In their experiments, proteomics researchers routinely use immobilized metal affinity chromatography (IMAC) to enrich for phosphopeptides and phosphoproteins, but these resins are not specific for phospho groups. In fact, IMAC resins will bind to any negatively charged groups, including carboxylic, aspartic, and glutamic acids. To overcome this challenge, some researchers have used a methyl esterification method (Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233–6237) to methylate these acidic groups so that they cannot bind to IMAC resins. Therefore, the background of nonphosphorylated peptides is reduced. Another advantage of the method is that labeling methylated peptides for quantitative analyses is straightforward (Rapid Commun. Mass Spectrom. 2001, 15, 1214–1221). A researcher simply treats one methylated sample with deuterated methanolic HCl and the other with a nondeuterated version. “The phosphopeptide will at least have the carboxylic end, the C-terminus, and if it has any acidic residues on it, they will also get labeled,” says Daniel Figeys at the University of Ottawa (Canada). Compared with other, more popular isotopic labeling protocols, this one is much less expensive and can be applied to samples from bodily fluids or tissues. Although the reaction has its advantages, it is rarely performed for phosphoproteomics studies. Figeys explains that the required reagents are chemicals that typically are not found in biology labs. In addition, the reaction is a bit finicky—if any water is present, the reaction will not go to completion. Undeterred, Figeys, Jeffrey Smith, and colleagues at the University of Ottawa revisited the methyl esterification procedure. As they describe in this issue of JPR (pp 3174–3186), they applied the method to the study of the phosphoproteome of differentiating cells. To investigate phosphorylation dynamics in differentiating cells, Figeys and colleagues studied P19 cells, which are mouse cells that can be induced to specialize into different cell types, depending on the stimulus. For example, retinoic acid (RA) induces the cells to become neurons, and dimethyl sulfoxide (known as DMSO) induces the cells
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DANIEL FIGEYS
The phosphoproteomics of differentiation
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Differences in differentiation. Schematic of the protocol that researchers used to determine the changes in the phosphorylation states of proteins that occur during neuronal differentiation.
to become muscle. In this study, the researchers treated one batch of P19 cells with RA and extracted proteins at day 15. Another batch was left untreated; these cells became the day 0 sample. Several control experiments were conducted to ensure that the methyl esterification protocol would work reproducibly. After the researchers optimized the conditions, they extracted proteins from both samples. The proteins were digested, methyl esterified, differentially labeled, and mixed. Then, the mixture was run on an IMAC column, and enriched phosphopeptides were analyzed by LC/MS/MS. The identification of phosphopeptides is a challenge for proteomics researchers, says Figeys. “Interestingly, the scores that you get from the search engine, at least in terms of phosphopeptides, don’t mean much because we have observed some phosphopeptides with scores down to zero with Mascot that were right and others that have very high scores that are wrong,” he points out. “So you really need to have further data interpretation strategies when you are doing posttranslational modifications.” The scientists manually examined the data, and they eventually developed an algorithm (available at www.oisb.ca/downloads.htm) to speed up the analysis. The phosphorylation states of 56 proteins were different at day 0 and day 15, and most of the phosphorylation sites identified in the study were novel. The researchers also investigated whether these proteins had similar functions or interacted with one another. Therefore,
an interaction network was constructed, and 20 of the proteins were linked directly or through an interaction with one additional protein. “When you look at the proteins that are linked, you can see that many of them are associated with cell differentiation,” says Figeys. “From that point of view, that [finding] validated our hypothesis, which is that you can use phosphoproteomics to study those types of processes.” One interesting protein identified in the study is called Janus kinase (Jak3). According to reports, the level of this protein increases during differentiation. Figeys and colleagues observed a >7.5-fold decrease in the abundance of a Jak3 phosphopeptide with RA treatment, however. The two phosphorylations on this peptide are located near Jak3’s kinase domain. “So, we postulated that the function of the phosphosites is somewhat inhibitory for that protein,” he explains. Therefore, when Jak3 activity is needed, its level increases, and it probably becomes dephosphorylated. Figeys gives much of the credit for the eventual success of the optimized method to Smith, a postdoc in his laboratory. Figeys says that Smith’s careful analysis was a good demonstration of how taking the time to do the work properly pays off. Now that the researchers have optimized the process, they plan to perform a larger differentiation study. Figeys says that the group is gearing up to follow a time course to observe the changes in phosphorylation between days 0 and 15. —Katie Cottingham
Journal of Proteome Research • Vol. 6, No. 8, 2007 2919
