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German Team Develops Method for Making Peptide Chips Faster and Cheaper, They Say

Researchers in Germany have developed a method they claim will drastically reduce time and cost of making peptide microchips.
The team from the German Cancer Research Center and the Kirchhoff Institute for Physics, both in Heidelberg, Germany, had two goals: to reduce the number of coupling cycles to create a peptide microchip, and to increase the density of such a chip.
To create a high-density peptide chip takes 20 coupling cycles “for a complete combinatorial layer, which adds up in time, in consumables, and of course in artificacts of synthesis,” Volker Stadler, from the German Cancer Research Center, and one of the developers of the method, told ProteoMonitor this week. In comparison, the technique developed by Stadler and colleagues results in a high-density chip in one coupling cycle for every peptide mer.
Also, conventional methods for making a peptide microchip with thousands of peptides cost thousands of dollars. The newly developed method would cost significantly less, Stadler said.
“On the one hand, the amino particles as consumables are just employed with very low amounts, and on the other hand the chips can be recycled,” he said. “If you generate chips on a very high number, the costs drop significantly, so the costs will be in the range of cents per peptide.”
In addition, while peptide arrays currently available have a density of 22 peptides per square centimeter, the German team was able to produce a chip with 40,000 peptide spots per square centimeter.
Stadler and his colleagues have created a company, PEPperPRINT, to further develop the technology and commercialize it. They are in the process of seeking venture capital and anticipate their chip will hit the market in about five years.
The team is also designing a next-generation version of the chip with better integrated circuits and a larger active area, and plans to integrate a photodiode beneath each pixel electrode as implemented optical sensors instead of scanners, Stadler said.
Their technique is described in a study in the Dec. 21, 2007, issue of Science.

“Our method should be especially helpful in the field of proteomics because it allows for the translation of whole proteomes into arrays of overlapping peptides.”

While the use of protein chips is on the rise in the proteomics field, peptide chips are still a niche technology and available only from a very small number of companies, including Jerini, headquartered in Berlin, and LC Sciences, based in Houston.
According to Xiaolin Gao, founder of LC Sciences and a professor of biology and biochemistry at the University of Houston, while cost is one factor holding back greater use, the diversity of applications for peptide arrays makes designing a one-size-fits-all peptide chip impractical.
“For DNA studies or for RNA profiling, you can develop really one method to reach hybridization specificity, and that pretty much applies across all different kinds of samples,” said Gao. “Your experimental methods have [greater] uniformity. For peptides there is no universal way [to have] a few protocols cover diverse applications.”
Moreover, “making the chips is a lot more technically challenging,” she said. “Peptide synthesis is a lot more difficult than … oligonucleotide“ synthesis.
Less Coupling, Greater Density
In the Science paper, the authors assembled 20 different kinds of chargeable amino acid particles onto the microchip with the help of electric field patterns from individual pixel electrodes.
Because the solid particle matrix “freezes” the activated amino acid particle, coupling reaction occurred only when a completed layer of all 20 particles is melted simultaneously.
“This releases activated amino acids to diffuse to free amino groups incorporated into the chip’s coating,” resulting in only nine repeated coupling cycles being done for an array of nonameric peptides “with the density only restrained by the size of the particles and pixel electrodes.”
They then synthesized on the surface of the microchip an array of peptides — Tyr-Pro-Tyr-ASP-Val-Pro-Asp-Tyr-Ala, or hemaglutinin, and Asp-Tyr-Lys-AspAsp-Asp-Asp-Lys, called FLAG. The peptides were differently labeled with HA- and FLAG- specific antibodies, revealing an epitope-specific staining pattern with a density of 40,000 peptide spots per square centimeter.
According to the researchers, the chips have a number of potential applications. In their own cancer-related work, they have used their technology to screen for SEREX-defined proteins. Other applications include targeting highly expressed protein in cancer cells with D-peptides that specifically bind to the target protein; screening for catalytically active peptides; and screening for novel antibiotics.
“Our method should be especially helpful in the field of proteomics because it allows for the translation of whole proteomes into arrays of overlapping peptides,” the team writes in the Science paper.
However, Stadler and F. Ralf Bischoff, also at the German Cancer Research Center and a co-author of the article, stopped short of saying the chip would increase the use of peptide chips even if their method could bring down costs.

LC Sciences’ Gao said the technique is “very neat,” though added it is unclear whether it could be scaled up and reproduced to become a “routine method [for] generating peptide arrays” on a commercial level.

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