An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

The
tyrosine residue of proteins participates in a wide range of
activities including enzymatic catalysis, protein–protein interaction,
and protein–ligand binding. However, the functional annotation
of the tyrosine residues on a large scale is still very challenging.
Here, we report a novel method integrating azo coupling, bioorthogonal
chemistry, and multiplexed proteomics to globally investigate the
tyrosine reactivity in the human proteome. Based on the azo-coupling
reaction between aryl diazonium salt and the tyrosine residue, two
different probes were evaluated, and the probe with the best performance
was employed to further study the tyrosine residues in the human proteome.
Then, tagged tyrosine-containing peptides were selectively enriched
using bioorthogonal chemistry, and after the cleavage, a small tag
on the peptides perfectly fits for site-specific analysis by MS. Coupling
with multiplexed proteomics, we quantified over 5000 tyrosine sites
in MCF7 cells, and these quantified sites displayed a wide range of
reactivity. The tyrosine residues with high reactivity were found
on functionally and structurally diverse proteins, including those
with the catalytic activity and binding property. This method can
be extensively applied to advance our understanding of protein functions
and facilitate the development of covalent drugs to regulate protein
activity.