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HUPO, Invitrogen to Create Protein Standards As Sigma-Aldrich Prepares to Launch ABRF Standard

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The Human Proteome Organization and Invitrogen plan to co-develop two sets of experimental protein standards â€" mixes of proteins â€" for proteomics platforms, ProteoMonitor has learned.

Separately, Invitrogen rival Sigma-Aldrich said it plans to launch in a few months protein standard mix it co-developed last year with the Association of Biomolecular Resource Facilities' Proteomics Standards Research Group.

Invitrogen's and HUPO's standards, which the company also plans to turn into a commercial product, will join other efforts in the field to create standardized mixtures of proteins or peptides for benchmarking proteomics platforms, a dynamic that underscores the need among scientists to test the capabilities of their platforms before comparing their data.

"You need to know the limitations of what you do and how you do it. Without those [standards], it's very difficult to do," said Eugene Kolker, president and director of the Biatech Institute, a nonprofit research organization that has developed and distributed two standard mixes for proteome studies in the past. "[When] you look at other fields, like in the array area, there are still so many open issues, in part because not very many experimental and computational standards have been developed," he said.


"It seems like [HUPO and Invitrogen] want to walk when other groups are already running."

HUPO's project with Invitrogen resulted from a meeting last week in Montreal by its committee on test standards, co-chaired by HUPO president John Bergeron and past president Sam Hanash. Other members include scientists, editors of proteomics journals, and representatives of funding agencies such as the NIH, the Canadian Institutes of Health Research, and the European Commission.

The first standard will comprise 20 proteins in equimolar amounts selected from a list of 100 for their "extremely high purity, quality, stability and robustness," according to Bergeron. These proteins, whose identity will be kept under wraps for now, "cover various criteria which the committee put together," he told ProteoMonitor this week.

The second standard will contain 20 proteins in four different samples at varying concentrations spanning either three or four logs, with some kept constant at high abundance, some at low abundance, and others mixed in.

The standards, Bergeron added, were designed for proteomics based on both 2D gels and mass spectrometry.

HUPO plans to send these protein mixtures to 24 as-yet unselected laboratories for analysis. These labs will receive "not only the proteins but also a database for them to match the mass spectra data to," he said. The results from this study, which will not be anonymized, will be reported at HUPO's annual meeting in Long Beach, Calif., in late October.

After that, HUPO intends to distribute the standards more widely in a manner that is yet to be decided by the committee, Bergeron said. HUPO's education and training committee also plans to make the standards available to whomever "wishes to take advantage of it to help it benchmark its proteomics platform."

The aim is "to ensure that our proteomics platforms are operating the best that they can," Bergeron said.

Invitrogen intends to turn the protein standards into a commercial product, according to Charles Piazza, the company's vice president of proteomics. For now, the company is helping to design, manufacture, and supply the proteins according to HUPO's specifications to "make these standards a reality," he said. The intention is "to improve the quality of standards and to improve the ability to compare across laboratories."

But HUPO has even more ambitious goals for the future, planning to develop more complex protein mixtures, he said. Within a year, the organization hopes to create a standard made up of 200 proteins, and maybe move on to 1,500 different proteins within two years. It was not immediately clear whether Invitrogen will be involved in the development of these standards as well.

In addition, HUPO is currently "setting up mechanisms" to develop and make available heavy-isotope labeled test proteins and peptides, glycosylation standards, and phosphorylation standards, according to Bergeron. He said the group plans to talk about these more at the October meeting.

Besides protein mixtures, HUPO's standards committee is also considering antibody standards that it plans to discuss at the Long Beach meeting, Bergeron said, including a quality-control standard and paired antibodies for benchmarking multiplexed ELISAs.

Sigma to Sell ABRF Standard

HUPO's efforts to develop experimental protein standards for proteomics is neither the first nor the only one. Earlier this year, the Association of Biomolecular Resource Facilities' Proteomics Standards Research Group (sPRG) presented results from its 2006 study, which involved the analysis of a mixture of 49 human proteins in equimolar amounts by participating laboratories (see PM 2/23/2006).

The sPRG developed this protein standard in collaboration with Sigma-Aldrich, which plans to launch the mix commercially worldwide this fall, according to Dale Peluso, the company's market segment manager for quantitative proteomics.

"It's really the same standard that [ABRF] used in their study," he said, except for a greater level of purity. "In the study, they showed what they called 'bonus proteins,' and we managed to eliminate most of those," he said. While the price has not yet been determined, "we are going to price it such that it is accessible to the labs that need them," he said.

It was not immediately clear why HUPO decided to develop a separate set of 20 proteins with Invitrogen, rather than adopt the ABRF/Sigma-Aldrich set that already exists. "It seems like they want to walk when other groups are already running," said Peluso who also attended the HUPO meeting in Montreal last week.

However, Peluso did not have the impression that other standards were being excluded at the HUPO meeting. "I don't know that they are partnering with Invitrogen to exclude everyone else, it doesn't seem that way," he said. "It seems that they are very open to anyone who has techniques and solutions that may help."

Several standards are probably a good thing, Jeff Kowalak, the current chair of the ABRF's sPRG, agreed. "We applaud anybody who wants to bring reference materials to the science of proteomics," he said. "There is clearly a need for multiple standards. As far as I am concerned, it's a wonderful thing."

However, Biatech Institute's Kolker cautioned that whoever develops standard protein mixes needs to make sure they remain available for a long time. His group has recently developed another protein mixture that aims to mimic real-life samples, which contain proteins at a vast dynamic range. His latest mix, which he plans to describe in a publication soon, contains 54 proteins that are divided into three groups of different concentrations. Following the publication, Kolker plans to disseminate datasets from the analysis of the protein mix. He said he also intends to work with HUPO in the future to develop more complex standards.

Also interested in developing protein mixes for standardization is the NCI's Clinical Proteomic Technologies for Cancer Initiative, which aims to optimize existing and develop new proteomic technologies and reagents. On its website, under "future funding opportunities," the initiative lists a "clinical proteomic reagents resource." This resource will include, among other reagents, "standard protein and/or peptide mixtures," according to the website. The NCI has allotted $12.5 million over five years for the proteomic reagents resources, with $2.5 million for each year [see ProteoMonitor 03-21-06]

â€" Julia Karow ([email protected])

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