Harvard University researchers have developed a mass spectrometry-based technique for providing detailed information on patterns of protein phosphorylation.
The method, detailed in a study published in the current edition of Nature Methods, uses full-length SILAC-labeled proteins and high-resolution mass spec for site-specific quantification of multiple phosphorylation events, enabling in-depth analysis of protein kinases and protein signaling mechanisms.
In addition to being a tool for basic phosphoproteomic work, the technique could prove particularly useful in pharmaceutical research, where kinase inhibitors have emerged as a significant area of interest, Judith Steen, a Harvard researcher and leader of the study, told ProteoMonitor.
Traditional kinase assays use 32P-labeled phosphate to image protein phosphorylation levels after incubation with a given kinase. This approach offers little information about the particular phosphosites affected, however, Steen noted.
"It just tells you whether [the protein] is phosphorylated or not," she said. "But it doesn't tell you anything about the mechanisms of the phosphorylation."
Increasingly, Steen said, researchers are interested in not just levels of post-translational modification, but the patterns of modification as well.
"It's becoming very clear to us that modifications work in concert with each other," she said. "It's not just one particular site that is important, but it's a consort of sites that makes [a protein] perform a particular function or interact with a particular protein. So … you have these patterns of modification on a protein, and I think that is the next frontier."
Called FLEXIQinase, the Harvard team's technique relies on parallel reactions – one using an active form of the kinase of interest and a "light" SILAC-labeled protein; and the other, the control, using an inactivated form of the kinase and a "heavy" SILAC-labeled version of the same protein.
These reactions are followed up by mass spec analysis – on a Thermo Scientific LTQ Orbitrap Classic in the case of the Nature Methods paper – to determine the changes in phosphorylation. If there is no change in the peak ratio of unphosphorylated peptides between the two samples, it can be said that the kinase doesn't phosphorylate that peptide. If the kinase does phosphorylate a given peptide, there should be decreased or absent signal from the unphosphorylated version of the heavy peptide.
Because the technique provides information on specific peptides, it enables researchers to investigate specific patterns of kinase activity and the resulting patterns of protein phosphorylation.
"The method could be used for multiple types of assays," Steen said. "You could do it for a one-kinase assay, for a two-kinase assay. You can imagine using it for time-course assays where a protein is being phosphorylated by a kinase and you want to understand which sites are phosphorylated first … you could actually measure that quantitatively over time."
In their study, Steen and her colleagues used the method to investigate JNK-dependent GSK3β activity on the doublecortin, or DCX, protein, identifying patterns of crosstalk between these two kinases as well as details on patterns of DCX phosphorylation and their influence on GSK3β activity.
"DCX is a very important protein in the brain that is involved in stabilizing microtubules," Steen said. "And this basically allows us to understand the mechanisms of [DCX] phosphorylation by JNK and GSK3βm." She added that her lab is also using the technique to study phosphorylation of tau protein, which has been implicated in diseases including Alzheimer's.
"One particular example where we are trying to apply this method right now is looking at aggregates in the brain such as tau, which can be highly phosphorylated," Steen said. "We know that there are some kinases that might be involved and there are some small-molecule inhibitors [to those kinases]. So, basically, you could treat a mouse with a particular inhibitor and [use the method to] see how the phosphorylation patterns change and how the purified aggregates change in those particular samples."
"There are references in literature that suggest that proteins such as tau can change their function based on modifications – whether it be phosphorylation or acetylation – so we are interested in finding out which modifications are actually affecting their aggregation," she said.
Beyond this neurological work, the technique could prove useful for kinase inhibitor research generally, Steen said.
"We hope this method will be used in drug studies to understand the mechanisms of the kinases and how inhibitors change phosphorylation patterns of a particular protein," she said. "You can imagine how it might be quite powerful as a tool because if you think about how people have been looking at phosphorylation of proteins using just a basic kinase assay, it's really been a very simple approach."
Although the Nature Methods research was in vitro, the technique could also be used for in vivo work, with SILAC-labeled cells, for instance. The scientists are currently refining it for use across large signaling pathways, Steen said, noting that her team is working with developers of the University of Washington's Skyline mass spec software to develop mass spec workflows for this type of research.
The technique, she added, could be applied to other post-translational modifications, as well.
The researchers have patented the technique and are discussing potential licensing deals, Steen said, though she didn't name any potential partners.
The FLEXIQinase assay stems from the researchers' previous work on a mass-spec based PTM quantification system called FLEXIQuant (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification) that they detailed in a 2009 paper in the Journal of Proteome Research.
That technique, as well as the FLEXIQinase method, relies on a wheat germ extract-based protein expression system that, Steen said, allows for cheap, rapid expression of the proteins used in the assays.
"That [expression system] made [the assays] much easier and cheaper to do," she said. "So now we've made libraries of these expression vectors and since with mass spectrometry you don't need much protein you can use very small amounts of wheat germ extract, so it costs a few dollars per protein and you can do it overnight."
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