Labeling Preferences of Diazirines with Protein Biomolecules

Abstract

<p>Diazirines are widely used in photoaffinity labeling (PAL) to trap non-covalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Here, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that aryl-fluorodiazirines react primarily through a carbene intermediate, while alkyl diazirines generate a reactive alkyl diazo intermediate on route to the carbene. The generation of a reactive diazo intermediate leads to preferential labeling of acidic amino acids in a pH-dependent manner. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why these probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive-charge tend to produce higher labeling yields. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome, and will facilitate probe design and interpretation of biomolecular labeling experiments with diazirines.<b></b></p>

pChem: a modification-centric assessment tool for the performance of chemoproteomic probes

“…6, Supplementary Tables 1 and 4) . Notably, during the preparation of this manuscript, a recent study also systematically uncovered the labeling preference of diazirine probes toward acidic amino acids 31 , in accordance with our observation to some extent.…”

Development of photo-lenalidomide for cellular target identification

“…Additionally, the strategy will not report on target proteins that lack an appropriate amino acid environment due to insufficient labeling of the probe. 22 Finally, the proteome enriched by photo-lenalidomide requires validation by competition with lenalidomide, as photolenalidomide and PAL can potentially label non-specific targets. Evaluation of whether an identified target protein is part of a ternary complex with CRBN, or it is CRBN-independent additionally requires followup studies.…”

“…Proteins that are selectively enriched represent direct binding events with the chemical probe, which can yield new targets of small molecules in combination with competitive displacement. We have been characterizing diazirine chemistry for its ability to yield novel targets and binding site measurements of small molecules, 22 with the objective of expediting the discovery of novel molecular glues, like rapamycin, 23 sanglifehrin, or lenalidomide, and the protein complexes that underpin their biological activities. However, to date, the propensity of PAL to report on protein complexes mediated by a molecular glue within cells remains undetermined.…”

Protocol for clickable photoaffinity labeling and quantitative chemical proteomics

“…These controls include the probe vs. probe, probe vs. no probe and UV vs. no UV controls detailed in steps 9–12 during live cell photoprobe labeling. In addition, in some cases, certain photoreactive groups, such as alkyl diazirine, may have preferential reactivity rather than non-specific labeling ( West et al., 2020 ). Because the data analysis uses MS-based proteomics, the limitations of proteomics also hold for the overall protocol.…”