Supplementary Figure 13: Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms diselenide bonds can replace essential structural disulfide bonds in therapeutically relevant protein families.
From: Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains
(a) Mass spectrum of intact seleno-anti-MS2 scFv. (b) Deconvoluted mass spectrum of seleno-anti-MS2 scFv. (c) Experimentally determined monoisotopic mass (31855.08 Da) matches the theoretical mass (31855.06 Da) of seleno-anti-MS2 containing a two diselenide bonds. 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 24+ charge state. (e) UVPD fragment map shows a lack of detectable fragments between U42 and U116 in the VH domain, and U179 and U249 in the VL domain, which confirms correct formation of both diselenide bonds. (f) Mass spectrum of intact seleno-hGH. (g) Deconvoluted mass spectrum of seleno-hGH. (h) Experimentally determined monoisotopic mass (23400.27 Da) matches the theoretical mass (23499.27 Da) of seleno-hGH containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 16+ charge state. (j) UVPD fragment map shows a lack of detectable fragments between U54 and U166 and U183 and U190 which confirms correct formation of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.
