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Mouse serum proteomics
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teins in the fractions are digested into peptides, which are then run on strong With scientists racing to identify the cation-exchange columns and analyzed components of various human proteby micro-reversed-phase LC/MS/MS. omes, little attention has been given to According to Conrads, the key to the proteins in the model organism Mus analyzing whole serum is the high-resomusculus—the mouse. “I think the hope lution separations strategy. “Separation for a [diagnostics] home run has really is really what will get you over the dymotivated researchers to go directly to namic-range constraints in any samhumans,” says Tom Conrads at the National Cancer Institute (NCI) in Frederick, Md. “Everybody wanted to move to [humans], hoping that study design and sample size would transcend the variability due to individual lifestyle and genetics.” He points out that the discovery of biomarkers and disease mechanisms is much easier in mouse models because one can control these factors, which are impossible to control in humans. As described in this issue of JPR (pp 1561–1568), Conrads and his co-workers used a new separations strategy to identify 4567 unique proteins in the mouse serum proteome. Because a few proteins in serum constitute the bulk of the total protein content, many researchers remove a few of the most highly abundant proteins from serum and plasma samples before performing separation steps. But Conrads points out, “The worry in any depletion scheme is that you may actually be depleting [potenPartitioning the proteome. Schematic of the new septial biomarkers] from serum.” arations strategy. Some high-abundance proteins, such as albumin, may bind to disple,” he says. “We went to great lengths ease-related proteins, pulling them out to optimize the separations that we of the sample in a depletion procedure. were doing, both at the protein level Conrads says that he and his coand at the peptide level.” workers devised a method that uses After performing the LC and MS conventional instrumentation and steps, the researchers were left with a “doesn’t involve depletion beforehand, mountain of data. “The greatest chalso we can be more inclusive rather lenge is always bioinformatics,” says than potentially exclusive.” In the new Conrads. The researchers carefully separations scheme, the researchers sorted through all of the peptides that load whole serum onto a weak anionthey had identified to ensure that each exchange column that is connected in one matched only a single unique protandem to a weak cation-exchange coltein in the database. “A lot of fragments umn. They separate the columns and are floating around in serum, and it’s elute fractions from each one. The pro-
© 2005 American Chemical Society
important to know whether you have identified a unique protein or whether you’ve identified a [member of a protein class],” says Conrads. Therefore, each of the 12,389 peptides that they reported uniquely matched one of 4567 proteins. Many different types of proteins were identified in mouse serum with the new method. According to Conrads, the number of membrane proteins was striking, however. “I don’t think we are identifying holoproteins that are floating around in serum that we’ve digested,” he says. “We’re actually seeing the action of shedding and clipping of membrane proteins that are released into the serum. We are identifying [those fragments].” He adds that such clipped proteins could be disease biomarkers, and if that’s the case, then the new method is ideal for their detection. The researchers also analyzed their results in terms of signal transduction pathways, which could be useful for discovering disease mechanisms. “Ultimately, when you’re studying cancer . . . you need to be able to understand the output of a proteomics experiment in the context of pathways because that’s truly what cancer is; it’s a function of deranged signal transduction pathways,” Conrads explains. For example, they identified 16 members of the -catenin/Wnt pathway, which has been widely implicated in cancer. Now that HUPO’s Plasma Proteome Project has released its database of human plasma proteins (Proteomics 2005, 5, 3226–3245), Conrads and his co-workers are starting to compare these proteins to their list from the mouse serum proteome. They are also working on mouse models of human cancer, which is a high priority for the NCI. “The hope is to take a very discovery-based approach in mouse [models] and use [these] discoveries to do targeted investigations in humans,” says Conrads. —Katie Cottingham
Journal of Proteome Research • Vol. 4, No. 5, 2005
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