Proteins can undergo an estimated 200 different post-translational modifications. From the perspective of human disease, one of the most important of these is the enzymatic addition of farnesyl, a precursor in the cholesterol synthesis pathway, as farnesylation of the signaling protein Ras is involved in oncogenic transformation for many cancers. Many other proteins are farnesylated, typically at a conserved 'CAAX box' motif, but not all such motifs are farnesylated, complicating target identification.

Inspired by Carolyn Bertozzi's Staudinger ligation (see box, “Chain Reaction”), University of Texas Southwestern Medical Center (Dallas, TX) researcher Yingming Zhao and his colleagues developed a Staudinger-based tagging strategy. A recent article from the Proceedings of the National Academy of Sciences, Zhao's team demonstrates the first test of their technique — which they call 'tagging-via-substrate,' or TAS — for the identification of farnesylated proteins.

They began by treating cells with farnesyl that had been modified to include an azide group. The cells were lysed, and the azidofarnesylated proteins subjected to Staudinger ligation with a phosphine-biotin probe, then affinity-purified and identified by mass spectrometry.

After confirming that TAS can efficiently label known farnesylation targets such as Ras and Hdj-2 in vivo without impeding their function, Zhao's team followed up by examining global protein farnesylation in COS-1 cells. They identified 17 'CAAX box' proteins, including several lamins, DnaJ proteins and Ras superfamily members, with only minor interference from a few endogenously biotinylated proteins.

Although this is far from a complete index of farnesylated cellular targets, Zhao's group is encouraged and is currently working to optimize reaction efficiency. Eventually, says Zhao, “we want to scale up in order to catalog all of the farnesylated proteins and to identify the true targets for farnesyltransferase inhibitors under clinical evaluation.” He suggests that TAS could one day become a generalized tool for proteomics, and notes, “I think that this strategy will have a lot of applications with other modifications outside of farnesylation.”