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For protein biochemists, 1DE has been the “go-to” method for protein analysis. But as proteomics gained momentum, many researchers switched from this labor-intensive method to chromatography- and IEF-based fractionation approaches. As two recent JPR papers show, however, the old 1DE workhorse still may be the best overall fractionation method for many proteomics applications (DOI 10.1021/pr901063t; DOI 10.1021/pr901072h). In both papers, 1DE was integrated into a typical LC/MS/MS workflow (GeLC/MS/MS), and its performance was compared with that of other methods. “What’s interesting is that the two studies have some differences in their individual sampleprocessing steps and use rather different starting samples, and yet they both concluded that the greatest depth of analysis comes from GeLC/MS/ MS,” says David Speicher 1DE reigns of the Wistar Institute. Leonard Foster of the University of British Columbia (Canada), the corresponding author of one of the JPR studies, got into the methods comparison game after he noticed that certain fractionation methods were becoming popular. He and his group began to wonder how these techniques stacked up against each other. “There were a few sporadic reports in the literature comparing one method to another, but we felt that nobody had really done a rigorous comparison of all the popular methods,” he explains. For their comparison study, Foster and colleagues analyzed lysates from honey bee heads with 1DE, strong-cation exchange (SCX) chromatography, an IPG-based IEF system, and a solutionbased IEF system. Proteins and peptides were fractionated with IEF methods. However, 1DE is typically applied only to proteins, so a 10% SDS-PAGE gel was used as the third protein-fractionation method. Similarly, SCX was implemented for peptide separation only. All analyses were performed in triplicate.
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To draw meaningful conclusions, the researchers controlled for several parameters, such as differences in sample handling among the methods. They also controlled for the amount of instrument time, a factor that Foster says is often overlooked. For example, an approach that produces 20 fractions will identify more proteins than an approach that produces only 12 fractions
The team already routinely fractionated samples with 1DE or SCX, but they also noticed that some groups fractionated secretomes with reversedphase LC. So, the researchers conducted a comparison study to determine which method was best for secretome analyses. They analyzed the performances of these methods in triplicate with the secretome of a lungcancer cell line. Two types of 1DE gels were assessed: a 10% acrylamide gel and a 4-12% acrylamide gradient gel. “Normally, it would not matter that much, but a number of important secreted proteins involved in signaling are in the lowmolecular-weight range,” says Piersma. These proteins are not resolved well on a standard 10% gel but are easily visualized with the gradient gel. Again, 1DE provided the most protein identifications, and it was the most reproducible method. The team also went a step further and applied the gradient 1DE method to an experimental case in a collaboration with scientists at ProQinase (Germany). With 1DE, they identified several potential biomarkers in the secretomes of mouse embryonic fibroblasts that express a particular receptor (IGF1R). The group verified two of these candidates with ELISAs. Finally, serum samples of mice that express the receptor in tumors revealed that the levels of one of these proteins, osteopontin, correlated with tumor growth in vivo. Although he says comparison studies are important, Jacek Wis´niewski of the Max Planck Institute for Biochemistry (Germany) faults these papers for pitting a tried-and-true method (1DE) against newer methods that may not yet be optimized. However, Speicher applauds the groups for performing replicates and comparing so many diverse methods. He adds that these projects, which were massive undertakings, will give investigators useful information and will likely motivate others to perform similar studies in the future. —Katie Cottingham LEONARD FOSTER
1DE proves its worth... again
supreme. A representative 1DE gel is shown.
simply because it uses more of the mass spectrometer’s analysis time, he explains. 1DE provided the best coverage and was the most reproducible method. A whopping 90% of all of the proteins identified in the study were identified with 1DE. The IPG-based IEF system had the best recovery rate (92%), but 1DE wasn’t far behind (81%). 1DE’s good recovery surprised Foster, who had always figured that about half of the starting material would not be recovered with the technique. On the basis of these results, Foster and colleagues deemed 1DE the best overall method. In the second JPR paper, Sander Piersma, Connie Jime´nez, and colleagues at the VU University Medical Center (The Netherlands) tested fractionation methods for secretome analyses. Piersma explains that as tumors form and progress, they may secrete proteins into the circulation system. Such proteins could serve as biomarkers.
Journal of Proteome Research • Vol. 9, No. 4, 2010
10.1021/pr100103x
© 2010 American Chemical Society
