Small molecule probes with distinct reactivities are useful tools for the identification and characterization of protein modifications and function. Herein, we show that hydrazone probes with an N-carbamate structural motif react differently from N-carbamates within the human proteome. Mass spectrometry analysis of probe-treated mammalian cell lysates identified several proteins that were covalently modified by the hydrazone probes, including the cytidine deaminase APOBEC3A. We used this enzyme as a model to explore the reactivity of the probes with amino acid residues using LC-MS/MS. Both reactive serine and cysteine residues outside of the enzyme active site were covalently modified. A 1-napthol leaving group provided the most extensive reactivity. These results confirm a unique chemotype for hydrazone probes which can be further optimized to target distinct targets of the human proteome.
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