Acid-Cleavable Guanosine Triphosphate-Photoaffinity Probe for Global Profiling of Guanosine Triphosphate-Binding Proteins and 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.