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Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.Protein-protein interactions are vital to biological processes, but the shape and size of their interfaces make them hard to target using small mo