As fast as Reddy's approach might be, it still isn't nearly fast enough to tackle the proteome en masse. Many researchers study well characterized proteins for which high quality affinity reagents already exist. Yet the majority of human proteins have no available reagents at all. In a Nature editorial earlier this year, Aled Edwards of the University of Toronto, and colleagues surveyed the biomedical literature to show that research is skewed towards those genes for which antibodies or chemical inhibitors exist, leaving the bulk of genes understudied.
“Much of the work that has emerged from exploring the human genome over the past ten years lies fallow,” Edwards wrote. “Challenges notwithstanding, making protein-based research tools readily available must be a major objective in the decade to come.”
Work is already progressing on this front. The latest version (version 8) of Mathias Uhlen's Human Protein Atlas (proteinatlas.org), for instance, includes some 14,500 antibodies covering more than 11,250 human genes. But those are polyclonals, a non-renewable resource.
This past July Colwill, Gräslund, and a collection of researchers calling themselves the “Renewable Protein Binder Working Group,” published a study in which they challenged researchers to generate antibodies to 20 soluble SH2 domain proteins. The report, entitled “A roadmap to generate renewable protein binders to the human proteome,” details how five groups, including Sidhu's, Dübel's, and McCafferty's, produced some 1,040 antibodies, 340 of them unique.
Put through their paces via surface plasmon resonance, immunoblot assays, immunofluorescence, and ultimately immunoprecipitation, these antibodies fared well, according to Colwill, with both hybridoma and display-based approaches producing high quality reagents. “They all had reasonable successes,” Colwill says, “there wasn't a clear winner.”
At the same time, though, it's obvious a proteome-scale resource requires in vitro technology, says Dübel, who delivered 362 antibodies of his own in about two months; if nothing else, making 100,000 monoclonals would require millions of animals, he says.
The SGC, says Gräslund, has already begun building on this pilot study with a new effort to develop in vitro antibodies to some 300 epigenetics-related proteins. And proteome-scale efforts are in the works, both in the US and Europe. EU-sponsored programs like the AffinityProteome and Affinomics, Dübel says, are already working to produce a “couple hundred to a couple thousand proteins.” This past December, the NIH issued RFA-RM-10-017, a $20 million call for applications for “centers that can produce renewable, high quality affinity reagents against all human transcription factors.” From there, the proteome itself is just a matter of scale.
Ultimately, says Dübel, researchers may be able to sift through a whole-proteome antibody catalog, representing not just every one of the 25,000 or so human genes, but alternative splice variants, modified forms, and so on. With existing technology, he estimates the job could be completed for perhaps $50 million to $100 million.
“The method is there,” he says. “You just have to pay for it.”