Labeling Preferences of Diazirines with Protein Biomolecules

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.