Protein microarrays are many things to many people. Like the more mature DNA “chips”, they are often patterns of tiny spots arrayed on a microscope-type slide, used to determine the makeup and amount of protein in a sample in a multiplex fashion. But not always.
Just as there are a wide variety of uniplex protein assays, there are a variety of multiplex protein assays as well. Chips, yes—but they’re also found arrayed on the bottom of microwell plates and as beads in a tube. These, and others, were hot topics at BIT Life Sciences’ recent “PepCon” protein and peptide conference.
Protein chips are typically laid out by using a contact pin spotter or a noncontact microarrayer to immobilize ligands on a substrate. These can be antibodies, aptamers, peptides, or other molecules whose binding affinity is used to query samples. In the case of peptides, arrays can be created from random libraries for biomarker discovery, for example, or from series of overlapping protein sequences for epitope mapping.
For PEPperPRINT’s PEPperCHIP® microarrays, peptides are synthesized directly on a functionalized glass slide with a 20-color laser printer. The activated amino acids, which are embedded in the toner, are released from the solid matrix material upon melting. “The toner matrix liquefies and the activated amino acids can approach amino groups on the surface of the chip,” explained CEO Volker Stadler, Ph.D., “and that way they can couple covalently to form the peptides on the chip.” Up to 20 mers are produced by sequential rounds of heating, washing, and deprotection.
Toner is heated to just above the melting temperature of the amino acids so there is little or no evaporation. “And because the toner matrix has an oily consistency, it doesn’t spread on the slide surface. This is a key feature that allows us to achieve much higher spot densities compared to other technologies.”
Another major advantage of this approach, Dr. Stadler added, is that the combinatorial laser-printing process allows for one-off microarrays to be easily produced. “It’s the same effort to synthesize hundreds of different or hundreds of the same peptide microarrays.” Thus the Heidelberg-based biotech can offer custom microscope slide-sized arrays with about 9,000 peptides on a slide for around c900, or about c0.10 per feature. These high-density arrays allow for low per-feature reagent consumption as well, typically requiring only about 5–10 microliters of sample per chip.
Standard PEPperCHIPs can be processed and read with standard lab equipment. PEPperPRINT also offers larger slide formats with up to 275,000 features.
The company, which has been developing its technology for the past decade, began offering screening services in early 2010. In March of this year it started marketing chips for its customers to use in-house. It is currently focused on serology and antibody analysis—“it’s the most straightforward way to work with peptide chips,” Dr. Stadler said.
The chips can also be used for kinase, acetylase, deacetylase, and other enzymatic analyses, “but we don’t have enough experience in the field to really know that they work as well as for standard applications,” Dr. Stadler confessed. “So we have collaborators testing the chips now” to see how they perform and to elicit feedback on how to improve them for enzymatic assays.
Robert Sack, Ph.D., SUNY College of Optometry professor, uses conventional commercial microarrays in slightly unconventional ways, and in the process has come to some disturbing conclusions about standard protocols.
Specifically, he uses planar arrays and microwell plate arrays, along with techniques such as HPLC and immunoprecipitation, to identify and characterize the chemokines and cytokines in lacrimal fluid.
“There is a tremendous difference in the host defense mechanism between the open and closed eye,” Dr. Sack says. While the eye is closed, there is a fluid layer with no place to go. Bacteria stimulate epithelial cells, which in turn recruit immune cells to digest the microorganisms, the result being high levels of peroxidases, proteases, interferons, interleukins, and TNF alpha. “You have a tissue, the cornea, which is lying right next to it and is extremely vulnerable to any change. Any neovascularization, any scarring, can have a devastating effect. Yet the eyes of a person in a coma can stay closed for years without any major effect.”
Earlier work had shown that protease inhibitors were released from the ocular surface, and Dr. Sack wanted to know how the cytokines were kept in check. In his research, he found that “virtually all the inflammatory cytokines and immune modulating cytokines were in the form of macromolecular inactive complexes.”
Many of these were not detectable using standard microarray protocols, or they gave wildly varying results. Yet under the right conditions, Dr. Sack was able to show that the cytokines were complexed with soluble receptors and another protein that tags it for destruction.
Tears contain various surfactants and other substances with a high affinity for plastic, and thus show a high nonspecific reactivity in many assays. Using the correct blocking agents and “running proper controls, such as spiking samples with different types of tears from different individuals (because the levels of these interfering factors vary enormously from one sample to another),” are only part of the solution.
Because the cytokines are found in complexes, they may not be detectable using the antibodies supplied by the assay manufacturer. “The way I was able to show these complexes was to take standard arrays and substitute antibodies for the complexing proteins in place of the normal secondary antibody,” Dr. Sack explained. Complexes were isolated by molecular sieve HPLC and characterized.