MEETING NEWS
Katie Cottingham reports from the Association of Biomolecular Resource Facilities 2007 Meeting—Tampa, Fla. ABRF proteomics studies show that experience is key Whether the task is determining the relative amounts of proteins in a complex mixture, identifying phosphorylation sites, or conducting a bioinformatics analysis, the results from three studies sponsored by the Association of Biomolecular Resource Facilities (ABRF) demonstrate that the most critical factor is the expertise of those performing the analysis. This intriguing conclusion was trumpeted by all three proteomics research groups at the ABRF 2007 meeting held March 31–April 3.
PRG and relative quantitation: round 2 Capitalizing on the momentum from last year’s study, the Proteomics Research Group (PRG) revisited the topic of relative quantitation. In the previous study, participants determined the relative abundances of eight proteins in two samples. “A lot of people felt that while it was a challenge for some people, it wasn’t as realistic as they’d like it to be,” says Mike MacCoss, who is at the University of Washington and is a member of PRG. So, to simulate a real-world proteo mics problem, PRG members spiked a total of 12 proteins (2 E. coli proteins and 10 from other organisms) into an E. coli lysate at different levels and ratios in two samples, A and B; a third sample, C, was identical to sample B. A database with coded accession numbers and descriptions also was provided to participants as an attempt to blind the study and ensure that the results were reported in a consistent fashion. Most researchers used isobaric tags for relative and absolute quantitation (known as iTRAQ); others used 2DE-based or label-free methods. “One of the things that we felt was quite clear was that this was a very tough study for most labs,” says MacCoss. “However, the good news was that some labs can do this very well.” The E. coli proteins and those present at the lowest abundances were the most
difficult ones for the researchers to identify and quantitate. The number of participants was too small for PRG members to draw definite conclusions about techniques, but none drastically outperformed the others. According to MacCoss, the experience of each participant was the main predictor of success in this study.
sPRG investigates a phospho protein standard With a similar goal of creating a realworld sample, the Proteomics Standards Research Group (sPRG) developed a pilot phosphoprotein standard mixture. The standard consisted of one nonphosphorylated protein and six phosphoproteins, most of which were present at low phosphorylation stoichiometries. Most phosphoproteins are involved in signaling events, and only a small percentage of a particular protein typically is phosphorylated in vivo, says Jeff Kowalak, who is at the U.S. National Institutes of Health and is a member of sPRG. In total, 44 labs submitted 68 data sets. Kowalak says that most of the proteins were identified correctly, but few of the phosphorylation sites reported in the literature for these proteins were pinpointed. Compounding the problem was the fact that many participants reported additional, “unverified” phosphorylation sites for several proteins. sPRG members included these if they were listed by ≥2 labs. “This isn’t a definitive standard where we knew 100% of what we had going in, so we have no way of saying that [the unverified sites] are wrong,” Kowalak explains. He says that the time crunch was the main reason the sPRG members didn’t have an absolute answer key of phosphorylation sites that were present. Again, the number of participants was too low to enable sPRG members to draw significant conclusions, but Kowalak says that no one technique shone. “Much like last year’s study, the operator’s skill and experience level was the best way to correlate success rate,” he points out.
A PRG is born Last year’s sPRG2006 study, in which researchers were charged with identify-
2054 Journal of Proteome Research • Vol. 6, No. 6, 2007
ing 49 human proteins in a standard mixture, resurfaced at the meeting as the basis of an in-depth bioinformatics study conducted by a subgroup of sPRG called the Bioinformatics Committee (BIC). According to Kowalak, who also is one of the six members of sPRG BIC, the subgroup has spun off to form the Proteome Informatics Research Group (known as iPRG). Kowalak explains that in addition to the 49 proteins intentionally included in the standard, ~84–87 “bonus” proteins were reported by various labs in the original study. Because some of the 49 proteins did not meet the goal of being 95% pure, extra contaminating proteins were present. However, some of the extra proteins reported by participants could have been contaminants introduced in their own labs during sample analysis. The initial goal of BIC was to reevaluate the submitted data and determine the official number of proteins that were present in the master standard. “As the study progressed, a second goal naturally accrued, and that was: can a committee of people who are bioinformatically competent take the data sets and come down to a consistent agreement on what proteins were in those data sets?” he says. Working independently, BIC members applied various search engines to the data and found that they could whittle down the list of bonus proteins to ~20. Some of the rejected protein identifications were, in fact, probably due to contaminations within an individual lab. For example, nine trypanosome proteins were reported by a single lab that happens to conduct research on this organism. BIC members also discovered that although they used several different bioinformatics methods, they still arrived at consistent results at the end of the day. “Again, I attribute that to the experience level of the analyst,” says Kowalak. Currently, all of the groups are contemplating topics for next year’s studies. In the meantime, researchers can view the presentations and posters describing the current studies at the ABRF website (www.abrf.org). In addition, the results will be published in an upcoming issue of ABRF’s Journal of Biomolecular Techniques.
