Labeling Preferences of Diazirines with Protein Biomolecules

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, 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 alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine 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 in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Automated summary

The reactivity of alkyl and aryl diazirines with amino acids, proteins, and the cell proteome was systematically evaluated, revealing different labeling patterns. Alkyl diazirines preferentially label acidic amino acids in a pH-dependent manner, while aryl-fluorodiazirine labeling occurs mainly through a carbene intermediate. These findings help rationalize the enrichment of specific proteins by alkyl diazirine probes and the higher labeling yields of positively charged probes in cells and in vitro.