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Researchers Use Ciphergen s Equalizer Beads to Detect More Proteins in Urine

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A new study of the urinary proteome showed that Ciphergen's new Equalizer bead technology enabled researchers to discover about 250 proteins that had previously not been seen in urine.

The study, published last week in the online version of the Journal of Proteome Research, was a welcome spot of good news for Ciphergen. Earlier this week, the company said an internal investigation into second-quarter revenues caused it to postpone its third-quarter earnings report (see sidebar).

In the urinary proteome study, researchers were able to find 471 unique proteins "whereas previously, the people who could do the best — the Anderson group — were able to find 150 unique gene products," said Pier Giorgio Righetti, the last author of the study, who recently moved from the University of Verona to the Polytechnic Institute of Milan to head up a new proteomics facility. He was referring to a study led by Rembert Pieper which was published in 2004 in Proteomics.

"To go straight from 150 to 471 is very remarkable," Righetti said.

Accounting Investigation Delays Ciphergen's Q3
Financials; Issue Not Likely 'Subversive'

Ciphergen disclosed this week that an internal audit committee is investigating how and why the company recognized around $500,000 in sales in the second quarter. As a result, the company has delayed the release of its third-quarter financial results, which were originally scheduled for Nov. 3, according to a posting on the company's web site that has since been removed.

Ciphergen said it has enlisted the help of undisclosed "external accounting advisors" to help with the investigation.

Ciphergen declined to comment on the investigation. But a former Ciphergen official who spoke on the condition of anonymity because of his proximity to the company said Ciphergen could be struggling to deal with the Sarbanes-Oxley accounting laws.

"To satisfy the auditors, you can't recognize a sale until all the i's are dotted and all the t's are crossed," said this source. "It could be as simple as saying, 'We'll train six people if you buy the instrument,' but until that training was done, they couldn't recognize the sale."

The source added that he doubted that Ciphergen was dealing with "subversive stuff" where they were trying to get around the act. "Dan Caserza, the accountant for the group, is pretty squeaky clean about that stuff," he said.

In principle, the new Equalizer bead technology, which is not yet on the market, works by having a library of 64 million beads attached to different hexomeric peptide ligands bind to proteins within a biological sample. According to theory, the highly abundant proteins should quickly saturate their peptide ligand, while low-abundance proteins should have ligands to spare. When the beads are washed, the highly abundant proteins are washed away, while low-abundance proteins are enriched, resulting in protein concentrations being somewhat "equalized."

"One of the real advantages of this is there's no pre-fractionation whatsoever," said Righetti. "Often when you do pre-fractionation, you lose components. The more pre-fractionation you do, the more components you lose."

Once the proteins have been "equalized", they are washed under denaturing conditions so that they detach from their hexomeric peptide ligands. The proteins are then analyzed using mass spectrometry techniques.

Proteins found included plenty of low-abundance proteins with concentrations below 1 nanogram per milliliter, Righetti said.

"With proteins that you haven't seen before, I would guess that it is almost understood that these are very low abundance proteins," said Righetti.

There is an important potential drawback to using the Equalizer beads, Righetti pointed out: If protein concentrations are "equalized," then it may not be possible to do quanititative comparisons of proteins, for example in healthy versus diseased states. Quantitative comparisons between different physiological states have been a cornerstone for doing disease biomarker discovery work.

"We still do not know after equalizing if we will still be able to see quantitative differences," said Righetti. "With whatever saturates the hexomeric peptide ligands, you will have lost the quantitative differences. But with rare proteins that do not saturate the beads, in principle, you could still see the differences."

Sunny Tam, the director of the Protein Fractionation Group at the University of Massachusetts Medical School, said that overall, Ciphergen's bead technology can be a good complementary technique to existing approaches, but it requires more testing.

"Many protein identifications from mass spec were made with just one peptide from a database search," Tam pointed out. "The conclusion of the paper that novel proteins have been identified, and that an increased number of proteins have been found in urine, will require more validation and testing by other groups."

Both Tam and Righetti also pointed out the 'efficiency' or 'completeness' of protein capture by the beads remains a question.

"We have to try to figure out what fraction of the intact protein we are losing," said Righetti. "It would be naïve to think that we capture 100 percent of the proteome."

Currently, the Equalizer beads are only being used internally at Ciphergen, and it is unclear when and if the beads will be released onto the market.


"One of the real advantages of this is there's no pre-fractionation whatsoever. Often when you do pre-fractionation, you lose components. The more pre-fractionation you do, the more components you lose."

"We are using this technology for some projects with pharma companies and with diagnostic development internally, and we offer it as a service within our biomarker discovery centers," Carol Berry, senior director of marketing at Ciphergen, told ProteoMonitor. "I can't give a date when it will be released [to the public]."

Aside from studying the urinary proteome, Righetti and his colleagues have also used Ciphergen's Equalizer beads to study the serum proteome. According to Righetti, the investigators found 1,300 unique gene products, which they are still analyzing.

Righetti said he has plenty of projects involving Ciphergen's beads planned for the future.

"We have a project with cerebral spinal fluid, and we are also collecting bile, tears, sweat — all kinds of biological fluids," said Righetti.

Other researchers who were involved in Righetti's urinary proteome study were Annalisa Castagna and Daniela Cecconi from the University of Verona; Egisto Boschetti, Luc Guerrier, Frederic Fortis, and Lee Lomas from Ciphergen; and Lau Sennels and Juri Rappsilber from the Italian Foundation for Cancer Research Institute for Molecular Oncology Foundation.

— Tien-Shun Lee ([email protected])

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