FIGURE 7.

Deconvoluted ESI-MS spectra of sfGFP containing ncAAs at position 27. Full-length sfGFP-3, sfGFP-4, and sfGFP-5 were expressed using the N-PylRS⋅tRNA^Pyl pair in the presence of ncAA 3, 4 (2 mM) or 5 (1 mM), respectively, in GMML medium (sfGFP-3 and sfGFP-4) or LB medium (sfGFP-5). The purified protein samples were subjected to ESI-MS for identification of the incorporation of defined ncAA. Deconvoluted ESI-MS of ncAA 3–5 incorporated sfGFP at position 27 are shown above. sfGFP-X in which X represents one of the ncAA 3–5 was incorporated into sfGFP at position 27. The calculated molecular masses of sfGFP-3 are 28,030 and 27,899 Da (-Met); the observed molecular masses are 28,030 and 27,899 Da (-Met). The calculated molecular masses of sfGFP-4 are 28,046 and 27,915 Da (-Met); the observed molecular masses are 28,046 and 27,915 Da (-Met). The calculated molecular masses of sfGFP-5 are 27,974 and 27,843 Da (-Met); the observed molecular masses are 27,974 and 27,843 Da (-Met). The detailed electrospray and deconvoluted mass spectra are shown in Supplementary Figures S1–S3.