This affinity probe captures GTP-binding proteins for proteomics analysis. Image by Beth Cisar.
GTP-binding proteins make up huge and ubiquitous portions of proteins that have essential functions in cell signaling, trafficking, cytoskeletal structure, nucelotide metabolism and translation. In a recent paper in the Journal of American Chemical Society, Hugh Rosen and colleagues at The Scripps Research Institute described a GTP affinity probe for proteomics. The probe, say the investigators, should help researchers identify a variety of GTP-binding proteins in a single shot by mass spectrometry.
The probe was designed to solve a problem in the Rosen laboratory, explains Beth Cisar, the first author on the paper. The laboratory is interested in the sphingosine-1-phosphate (S1P) receptors, which are five G-protein coupled receptors (GPCRs) that are needed for the proper functioning of many systems, including the cardiovascular and lymphatic systems. GPCRs work in concert with a variety of GTPases. But the investigators didn’t have a tool that would let them comprehensively pull out, in one shot, all the GTP-binding proteins involved in S1P receptor signaling pathways.
So the investigators designed the GTP-BP-yne probe. The molecule covalently binds to GTP-binding proteins through a photocrosslinking reaction and has an alkyne handle that lets reporter tags, such as avidin and rhodamine, latch onto
it. This allows the investigators to analyze GTP-binding proteins either by mass spectrometry or in-gel fluorescence.
With their probe, the investigators pulled down many GTP-binding proteins found in human embryonic kidney cells, which were their test case, by mass spectrometry. Rosen and colleagues got 33 proteins, including members of several known classes of GTP-binding proteins.
But, much to their surprise, they also found ATP-binding proteins, including three related kinases called Src, Lyn and Yes. Src had been shown previously to bind GTP, but Lyn and Yes’ ability to do so was unknown until this study. This finding highlights “the idea that purine nucleotide selectivity is often not as strict as it is thought to be,” notes Cisar.
There are fluorescent or radioactive GTP analogs to study GTPases and other
GTP-binding proteins. There is a commercial GTP probe for proteomics, but Cisar says that their GTP-BP-yne probe labels targets via a different mechanism that allows identification of not only GTP-binding proteins but also proteins that bind GTP-binding proteins.
With their probe now in hand, Cisar says, the investigators are going back to their original problem, which was the study of S1P receptor signaling pathways. “We are particularly interested in determining whether the probe can distinguish between targets’ active and inactive states,” she says.
