C-terminal modification of VesB.
VesB was overexpressed and purified from supernatants of LB-grown V. cholerae culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D). A. Left panel: Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser378 (*) or Ser380 (**) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass. Right panel: The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration. B. C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser378 (*) and Ser380 (**). C. Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. D. MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y7-172 at m/z 587.3 and y10-172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C3H9O6P, 172.013 Da). E. VesB was extracted from WT V. cholerae with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. F. MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y7-172 at m/z 587.3 and y10-172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C3H9O6P, 172.013 Da). G. VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.
