Figure 2. MS/MS spectra of crosslinked peptides of WT and F₂-Tyr157 CDO proteins and ¹⁹F NMR detection of leaving fluoride.

a, Proposed crosslink of peptides Gly80–Phe94 and His155–Phe167 through Cys93 and F₂-Tyr157. b, Mass spectrometry of the crosslinked peptide Gly80–Phe94/His155–Phe167 including one carboxyamidomethyl cysteine at m/z 3,226.4 for WT and m/z 3,244.5 for F₂-Tyr157 CDO. c, Collision-induced dissociation (CID)–MS/MS spectrum of precursor ion m/z 3,226.4 and m/z 3,244.5. d, In situ detection of fluoride during cofactor biogenesis in F₂-Tyr157 CDO. ¹⁹F NMR spectra of the fluoride ion in solution from the cross-linking reaction of F₂-Tyr157 CDO after acid precipitation of the protein. Top: ¹⁹F NMR spectrum after the conversion of F₂-Tyr157 CDO to the all-cross-linked form after 12,480 transients. Bottom: ¹⁹F NMR spectrum after spiking in aqueous KF to confirm that the signal is F^– after 128 transients. Both spectra are referenced to internal trifluoroacetic acid. Mass spectrometry experiments were repeated one time, and ¹⁹F NMR experiments were repeated two times with similar results. The F₂-Tyr157 CDO was also recorded once by ¹⁹F NMR before the autocatalytic processing reaction (a negative control) and the resulting spectrum is shown in Supplementary Figure 7.