ZURICH—To get a clear picture of the complexity of a given proteome, researchers have to be confident they are seeing all the participating proteins and peptides. Unfortunately, different isolation methods are biased toward different proteomic subpopulations. With this in mind, researchers at the Swiss Federal Institute of Technology and Seattle's Institute for Systems Biology performed a systematic analysis of three phosphoproteomic isolation methods to determine how much the three methods overlap.
As they report in Nature Methods, the researchers used phosphoramidate chemistry, immobilized metal affinity chromatography, and titanium oxide separation to isolate phosphopeptides from a trypsinized Drosophila cell extract.
Following the isolations, the researchers used LC-MS to compare protein patterns, finding modest overlap—about one-third—between the three isolation methods. They then used LC-MS/MS to identify specific peptides and found that the ~33% overlap was consistent at the sequence level.
The researchers were unable to make a definitive statement regarding the three methods because a "gold standard" complete phosphoproteome does not exist. However, they noted their results implied "that none of the methods by itself is currently able to comprehensively analyze a phosphoproteome."