Supplementary Figure 12: Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms highly efficient selenocysteine incorporation and correct formation of a diselenide bond.
From: Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains
(a) Mass spectrum of intact EcDHFR U39-U85. (b) Deconvoluted mass spectrum of EcDHFR U39-U85. (c) Experimentally determined monoisotopic mass (19054.13 Da) matches the theoretical mass (19054.14 Da) of EcDHFR U39-U85 containing a diselenide bond. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (d) UVPD mass spectrum of the 17+ charge state. (e) UVPD fragment map shows a lack of detectable fragments between U39 and U85 which confirms diselenide bond formation. (f) Mass spectrum of intact GFP U135-U177. (g) Deconvoluted mass spectrum of GFP U135-U177. (h) Experimentally determined monoisotopic masses (28013.64 Da and 27882.61 Da) match the theoretical masses of (28013.66 Da and 278861 Da) of GFP U135-U177 containing a diselenide bond ± the N-terminal methionine residue. The experimental masses have been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 28+ charge state. (j) UVPD fragment map shows a lack of detectable fragments between U135 and U177 which confirms diselenide bond formation. The lack of fragments near Y66 corresponds to the mature GFP chromophore. All spectra are representative from a minimum of two technical replicates.
