Fig. 2: Mass spectrometry evidence for the formation of a WbbB_GT99 D232-Kdo adduct in different WbbB_GT99 active site variants.

All mass spectra were acquired in positive mode with a UHMR Orbitrap mass spectrometer, and all except c were collected for aqueous ammonium acetate solutions (200 mM, pH 7, and 25 °C) and represent the +21 charge state of the dimer of WbbB_GT99 variants. The N-terminal methionine is removed from WbbB_GT99 with variable efficiency during expression, giving three major peaks for the unlabeled protein dimer. The addition of a single Kdo residue is calculated to add 220.06 Da to the protein’s mass. a Mass spectrum of WbbB_GT99-E158Q variant purified from ClearColi^TM. b Mass spectrum of the WbbB_GT99-E158Q variant after reaction with CMP-Kdo reaction mix. Note that for peaks where both CMP and Kdo are present, this method cannot resolve whether Kdo is covalently attached to CMP or WbbB. Source data are provided as a Source Data file. c MS/MS spectrum of the singly charged peptide (Q₂₂₈VED(Hse)SNL₂₃₅) precursor ion (m/z 905.42) acquired with a Q Exactive Orbitrap mass spectrometer at a collision energy (CE) of 40 V. The homoserine (Hse) in this peptide was generated by reducing the CMP-Kdo reacted wild-type WbbB with sodium borohydride, which will only reduce carboxylate residues which have been glycosylated. d Mass spectrum of WbbB_GT99-D232C variant after reaction with CMP-Kdo reaction mix. e Mass spectrum of WbbB_GT99-D232N variant after reaction with CMP-Kdo reaction mix.