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Beta Tester Says Sigma s New Top-20 Depletion Kit Improves Detection of Low-Abundance Proteins


Sigma-Aldrich's newly released ProteoPrep 20 Plasma Immunodepletion Kit depletes the 20 most abundant proteins in plasma with good specificity, and allows for the detection of more low-abundance proteins, according to a beta tester of the kit.

David Speicher, the director of the Wistar Institute's Proteomics Laboratory, who analyzed the bound and unbound fractions of plasma following ProteoPrep 20 depletion, said that only a small portion of proteins that are not targeted by the depletion kit end up in the bound fraction of plasma.

"There are a few non-targeted proteins that stick [to the 20 proteins]. Most are not sticking exclusively [to the bound fraction]. A few are sticking exclusively," said Speicher, who has been beta testing Sigma's product since last fall.

Before he gained access to Sigma's ProteoPrep 20 kit — the first immunoaffinity kit to deplete so many abundant plasma proteins — Speicher was using a column from Agilent that depletes the top six most abundant proteins in plasma.

Speicher's research team depleted the top six proteins as part of a four-dimensional fractionation technique that allows for the identification of proteins that differ in concentration by a range of nine orders of magnitude: The highest concentration proteins are present in milligram/milliliter ranges, while the lowest concentration proteins are present in picogram/ml ranges (see ProteoMonitor 4/8/2005).

"My feeling is that if you need to isolate and purify too many proteins, that [the product] is probably going to reach a point of diminishing returns."

Using his 4D-fractionation method, Speicher was able to identify about 2,000 proteins in human plasma samples sent out as part of the pilot phase of the Human Proteome Organization's Plasma Proteome Project. Speicher said that he expected to be able to increase to 6,000 the number of proteins identified in those samples by using the ProteoPrep 20 kit, combined with other enhancements to his 4D fractionation system.

"I expect that the top-20 column is going to give us pretty robust coverage at the low ng/ml level, and to expand the pg/ml level," said Speicher.

Speicher said he doesn't think that there are disadvantages to depleting more of the most abundant proteins.

"Obviously, the top 20, and 98 percent of total protein removed, is much better than the top six proteins removed," said Speicher. "In general, I would argue that more is always better."

Depleting the most abundant proteins upfront allows for more protein to be loaded downstream into gels or HPLC columns, Speicher said. By depleting the top 20 most abundant proteins as opposed to the top six, the volume of original plasma that can be analyzed downstream is increased by roughly an order of magnitude, he added.

"If you deplete 85 percent of proteins using a top-six column, you can load 10 to 20 times more protein downstream," said Speicher. "If 97 to 98 percent of proteins are depleted using a top-20 column, you can load 100 to 200 times more protein downstream."

The one disadvantage to depleting more of the most abundant plasma proteins is cost, Speicher said.

"My feeling is that if you need to isolate and purify too many proteins, that [the product] is probably going to reach a point of diminishing returns," he said.

However, the price of the ProteoPrep20 kit seems to be in the same ballpark as the top-six depletion columns that are out on the market.

According to Dale Teluso, the market segment manager for quantitative proteomics at Sigma, the ProteoPrep 20 kit costs $2,500 for three spin columns that can be used at least 100 times each.

Agilent's top-six depletion column costs about $2,500 for the 4.6 mm by 5 mm size, and about $3,400 for the 4.6 mm by 100 mm size. Sigma's Prot IA kit, which depletes the top two proteins — albumin and IgG — costs around $800.

An immunoaffinity column that could deplete even more than the top 20 most abundant proteins — say, the top 40 or 50 proteins — would probably eliminate 99.5 to 99.8 percent of the original protein, Speicher said. Such a column would be useful, but it would probably be hard for a company to produce such a column and have it work well and be profitable, he added.

"Having done a lot of work with antibodies and specific proteins ourselves, we know that when you try to produce an antibody, it doesn't always work, and when you affinity couple, it doesn't always work," said Speicher. "If you have a handful of proteins you need to deplete, you just keep working until you get it right. Some percentage of the proteins is not going to work well, and you're going to get some incomplete depletion, and a little bit of non-specific binding. The more proteins you're dealing with, the more those complicating factors increase."

Teluso said the ProteoPrep 20 kit is Sigma's "first serious entry" into the area of biofluid depletion. The company is currently developing other depletion columns that can be used with mouse plasma, and with other types of biofluids, he added.

"[The ProteoPrep 20] is the first of probably many [depletion products] that will be forthcoming," said Teluso. "But we're not going to rest on our laurels. We're looking to provide solutions to our customers, whether it's in mice or other fluids."

Sigma's competitors in the biofluid depletion field include Agilent, which has its top-6 column for human serum, as well as a top-3 column for mouse serum samples; Beckman Coulter, which has a top-12 column for human serum; and Genway, which has top-6 and top-12 columns for human serum.

Speicher said that from his experience, all of the immunodepletion columns from various companies perform roughly similarly.

"What it really boils down to is the number of proteins depleted that's most important," he said.

Graham Scott, the research and development manager of proteomics at Sigma, said that developing the ProteoPrep 20 kit was a "significant technological challenge."

He said he expected that competitors might want to produce a similar top-20 depletion product, but was confident that Sigma would remain a leader in the field.

"We've put a lot of effort into this product, and we're going to build on it," said Teluso. "We're proud of our technology. We were able to [engineer] this in a high-quality fashion."

— Tien-Shun Lee ([email protected])

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