Acid-CleavableGuanosine Triphosphate-PhotoaffinityProbe for Global Profiling of Guanosine Triphosphate-Binding Proteinsand Their Active Sites

Guanosine triphosphate (GTP)-binding proteins function as molecular switches in cell signaling, playing critical roles in various biological pathways. Their dysregulation is associated with the causes and progression of many diseases. Systematic analysis of GTP-binding proteins would facilitate studies of related signaling pathways and drugs. Previously reported acyl-phosphate GTP-affinity probes, which react with and label lysine residues near GTP-binding pockets, have proven efficient in identifying labeling sites but suffer from poor stability due to their high reactivity. We report here new GTP-photoaffinity probes that employ a UV-triggered photoreactive group for covalent labeling of proteins, greatly improving probe stability. The inclusion of a terminal alkyne group allows labeled proteins to be tagged either with a fluorophore for fluorescence analysis or with a biotin group to enrich for LC–mass spectrometry (MS)/MS analysis. We further designed a GTP-N probe featuring an acid-cleavable P–N bond. The P–N bond enabled the release of GTP from labeling sites upon incubation under acidic conditions after labeling and enrichment, which reduced protein-modification mass shift and facilitated MS-based modification-site identification. This new method demonstrates good potential for identifying new GTP-binding proteins and systematically analyzing GTP-binding sites. These novel GTP-photoaffinity probes could be further applied in studying related biochemical mechanisms and in evaluating GTPase inhibitors.