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

Abstract

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.

Triazine-pyridine chemistry for protein labelling on tyrosine

“…29 The biological significance and chemical properties make it an attractive residue for covalent probe development and bioconjugation. In recent years, several strategies for tyrosine labelling have been reported, [30][31][32][33][34][35][36][37][38][39][40][41] among which diazonium alkyne 42 and sulfur-triazole exchange (SuTEx) chemistry 6 showed very promising efficiency and chemoselectivity for protein tyrosine labelling in the whole proteome (Scheme S1, ESI †). SuTEx chemistry has been further applied in functional tyrosine profiling and phosphotyrosine site monitoring.…”

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation

“…This claim is strengthened by the selectivity observed in our mass spectrometry experiments, providing support for a model where the resulting BDI from 2 , or variants thereof, could be used as a small molecule covalent probe for activity-based protein profiling (ABPP) . Functional side chain profiling has been done with cysteine, lysine, and tyrosine, additional methods need to be explored in order to profile functional histidine residues. − Recent reports of alkyne-containing diazonium ions being used for proteomic profiling of cell lysate underscore the potential for aryl diazonium ions being used in this way . Future investigations into the global cellular effects of treatment with the BDI using pull-down probes and whole-cell proteomics are underway.…”

“…Curious as to why these residues were modified over the others, we looked to the sequence of PTP1B. Given recent reports of BDI derivatives modifying highly reactive tyrosine residues, we hypothesized that the unique microenvironment of these residues renders them prone to reactivity. Starting with the most statistically significant residues, H54 contributes to a magnesium binding site for crystallization, and is also implicated in celastrol binding, a known inhibitor of PTP1B .…”

“…Aryl diazonium ions have undergone a renaissance in the bioconjugation literature, ,− but their chromophoric properties enabled them to be one of the first reliable tools to modify proteins dating back to the early 20th century. ,, While useful, many aryl diazonium ions suffer from the requirement of generation immediately prior to the experiment due to their short half-lives in solution and general instability. Recognizing the promise of aryl diazonium ions as robust small-molecule covalent probes, triazabutadienes were developed as a new biochemical tool for their generation .…”

Protein Modification via Mild Photochemical Isomerization of Triazenes to Release Aryl Diazonium Ions

“…Alkynes have become a popular tool within bioconjugate chemistry because of their efficiency and bioorthogonality. ,, To synthesize a suitable ethynyl triazene, we chose to use 3-ethynyl aniline as the alkyne is only moderately withdrawing. Furthermore, others have already vetted the use of ethynyl aryl diazonium ions for protein labeling and orthogonal modification. − Starting with 3-ethynyl aniline, triazene 3 was synthesized in 83% yield (Supporting Information Figure S5). With 3 in hand, we next irradiated resorcinol in the presence of equivalent amounts of 3 to determine if it was capable of labeling.…”