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HUPO Plasma Proteome Project’s first fruits The publication of a special issue of the journal Proteomics marks the end of the HUPO Plasma Proteome Project (PPP) pilot phase. Begun in 2002, the international effort of 31 participating laboratories has released its preliminary results, including the identification of 3020 plasma proteins. Two important recommendations from the PPP are to use plasma instead of serum and to deplete samples of the most abundant proteins. Among those researchers studying blood, the question of whether to analyze serum or plasma is a hot topic. Alex Rai and his co-workers from eight laboratories in the U.S., Germany, and France compared serum and plasma samples and also examined the effects of storage conditions and protease inhibitor (PI) treatment (Proteomics 2005, 5, 3262–3277). “Everyone wants a onesize-fits-all approach, but I don’t think we’re going to have that,” says Rai. He adds that the choice of sample and handling methods depends on the purpose of the experiment and the analysis techniques that will be used. Although a standard operating procedure for sample handling is not in the cards for now, Rai and his collaborators list a few recommendations and caveats. The group suggests that researchers use plasma, especially when searching for low-molecular-weight species, such as peptides. They found that serum contained clotting-induced peptides that interfere with the identification of other peptides. Although freezing samples in liquid nitrogen is suggested, this
PPP bioinformatics Richard Simpson and colleagues at six laboratories in Australia and the U.S. evaluated five MS/MS search algorithms (Proteomics 2005, 5, 3475–3490). Mascot, X!Tandem, and Sonar were the most specific, although Spectrum Mill and Sequest were the most sensitive. Mascot outperformed the other programs when identifying true positives at a specified false-positive rate, but use of the rescoring algorithm Peptide Prophet increased the number of correct identifications generated by Sequest. The researchers recommend the application of two or more search algorithms to proteomics datasets.
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method is not practical for most clinical laboratories. In that case, Rai says that a good compromise is to store samples at –80 °C. Rai says that researchers must be careful about which PIs are used and how those inhibitors act, especially when separating proteins by 2DE. PI cocktails containing the inhibitor 4-(2aminoethyl) benzenesulfonyl fluoride resulted in smearing, shifts in isoelectric point, and the appearance of additional protein spots. Rai also says, “With any methodology that is going to be used to look at the low-molecular-mass proteome, you will overshadow endogenous components in your sample if you add high amounts of certain PIs, and other PIs can change the protein profile by covalently binding to endogenous proteins.” Many proteomics researchers routinely deplete a few of the most abundant proteins in serum or plasma before subjecting samples to further analysis. William Barrett at Agilent Technologies explains, “By removing the high-abundance proteins, you’re providing easier access to the lower-abundance proteins of interest. The secondary reason is that you can see and load more protein [on 2-D gels] when you remove the higherabundance ones.” Barrett and his co-workers at Agilent compared the performance of the company’s column—the multiple affinity removal system (MARS), which contains antibodies against the six most abundant proteins in serum and plasma— with that of a modified Cibacron blue dye that removes albumin (Proteomics 2005, 5, 3304–3313). David Speicher’s group at the Wistar Institute compared the Agilent column with two variations of the Cibacron blue dye and two antibody spin columns (Proteomics 2005, 5, 3292–3203). Both groups observed the greatest depletion of targeted proteins and the greatest specificity with the MARS column. According to the protein sponge theory advocated by some researchers in the field, highly abundant proteins may bind biomarkers. Therefore, depletion methods may remove important proteins and peptides. Because the MARS column was specifically designed to reduce protein–protein interactions before depletion, however, Barrett says that researchers can examine both the
Journal of Proteome Research • Vol. 4, No. 5, 2005
Mystery revealed. The PPP has identified
3020 proteins in plasma, which is a component of blood.
high- and low-abundance fractions. Researchers who are interested in albumin-bound peptides and proteins can use alternative methods. Despite the MARS column’s performance, Speicher and colleagues obtained a surprising result when they used 2DE and MS to identify the proteins in the flow-through fraction. “Many of [the spots contained] more than one protein, and the proteins we identified were minor forms of either high- or medium-abundance proteins,” says Speicher. He adds that researchers should, therefore, deplete even more proteins and that alternatives to 2DE should be used. For example, GenWay Biotech’s new microbead depletion technique removes 12 of the most abundant proteins (Proteomics 2005, 5, 3314– 3328). Although the pilot phase has ended, much work remains for the PPP participants. At a PPP workshop in Munich, Germany, two days before the start of the HUPO 4th Annual World Congress, the subject of future directions was actively debated. Possible projects include further work on sample handling procedures, subproteome analyses, informatics development, and diseaserelated studies. —Katie Cottingham
