Meeting News
Test standards for proteomics: multiple choices In a premeeting HUPO workshop, researchers described three independent efforts to produce standard mixtures of proteins for the proteomics community. The workshop was organized by John Yates, who is at the Scripps Research Institute and heads the HUPO New Resources and Technology Committee. According to Yates, the gathering was a chance for researchers who are developing protein test standards to share their progress and discuss the issue with interested scientists. Not to be confused with the standard data formats proposed by the HUPO Proteomics Standards Initiative, these standards are mixtures of proteins with various properties that can be used for calibration or for methods development. “The point is that we need standards for benchmarking purposes because right now, we don’t have any samples in which the compositions of proteins are absolutely known and known to be constant,” says Thierry Rabilloud of the Atomic Energy Commission (France) and the French Institute of Health (known as INSERM). At the workshop, Dale Peluso of Sigma-Aldrich unveiled the most mature of the standards, called the Universal Proteomics Standard. This 49-protein mixture was originally evaluated in a study conducted by the Proteomics Standards Research Group of the Association of Biomolecular Resource Facilities (J. Proteome Res. 2006, 5, 740). In the study, researchers used the mixture to assess the performance of current proteomics techniques for protein identification. After the results were announced earlier this year, researchers at Sigma-Aldrich continued work on the standard. They removed contaminants and ensured that the proteins could be reproducibly identified. The current version, which is commercially available, is composed of an equimolar set of 48 human proteins that span a wide range of molecular weights and pI values. HUPO also is getting into the act by coordinating the design of a protein test standard in collaboration with Invitro-
gen. According to Paul Predki of Invitro gen, HUPO representatives have been “developing the criteria for the proteins, and we’ve been selecting proteins on the basis of those criteria.” The first fruit of this collaboration is a 20-protein equimolar mixture. The proteins are expressed in E. coli from constructs that contain fully sequenced human open reading frames. Again, proteins in the mixture include those in various ranges of molecular weight, pI, and other properties. “Our intent is to mimic in some ERIC MILLER, NORTH CAROLINA STATE UNIVERSITY
Katie Cottingham reports from the HUPO Fifth Annual World Congress—Long Beach, Calif.
The next standard? Researchers are developing several standards for proteomics, including one based on the proteins of bacteriophage T4.
sense the complexity and diversity of a real proteomic sample,” says Predki. Soon, the standard mixtures will be sent to several proteomics laboratories for testing. Researchers will submit lists of protein identifications to the McGill University (Canada) laboratory of Alexander Bell, who will analyze the results. Depending on how this standard fares, it may become a commercial product. Predki says that any decisions regarding commercialization will be made jointly by HUPO and Invitrogen. A completely different standard was proposed by Rabilloud. Yates presented the results of a collaboration that his
22 Journal of Proteome Research • Vol. 6, No. 1, 2007
group undertook with Rabilloud’s laboratory in which they evaluated the 37 proteins of T4 bacteriophage as a possible standard mixture for proteomics studies. Rabilloud remembered an old paper that proposed the use of phage proteins as standards for 2DE gels. He thought these proteins also could be used as proteomics standards. He explains that an advantage of viral proteins is that they are easily distinguished from human proteins, so they can be spiked into human samples. Another benefit is that they comprise a defined complex mixture that occurs naturally. That fact also can be a disadvantage, however, because the dynamic range is predetermined. Rabilloud and colleagues tested the phage proteins by 2DE LC/MS and GeLC/MS/MS methods, and Yates’s group performed shotgun proteomics analyses on the proteins. “What’s amazing is that despite there only being 37 proteins, not a single technique can see all of them,” says Rabilloud. “The point is that even though you don’t see all of them, you know exactly which proteins are missing.” In the shotgun study, the researchers attempted to quantify the proteins by spectral counting, but the quantities determined with the method didn’t match the known abundances of the phage proteins. Yates notes that the results were preliminary and that the samples will be rerun. Yates and Rabilloud say that the proteins of T4 phage appear to be promising as a standard, although more work needs to be done. Ultimately, the researchers envision that T4 phage proteins will be commercially available as a standard protein mixture. At last, proteomics scientists will have protein test standards at their disposal. In fact, many choices may be available soon. According to Predki, HUPO and Invitrogen are planning to develop standards in which proteins are present at different abundances; this type of mixture could be useful for relative quantitation studies. Rabilloud also is thinking about quantitation and proposes that a mixture of T4 and T7 phage proteins could serve that purpose. In addition, Yates is considering even more complex standards, such as those that could mimic the makeup of an organelle or an entire cell. —Katie Cottingham
