Nitrilase-Activatable Noncanonical Amino Acid Precursors for Cell-Selective Metabolic Labeling of Proteomes

Cell-selective protein metabolic labeling is of great interest for studying cell–cell communications and tissue homeostasis. We herein describe a nitrilase-activatable noncanonical amino acid tagging (NANCAT) strategy that exploits an exogenous nitrilase to enzymatically convert the nitrile-substituted precursors to their corresponding noncanonical amino acids (ncAAs), l-azidohomoalanine (Aha) or homopropargylglycine (Hpg), in living cells. Only cells expressing the nitrilase can generate Aha or Hpg <i>in cellulo</i> and metabolically incorporate them into the nascent proteins. Subsequent click-labeling of the azide- or alkyne-incorporated proteins with fluorescent probes or with affinity tags enables visualization and proteomic profiling of nascent proteomes, respectively. We have demonstrated that NANCAT can serve as a versatile strategy for cell-selective labeling of proteomes in both bacterial and mammalian cells