Ironing Out The Wrinkles In Peptide Microarrays



Technology Spotlight

Monday July 27, 2009

by Caitlin Smith

With DNA microarrays a common tool now, proteomics researchers would like to introduce you to something analogous—peptide microarrays—with which you can interrogate samples for binding partners. Many hope that peptide microarrays can revolutionize medical diagnostics and peptide drug treatment. “The biggest challenges to improving peptide microarrays are to increase their flexibility to allow a broad range of applications and to implement robust applications,” says Chris Hebel, director of business development at LC Sciences. “Peptide array technology is currently at a development stage similar to where DNA microarrays were 10 years ago. Many applications for using peptide arrays for protein studies are yet to be developed. In a sense, peptides are ‘probes’ to target proteins, but these ‘probes’ do not have a universal answer, like hybridization between nucleic acid strands, to their targets. The protein–peptide interaction is far more complex.” Even though different protein-peptide interactions may require distinct conditions, here are some highlights of the peptide microarray field that may help you get going.

Peptide quality for success

A crucial element of peptide microarray experiments is using quality peptides. Many vendors offer custom peptide services, and some, such as New England Peptide, go the extra mile to provide quality in their product and their service. David Robinson, CEO of New England Peptide, says that they have “developed a post-array synthesis aliquotting technique which ... aliquots peptides down to picomole and even subpicomole scale.” They also offer their Peptides for Quantitative Proteomics (PQP) suite of services to support biomarker discovery and other proteomics applications. “PQP service includes synthesis of peptides in array format using our proprietary array synthesis,” says Robinson. “Synthesis is followed by aliquotting peptides array to picomole scale using our automated aliquotting system.” Robinson believes that “one of the major challenges in microarray synthesis on cellulose membrane or chip-bound is that peptides lack a well-defined three-dimensional structure, and therefore may not have appropriate orientation to promote the interaction with their target.”

Miniature works

Yet microarrays on chips can boast amazing advantages, too. LC Sciences’ PepArray combines the benefits of microarrays and microfluidics. In situ peptide synthesis and multiplexed assays all occur within the tiny PepArray™ device, a microfluidic microarray on a picoliter scale. One device can house thousands of titration and enzymatic reactions, effectively replacing laborious tubes or plates. “The technology allows us to synthesize thousands of custom peptides (sequences can be defined to each single amino acid residue) on an addressable array to act as kinase substrates, antibody epitopes, or protein binding ligands,” says Hebel. “We can perform enzymatic/binding reactions in a high-throughput format and generate quantitative results in a controllable, enclosed environment with minimal sample usage.”

The microfluidic design of the PepArray™ chip means lower sample volumes and better quality data. “The chip is a closed system, unlike an open glass slide as with typical microarrays, so dye oxidation and deterioration are not an issue,” says Robinson. “Additionally, the microfluidic technology enables active circulation of the sample throughout the chip, producing a uniform distribution of the sample solution on the array that enhances the binding kinetics and stringency wash processes. This results in increased signal-to-noise and decreased CV values.” He notes that using a microfluidic chip effectively miniaturizes at least 50 microwell titration plates. “Compared to a conventional microplate, 50 times the amount of data is obtained with one hundredth of the amount of the sample and in very short time,” says Robinson. “This, coupled with the in situ synthesis of custom peptide arrays, provides tremendous opportunities for proteomics studies of proteins and protein-interacting molecules.”

ArrayIt supplies platforms for the manufacturing, detection, and processing of peptide microarrays. With their NanoPrint and SpotBot Microarrayers, for example, ArrayIt can custom manufacture peptide microarrays for you, or they can support you in learning how to manufacture and process your own microarrays using their core technology. Todd Martinsky, co-founder and executive VP of ArrayIt, believes that improved diagnostics are one of the most exciting new developments to emerge from peptide microarray advances. “Just as oligonucleotides replaced cDNAs in many of today’s DNA microarray applications, our platform will empower the use of peptide microarrays to replace the proteins used in plate- or tube-based immunoassays,” says Martinsky. “The ability to miniaturize, multiplex and parallelize immunoassays with synthetic reagents will provide more robust, accurate, and sensitive diagnostics.”

Martinsky explains that to maximize the performance of their microarray products, they have incorporated the most advanced forms of technology for each step in the process, such as sample preparation, robotics, surface chemistry, and detectors. “We’ve worked with peptide synthesis experts to optimize the covalent bond between the peptide and our microarray surface to maximize its capture binding efficiency,” says Martinsky. “We’ve consulted with robotic experts and developed the NanoPrint microarrayer. With the nanometer resolution of this microarrayer, we can get more reproducible microarrays than ever before. In detection, the confocal autofocus architecture and cooled lasers on the Innoscan scanners assures superior signal-to-noise ratios and better reproducibility than inferior systems that do not have these advancements. Speed of detection is also important—we can easily detect 25,000 peptide microarray data points in about 3.5 minutes. The technology has really come a long way.”

Smart screening

Peptide drugs are a small but rapidly growing class of new pharmaceuticals. Mimotopes hopes that its peptide microarray libraries will hasten more of these to market by providing a faster and cheaper screening solution. The libraries also can be used for epitope mapping, particularly for immunology applications. “Understanding the basis of clinical disease related to the immune system may require testing of large numbers of individuals over many proteins or epitopes,” says Nicholas Ede, CEO of Mimotopes. “Peptide libraries are ideal for T-cell epitope searching, because T-cell epitopes are by nature short linear peptides from the primary protein sequence. They are also appropriate for scanning the primary sequence of proteins for linear, or ‘continuous,’ antibody-defined epitopes.”

In collaboration with GlaxoSmithKline and the University of Leeds, Mimotopes developed a rapid endopeptidase profiling microarray library (REPLi) that is a “‘small but smart’ generic screening tool for identifying protease substrates, which are increasingly viewed as valuable drug targets,” says Ede. “The microarray peptide library, PepSet, consists of fluorescently labeled tripeptide analogs spanning the sequence surrounding the cleavage site of the substrate. With just 512 distinct pools containing eight peptides in each, the library can be relatively small while still representing the residue requirements for the largest number of proteases.”

Peptide 2.0 is also interested in immunology applications, offering peptide arrays for T-cell and B-cell epitope mapping. Michael Xu, director of R&D at Peptide 2.0, says epitope identification and peptide mapping have been facilitated by “computerized peptide design for T-cell epitopes and 96-well high-throughput synthesis.” But Xu also notes that “maintaining the solution-like structure of a given peptide is the challenge. Progress is being made by incorporating longer spacers between the peptide and the solid surface, and by utilization of strepavidin-biotin interactions.” In a decade or so, perhaps the wrinkles in peptide microarrays will be ironed out—at least as smoothly as DNA oligonucleotide arrays are now.

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"THANKS AGAIN for all the intellectual support that NEP has provided to us over the last year. My colleagues have been overwhelmingly impressed with the quality of service and level of thought that you have put into researching and thinking about our particular peptide and antibody production needs. The level of scholarship went well beyond what we expected from any peptide and antibody company, and was crucial in shaping our decisions. It has truly been a pleasure corresponding with you."
Cliff N. PhD - Lawrence Berkeley National Laboratory