine development. Interestingly, these two applications exploit different advantages of the technology. While allergy screening benefits from huge economic savings in multiplexed isotype detection on modest-sized arrays, vaccine development takes advantage of extremely high-density arrays.”
As awareness of protein microarray technology continues to grow, and specialist providers advance its capacity into complementary technologies such as MS, MALDI-TOF and lateral flow, this powerful technique looks set to become a leader in proteomic studies.