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Should High-Abundance Proteins Be Removed? Plasma Proteome Pilot Generates Insights


Of the hundreds of lectures and posters presented at the HUPO Congress in Montreal last week, the latest data coming out of the HUPO Plasma Proteome Project’s pilot phase generated some of the greatest interest and industry buzz. For companies such as Agilent, Invitrogen, Thermo Electron, and Amersham (see story above), all eyes were particularly focused on the best way to get rid of those pesky high abundance plasma proteins.

This problem of how to remove high abundance proteins from plasma without removing other proteins with them — and of whether to attempt removal at all — is a debate that has been going on at least since HPPP launched (see PM 8-15-03). It gained considerable new momentum at the Congress. Gil Omenn, who heads up HPPP, said in his presentation at the Congress that determining whether the most abundant plasma proteins should be depleted was one of the five main aims of the HPPP pilot phase. In an interview this week, Omenn told ProteoMonitor that from what he has seen so far, “there’s little doubt that removing the most abundant proteins makes it much more feasible to detect other proteins,” but that “the data’s quite incomplete at this point … [and] we’ll see how this all plays out as we have more data.”

As attendees pored over the posters, suspense was definitely in the air. “Everyone wants to be like Celera was for the Human Genome Project — to serve that sort of leading role for HUPO,” an employee manning the Amersham exhibition booth told ProteoMonitor when asked about the role that Amersham hoped to play in the project. Amersham and Thermo conducted a joint lunch seminar and also presented a lecture showing data analyzing an HPPP pilot reference sample that they obtained using Amersham’s front-end platforms and Thermo’s LTQ ion trap mass spec. The experiments were conducted using variations on Amersham’s 2D gel and LC platforms, and its Blue Sepharose and Protein G Sepharose removal systems were used to remove albumin and IgG prior to sample separation. The group tested two workflows: one using a 2D gel followed by MALDI MS, and one using LC ESI MSMS. Although the scientists found over 300 proteins using the LC MSMS technique, Erik Forsberg from Amersham and Tori Richmond-Chew of Thermo both noted that other proteins did elute in the albumin fraction. “We looked at five fractions of albumin and one mostly was a big albumin smear, but others have other proteins in there too,” Richmond-Chew said.

The technologies with which the HPPP participating reference labs and other independent labs experimented for high abundance protein removal included: Amersham’s Sepharose products, GenWay Biotech’s chicken antibodies, Applied Biosystems’ POROS depletion kit, Millipore’s Montage depletion kits, Cibracon Blue, and Agilent’s Multiple Affinity Removal Column. Additionally, David Speicher, director of the proteomics laboratory at the Wistar Institute and Emma McGregor of Proteome Sciences tested out Invitrogen’s Zoom IEF fractionator — which Speicher originally developed — as a pre-fractionation alternative to depletion. They also tested it as a companion pre-fractionation step to depletion. Most groups found that depletion increased their ability to identify low-abundance proteins no matter what accompanying front-end and mass spec products they used, with Agilent’s column gaining the most praise.

Omenn said that HPPP would not pick one single platform for all member labs to use when they study plasma, but would come up with a set of recommendations for which technologies a lab should consider using if it has a particular set of resources and particular objectives. “For example, if you ask how do you detect very large numbers of proteins, it might be that the FT-ICR mass spec is the gold standard right now. But obviously, that’s not practical for most laboratories,” Omenn said. “And it may turn out that with some fractionation steps, much less expensive technologies leading into MSMS would be quite sufficient, and maybe do nearly as well.” He noted that a broader study would also likely carry different product recommendations with it than a more focused study. “I don’t think any company should feel that they are going to be winners or losers in this,” Omenn emphasized.

While the HPPP leadership will not make any conclusions about protein abundance removal or any other technologies until all the final pilot phase data — which is due from participating labs in draft form on Dec. 31 — has been presented at a final “Jamboree” to be held June 1-4, 2004, Omenn did express particular confidence in Agilent’s removal column. When asked whether it was better to deplete the high abundance proteins and accept that it cannot be helped that some low abundance proteins of interest might be lost, or to keep all the proteins and do extensive fractionation instead of depletion, Omenn said, “It’s likely that [losing proteins] can be helped, and the Agilent column appears to be a major step in that direction.”

But one presentation made in conjunction with the HPPP data suggested that any sort of depletion might not only be a bad idea — it might actually directly be at odds with successful biomarker discovery. Thomas Conrads, associate director of the mass spectrometry center and analytical chemistry laboratory at SAIC-Frederick in Maryland, presented data showing that high abundance proteins such as albumin may actually work as molecular “sponges,” sopping up low abundance proteins and peptides that leak into the plasma from organs. These leaked molecules are exactly the sorts of molecules that often act as biomarkers for disease. If this were the case, removing albumin would also mean removing just the proteins or peptides the scientist is looking for.

“If the goal is truly disease diagnostics, and the diagnostic route you are taking is based on mass spectrometry, it’s really dangerous to do depletion because everything that we find and everything that most of the folks in the literature are finding is that these are low molecular weight species that are bound to high abundance proteins,” Conrads said. “One would imagine that in serum, which is full of proteases and things like that, that these would be rapidly clipped up and cleared by the renal system, but in fact they aren’t, and the only real route that you can postulate is that this extension of half-life must be coupled with proteins that have long half lives in the serum, like albumin.” Conrads expressed skepticism that Agilent’s column would avoid retaining these bound proteins, but said that the column might have a use Agilent has not yet anticipated. “I’m actually interested in their columns, because if they’re as efficient as they say, that may be a multiplexed way for us to actually mine for biomarkers — to look at the proteins bound to the column, and then at who’s bound to the proteins,” Conrads said.

Omenn, however, was not convinced that Conrads’ suggested sponge effect was a deal-breaking problem. “This notion of using albumin or other proteins as sponges has some merit, and I think it will be interesting to see what that shows. … [Agilent’s] claim is that the buffer systems they use and resin features they develop reduces markedly the sponge effect.” He added that whether or not this claim was warranted would be a question for further pilot project experiments. “Some of the other pre-fractionation methods are also under conditions with lots of urea and reducing compounds that probably break most of that binding. So that’s an experimentally testable question that should be addressed.”

Other experimentally testable questions that will continue to be tried across technologies, according to Omenn, include sample handling and technologies for detecting post-translational modifications, quantitation, handling of dynamic range, and handling of low concentrations. Omenn emphasized that one strength of HPPP’s methods was that the same specimens were used across the different technologies.

When it comes to the question of what will follow the pilot study, Omenn said that the data generated in the initial pilot phase might generate a “pilot phase part II.” After that, the “grand scheme” for the project will be to look for disease-related biomarkers. At that point, Omenn said, HUPO might pull away from HPPP. “My present thinking is that we will have a chance to form and stimulate collaborations,” Omenn said. “Whether HUPO itself will organize to undertake such studies, that’s a much harder question.” Omenn explained that once big population studies and clinical trials for potential biomarkers come into play, there will be competition for proprietary applications, and HUPO may not want to get involved with that as an organization. He emphasized, however, that “the descriptive information and the characterization of specimens and technology and the development of proposed standard operating procedures will all be completely in the public domain and available as early as possible.”

Omenn also said that he hoped the NIH might “get its act together” to develop a trans-NIH human plasma initiative that would provide funding for future phases of the project. Six institutes — the National Cancer Institute, National Institute on Aging, National Institute on Alcoholism and Alcohol Abuse, National Institute on Diabetes and Digestive and Kidney Diseases, National Institute for Environmental Health Sciences, and National Institute of Neurological Diseases and Stroke — have committed funds to the HPPP pilot project. Corporate sponsors include Ciphergen, Agilent, Amersham, and Invitrogen.



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