Most proteomics researchers and industry watchers are probably familiar with ICAT, a tagging technology developed by Ruedi Aebersold’s former laboratory at the University of Washington. By extension, many in the field are probably familiar with the typical complaints — warranted or not — associated with the ICAT reagent technology: It requires a steep learning curve, its affinity selection criteria are arbitrary, and perhaps most importantly, its cost is high.
So where are the alternatives?
One would think, given the apparent market for alternative reagents, that another reagent or mass spectrometry manufacturer would have stepped in to offer a competing approach to the ICAT (isotope-coded affinity tagging) technology. But for several reasons, vendors have been slow to jump on the bandwagon, leaving Applied Biosystems, the exclusive licensee of the ICAT reagents, with a monopoly on the market for off-the-shelf tagging chemistries designed for quantitative proteomics experiments.
But that’s not to say people aren’t trying. Many labs, particularly in industry, have surreptitiously designed tagging chemistries for their own in-house use, and several academic groups have published variations on the general ICAT reagent theme in the last 12 months. And most recently, according to Peter James, a protein mass spectrometrist at the University of Lund in Sweden, Amersham Biosciences has begun investigating how to commercialize reagents developed in his lab.
The problem is that many researchers may find it difficult to buy these reagents and their associated protocols off-the-shelf. The patent awarded to the University of Washington for the discovery of the ICAT reagent technology — developed by Ruedi Aebersold’s group while at the university — is ostensibly broad enough to discourage others from commercializing competing reagents. Furthermore, many of the alternative technologies developed so far are in the hands of companies who would rather not see others use the technology for their own ends. Lastly, creating a reproducible method for tagging and analyzing proteins based on their abundance and identity is not easy, requiring considerable investments in protein chemistry expertise and software support.
Amersham takes on ICAT
Although intellectual property considerations may have diminished other vendors’ enthusiasm for competing with the ICAT reagents, Amersham appears to be making an attempt to circumvent the claims awarded to the University of Washington, and by extension, ABI. Amersham officials were not available to speak directly on their efforts to commercialize James’ labeling technology, but James said he and his partners at Amersham have applied for US and European patents on the method, and are currently waiting for them to be approved.
James is also reluctant to discuss the details of his method, but he has spoken about his general approach at conferences, and provided a few insights in an interview with ProteoMonitor. His technique relies on modifying the N-termini of all peptides in the two samples under comparison, with one sample labeled with a deuterium-enriched tag, and the other with a tag containing only hydrogens. James has created tagging protocols for performing differential protein expression experiments using both 2D gel and multidimensional liquid chromatography separations, and also for specifically tagging phosphopeptides in similar experiments.
While James does not rely on a purification step to simplify the mixture — the ICAT reagent technology currently allows researchers to study only cysteine-containing peptides — he said he has developed an undisclosed technique that uses “hardware, software, and chemistry” to selectively study only those peptide pairs from the two samples under comparison that differ significantly from one another in their expression levels. “That’s the absolute crux,” James said, adding that he is waiting for US patent approval before discussing the technique in detail.
But Aebersold is curious how Amersham expects to get around the patent on the ICAT reagent technology. The initial patent covers reagents with a broad range of protein reactive functionalities, isotope-coded tags, and affinity agents, Aebersold said, potentially making it difficult for another party to devise a reagent that didn’t fall under the claimed methods. “In the meantime there’s been a bunch of secondary patents filed for specific applications and implementations,” he added. “I’m not saying at all there’s no way around it, but if the lawyers did their job right, it should be fairly broadly covered.”
Industry Groups Are Secretive About Their Efforts...
IP considerations may provide one explanation for the scarcity of labeling chemistries generally available for quantitative differential proteomics, but competition between rival industry groups may offer another. ProteoMonitor has learned that researchers at Genentech and Millennium Pharmaceuticals have developed tagging chemistries for use in-house in quantitative differential proteomics experiments. Also, groups within big pharma, such as Roche, are coy about their internal efforts to develop alternative tagging chemistries.
In addition, industry groups have exclusively licensed rights to the commercial application of tagging approaches developed in academia. Fred Regnier, an analytical chemist and biochemist at Purdue University, has developed a technique called GIST, for global internal standard technology, that labels all N-termini of peptides in two samples with the heavy or light isotope version of a chemical tag. The advantage, Regnier said, is that the researcher has the discretion to then choose from a variety of purification strategies to simplify the peptide mixture. The ICAT reagents, he said, restrict the peptides under investigation to only those containing cysteine residues.
But while the GIST technique may offer researchers more flexibility, one company, Beyond Genomics, has an exclusive license to its commercial application. [Regnier happens to be a co-founder and member of Beyond Genomics’ advisory board]. Beyond Genomics is currently discussing the potential for commercializing the reagents with Micromass, its technology partner, but Steve Naylor, Beyond Genomics’ chief technology officer, said the talks are progressing remarkably slowly, given the perceived need for new reagents. “I don’t think people have an understanding of the market size and development costs,” Naylor said.
...and Abi Has a Head Start
Others also realize that developing a commercially viable reagent system is neither easy nor cheap. Aebersold, now at the Institute for Systems Biology, said that the high price of the ICAT reagents is not justified by the cost of chemical synthesis — it’s primarily a marketing decision on the part of ABI, he said — but ABI has made significant investments in product development and technical support. “It actually takes a long time to take these things to market,” he said. “You can’t just start selling reagents.”
Andrew Emili, a proteomics researcher at the University of Toronto, seconded this assertion. His group described a potential ICAT reagent alternative, called MCAT, in the February issue of Nature Biotechnology. MCAT, which stands for mass-coded abundance tagging, relies on two tags with different masses to distinguish between two peptides, as opposed to different stable isotopes. Although his chemical tag is considerably cheaper to synthesize than its ICAT counterpart, Emili’s method — like Regnier’s — labels all peptides in a sample, and requires a second purification step if a researcher is interested in simplifying the mixture.
But unlike Regnier, Emili said the primary challenge in designing a strategy for employing tags in differential proteomics experiments lies in the ability to process the large amounts of data that result from the mass spectrometry analysis. Emili, who said he is also hoping to build a company around his MCAT technology, said most of his academic lab’s current efforts are in developing algorithms and software to support the data analysis.
“MCAT is tractable to computing, but it’s not non-trivial to automate,” he said. “Chemistry is not the rate limiting factor,” he added. “It’s the data analysis that’s under-serviced, and the companies that solve that problem will take off.”