Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

Abstract

The chemical proteomic properties of five diazirine-based, fully-functionalized photoaffinity tags, including a newly developed, minimal tag, were compared. This study provides guidance for the development of new photoaffinity probes.

Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules

“…In addition, the luminescence signal is considerably diminished upon co-treatment of either terminal binding molecules (FKBP-c and p300-c) in a concentration-dependent fashion (Figure d,e and Figure S1c), confirming that observed ternary complex formation is dependent on the simultaneous binding of both BRD-p300 and FKBP12 F36V . To verify that AceTAG molecules can engage their protein binding partners in cells, we constructed “fully functionalized” photoaffinity probes of both the FKBP12 F36V (FKBP-p) and p300/CBP (p300-p) ligands and confirmed respective binding to recombinantly expressed p300 and FKBP12 F36V can be efficiently competed when cells are co-treated with increasing concentrations of AceTAG-1 (Figure f,g). Together, these biochemical data indicate that AceTAG 1–3 effectively engage FKBP12 F36V and p300/CBP and are capable of inducing complex formation.…”

Photo-Brook rearrangement of acyl silanes as a strategy for photoaffinity probe design

“…Similar diazirine probes have been used to capture specific interactions in proteins and whole cells. 31 Acyl silane (+)-JQ1 probe 16 , the analogous dimethyl-substituted probe 16-Me , and diazirine probe 16-DA were incubated in cell lysate and irradiated for 30 min on ice. Following click reaction, visualization by in-gel fluorescence shows intense fluorescence for irradiated samples treated with probes 16 and 16-DA , with minimal thermal labeling observed (Fig.…”

Evaluation of site-diversified, fully functionalized diazirine probes for chemical proteomic applications

“…Recent studies suggested practical instructions on tag design, such as utilizing different types of tags 25 and changing the electrostatic charge of the probe 26 . However, in our case of EGFR, it was found that the labelling efficiency of P5 is independent of its activity.…”

“…Recently, a comparative study of different "fully functionalized" diazirine tags found that tag has a substantial impact on labelling pattern of photoaffinity probes. 25 A series of studies focused on the diazirine reactivity indicated significant amino acid labelling preferences of diazirine. [26][27][28][29] Therefore, good binding between probe and target protein(s) cannot guarantee the accessibility of diazirine to protein residues, which might partially explain the false-negative labelling issue.…”