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Cleaning up serum
remove the majority of the high-abunThe 2-D DIGE signals from lowdance proteins,” explains McCutchenabundance proteins were enhanced by Biomarker discovery in serum would be Maloney. 76% after the researchers used an antia lot easier if only low-abundance proOne issue McCutchen-Maloney and body-based removal method to remove teins weren’t masked by high-abundance colleagues were concerned about was 6 high-abundance proteins from serum. proteins, such as albumin. In this issue the loss of low-abundance proteins Crude serum initially showed only 850 of JPR (pp 1120–1127), Sandra McCutthrough protein–protein interactions protein spots with 2-D DIGE. When only chen-Maloney and colleagues at Lawwith high-abundance proteins. Indeed, albumin was removed from the serum, rence Livermore National Laboratory albumin is known to be a carrier/trans1200 protein spots showed up on the report on a series of experiments that port protein that binds to other proteins gel. When all 6 high-abundance proteins they conducted to determine the best in blood. were depleted, the gel displayed >1500 technique for removing albumin and But when the investigators analyzed protein spots. other high-abundance proteins from the high-abundance protein fractions, The investigators compared two serum. The investigators found that they were unable to detect low-abuncommercial antibody-removal techantibody-based methods are more dance proteins. “To be honest, what we niques with two commercial ioneffective than those based on ionwere most surprised about was that exchange chromatography kits. One exchange chromatography. there weren’t more proteins Concentrations of albucarried over in the highmin and other high-abunabundance fraction,” says dance proteins can be 10 McCutchen-Maloney. “I think orders of magnitude greater it’s a statement to the specithan those of low-abundance ficity of the antibodies and proteins. “We’re interested in that antibody-based methods biomarkers for presymptomare really an improvement atic detection of plague and over previous methods to anthrax. We realized very early deplete serum of high-abunon that the proteomic view we dance proteins.” were able to obtain, without But McCutchen-Maloney the depletion of high-abundoes point out that just dance proteins, was pretty because they can’t detect limited,” says McCutchencarry-over low-abundance Maloney. Besides albumin, proteins in the high-abunthe researchers looked into dance protein fraction by removing five other hightheir current visualization abundance proteins: IgG, IgA, technique, it doesn’t mean transferrin, haptoglobin, and they aren’t there. She adds, antitrypsin. High-abundance protein removal. A 2-D DIGE image shows six high“As sensitivity and detection The investigators used a abundance proteins (red), serum after removal of high-abundance proteins capabilities improve, then we technique called 2-D differen(blue), and protein standards (green). would most likely want to tial gel electrophoresis (2-D look at the high-abundance DIGE) to characterize depletprotein fraction again to see if anything of the antibody-based methods only ed serum. In this variation of traditional else is being carried over.” removed albumin, while a second 2-D gel electrophoresis, three separate McCutchen-Maloney and colantibody-based kit depleted all six samples are each labeled with a differleagues are now using their high-abunhigh-abundance proteins. McCutent fluorescent dye. The three samples dance protein removal protocol to study chen-Maloney and colleagues found are separated on a single gel for multisamples of serum exposed to various that the antibody-based methods were plex analysis and co-detection of propathogens. “We’ve been able to detect more efficient than ion-exchange teins. McCutchen-Maloney says, “You proteomic differences that can not only chromatography. get quantitative differences between distinguish host response between difThe antibody-based method “is those samples because they are all [run] ferent pathogens, but [also among] more specific and more complete. Ion on the same gel. If one protein spot pathogens of the same species,” says exchange would leave some of the highincreased or decreased, you would see McCutchen-Maloney. abundance protein still in the sample, more or less of the fluorescent dye rela—Rajendrani Mukhopadhyay whereas the antibody methods would tive to the other samples.”
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Journal of Proteome Research • Vol. 3, No. 6, 2004
© 2004 American Chemical Society
