A novel photoaffinity probe for analysing the phosphatdylinositol 5-phosphate interactome

Phosphatidylinositol 5-phosphate (PI5P) plays a crucial role in cellular signalling, cell proliferation, the DNA damage repair response, and gene transcription. However, the underlying mechanism of PI5P function in these cellular pathways is poorly understood. This lack of understanding results, at least in part, from the dearth of available chemical tools to enable the investigation of PI5P interaction with target proteins in the corresponding biological systems. Here, we report the design and synthesis of a novel phosphatidylinositol 5-phosphate-based photoaffinity probe. We show that the probe binds, and photo-crosslinks, to the known PI5P-interacting proteins, hUHRF1 and hTAF3, and undergoes copper-catalyzed azide click chemistry with an azide-functionalized TAMRA dye, allowing visualization of these proteins. In addition, we demonstrate that this PI5P photoaffinity probe interacts with a range of proteins in the lysate of cultured human cell lines, through conjugated to a TAMRA-azide dye and subsequent fluorescent visualization. The data presented here validate the novel photoaffinity probe as an effective molecular tool to enable more detailed investigation of the PI5P interactome.